Mechanism of Action and Clinical Effects of Beta-Interferon in MS (Venice Workshop 1999)

Proceedings of the MS Forum
Modern Management Workshop

Venice, March 1999
Mechanisms of
Action and
Clinical Effects of
Beta Interferon in
Multiple Sclerosis
Chairmen: Kees Lucas; Barry Arnason
The MS Forum is supported by an unrestricted educational grant from

Schering AG, Berlin, Germany.
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Berlex Laboratories, Richmond, California, USA.
from Berlex Laboratories, Richmond, California, USA.
publication or Berlex Laboratories.
for the products.

Berlex Canada Inc., Lachine, Quebec, Canada.
from Berlex Canada Inc., Lachine, Quebec, Canada.
publication or Berlex Canada Inc.
for the products.
Proceedings of the MS Forum

Modern Management Workshop
Venice, March 1999
Mechanisms of
Action and
Clinical Effects of
Beta Interferon in
Multiple Sclerosis
Chairmen:
Kees Lucas
Leiden,
The Netherlands
Barry Arnason
Chicago, USA
Workshop Participants
Professor Barry Arnason (Chairman)
Professor Kees Lucas (Chairman)
Dr David Bates
Chicago, USA
Leiden, The Netherlands
Newcastle-upon-Tyne, UK
Professor Michel Clanet
Toulouse, France
Dr Frank Dahlke
Berlin, Germany
Professor George Ebers
Oxford, UK
Professor Cesare Fieschi
Rome, Italy
Dr Joseph Frank
Dr Sten Fredrikson
Professor Reinhard Hohlfeld
Bethesda, USA
Stockholm, Sweden
Munich, Germany
Professor Ioannis Milonas
Thessaloniki, Greece
Professor Mathias Mller
Vienna, Austria
Dr Lex Nagelkerken
Professor Chris Polman
Dr Richard Ransohoff
Dr Anthony Reder
Professor Nancy Ruddle
Dr Takahiko Saida

Amsterdam, The Netherlands
Cleveland, USA
Chicago, USA
New Haven, USA
Kyoto, Japan
THE MS FORUM
The MS Forum is an initiative funded by Berlex Laboratories
which has been established to improve the awareness and
understanding of multiple sclerosis on an international basis.
Founded in 1993, the MS Forum draws its direction from an
Executive Committee of internationally renowned opinion
leaders. It is committed to encouraging debate and exchange of
knowledge in all aspects of patient care, from which will emerge
clear and practical guidelines for the care of people with
multiple sclerosis.
ii
Dr David Bates
Chicago, USA
Toulouse, France
Dr Frank Dahlke
Berlin, Germany
Oxford, UK
Rome, Italy
Dr Joseph Frank
Dr Sten Fredrikson
Bethesda, USA
Stockholm, Sweden
Munich, Germany
Vienna, Austria
Dr Lex Nagelkerken
Dr Anthony Reder
Dr Takahiko Saida

Cleveland, USA
Chicago, USA
New Haven, USA
Kyoto, Japan
THE MS FORUM
The MS Forum is an initiative funded by Schering AG which has
been established to improve the awareness and understanding
of multiple sclerosis on an international basis. Founded in 1993,
the MS Forum draws its direction from an Executive Committee
of internationally renowned opinion leaders. It is committed to
encouraging debate and exchange of knowledge in all aspects of
patient care, from which will emerge clear and practical
guidelines for the care of people with multiple sclerosis.
ii
Dr David Bates
Chicago, USA
Toulouse, France
Dr Frank Dahlke
Berlin, Germany
Oxford, UK
Rome, Italy
Dr Joseph Frank
Dr Sten Fredrikson
Bethesda, USA
Stockholm, Sweden
Munich, Germany
Vienna, Austria
Dr Lex Nagelkerken
Dr Anthony Reder
Dr Takahiko Saida

Cleveland, USA
Chicago, USA
New Haven, USA
Kyoto, Japan
THE MS FORUM
The MS Forum is an initiative funded by Berlex Canada Inc
which has been established to improve the awareness and
understanding of multiple sclerosis on an international basis.
Founded in 1993, the MS Forum draws its direction from an
Executive Committee of internationally renowned opinion
leaders. It is committed to encouraging debate and exchange of
knowledge in all aspects of patient care, from which will emerge
clear and practical guidelines for the care of people with
multiple sclerosis.
ii
Contents
Page
ii
Introduction
iv
Chapter One
The Clinical Evidence for Beta Interferon in

Multiple Sclerosis
Chapter Two
Implications of Beta Interferon Clinical Trial Results
Chapter Three
Multiple Sclerosis Immunopathology and

Immunomodulation
Chapter Four
Biological Markers of Beta Interferon Activity
12
Chapter Five
Kinetics of Beta Interferon Treatment
16
Chapter Six
Clinical Significance of Magnetic Resonance Imaging

Findings with Beta Interferon
19
Chapter Seven
Beta Interferon-mediated Intracellular Signalling
23
Chapter Eight
Effect of Beta Interferon on Immune Regulation
27
Chapter Nine
Effect of Beta Interferon on Mediators of Inflammation
31
Chapter Ten
Effect of Beta Interferon on Immune Activation
34
Chapter Eleven
Effect of Beta Interferon on the Natural History of

Multiple Sclerosis
37
Concluding Remarks
41
iii
Introduction
Today, the clinical course of both relapsing/remitting and secondary progressive forms of multiple sclerosis (MS) can
favourably be altered using beta interferon. This is the result of almost 20 years of clinical development, beginning
with a study by Jacobs et al that was published in 1981.1 This report provided the first indication that natural human
fibroblast (beta) interferon, administered intrathecally, was capable of reducing relapse rates in people with
relapsing/remitting MS.
Since this initial report, numerous further clinical studies, including several large phase III clinical trials, have
confirmed the benefit of beta interferon in two clinical forms of MS. However, there is one important question that
has yet to be answered. How does beta interferon mediate its effects both on relapse rate and on disease
progression in MS?
The original rationale for exploring the effects of the interferons in MS was based on the premise that MS was a
virally mediated disease. Viraemic episodes frequently presage a clinical MS relapse, and it was hoped that
administering the bodys natural antiviral agents would reduce the impact of the viral episode and thereby influence
disease activity. However, this view turned out to be simplistic. A trial of gamma interferon, another innate antiviral
agent, dramatically worsened MS, suggesting that this agent has a role in the pathological processes underlying MS.2
Perhaps one of the greater challenges facing researchers trying to explain the mechanism of action of beta interferon
in MS is the complex pathological process that causes the disease. It is clear that MS has an autoimmune basis, but it
is less clear how these autoimmune reactions originate, nor exactly how the immune system causes the damage that
results in the disease. There are many possible mechanisms that could be involved, and therefore many possible
theoretical points for therapeutic intervention.
Similarly, researchers are showing that beta interferon has many diverse effects on the immune system. Some of
these may inhibit the pathological processes underlying MS, whereas others may do the opposite. The challenge is to
identify the points of interaction between MS pathology and beta interferon activity, thereby revealing the
mechanism by which beta interferon exerts its beneficial effects.
The following chapters examine the available evidence for clues that may reveal the most important of these
interactions. Under consideration are the outcomes of the major clinical trials, the MRI findings in these and other
studies, and investigations of the biological effects of the drug. Also examined are the underlying biology of beta
interferon, and its activity in relation to other inflammatory and anti-inflammatory immune mediators.
Understanding these many aspects of the biology of beta interferon, and the implications of the findings, may offer
the opportunity to enhance beneficial effects, minimise potentially deleterious ones, and, overall, improve the
treatments currently available for people with MS.
References
iv
1.
Jacobs L, OMalley J, Freeman A, Ekes R. Intrathecal interferon reduces exacerbations of multiple sclerosis. Science 1981; 214: 10261028.
2.
Panitch HS, Hirsch AL, Haley AS, Johnson KP. Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1987; i:
893895.
CHAPTER ONE
The Clinical Evidence for Beta Interferon

in Multiple Sclerosis
Beta interferon was the first agent proven to favourably alter the course of multiple sclerosis (MS). Several large
clinical trials, and many smaller trials, have demonstrated this effect using both clinical and magnetic resonance
imaging (MRI) parameters. Before considering the mechanisms underlying the observed clinical benefit, it is useful to
review recent trial outcomes to assess the magnitude of this effect in different types of MS.
Efficacy of Beta Interferon in Relapsing/Remitting MS

To date, three preparations of beta interferon have been studied in large Phase III clinical trials, and these are now
available for use in the clinics of many countries to treat people with relapsing/remitting MS. Numerous smaller trials
have also been conducted using these three agents.
Table 1 outlines the designs of three clinical trials of beta interferon.15 Each was unique in design, treatment
protocol, enrolled patient population and clinical outcomes. This prevents direct comparison of the trials.
Nevertheless, a review of the clinical trial outcomes may provide useful insights into the mechanism of action.
Duration
IFN MS Study Group13
MSCRG4
PRISMS5
2 years plus 1-year extensiona
Up to 2 years
2 years
Preparation
Interferon beta-1b
Interferon beta-1a
Interferon beta-1a
Treatment protocol
8 MIU versus 1.6 MIU

versus placebo
6 MIU versus placebo
12 MIU versus 6 MIU

versus placebo
Frequency and route

of administration
Every other day, sc
Once a week, im
Three times a week, sc
EDSS range
05.5
1.03.5
05.0
Primary outcome measures
Relapse rate
Proportion of patients relapse-free
Time to onset of
confirmed progression
Relapse rate
Secondary outcome measures
Time to first relapse

Relapse duration and severity
Hospitalisations
Change in EDSS
Proportion of patients with
confirmed progression
Relapse rate

Proportion of patients
relapse-free
Relapse severity
Steroid use
Hospitalisations
Ambulation index
Arm function tests
Change in EDSS
MRI outcome measures
Disease activity,
disease burden
Disease activity,
disease burden
Disease activity,
disease burden
rolling recruitment enabled 5-year data to be obtained in some patients
Table 1: Design of three clinical trials of beta interferon in relapsing/remitting MS
Relapse-related outcomes of the three clinical trials are outlined in table 2. These outcomes demonstrate the clinical
effect of beta interferon on disease activity, and on related clinically important consequences. The implications of
these findings will be discussed in chapter two.
Interferon beta-1a4
Outcome
Placebo
Interferon beta-1a 6 MIU
P value
Annual relapse rate
0.82
0.67 (18%)
0.04
Median time to first relapse (days)
253
331 (+31%)
ns
Proportion of patients relapse-freea (%)
26
38
nr
ns not significant; nr not reported; a subset of patients on study for 104 weeks
Table 2: Relapse-related outcomes in relapsing/remitting MS trials (continued overleaf)
ONE
Interferon beta-1a5
Placebo
Relapses per patient (mean)c
Proportion of patients relapse-freec (%)
Moderate and severe relapses per patientc
Number of steroid courses
Hospital admission due to MS (mean per patient)
2.56
135
16
0.99
1.39
0.48
Interferon beta-1a
6 MIU
12 MIU
1.82 (27%)
228 (+68.9%)
27
0.71 (26.5%)
0.97(30%)
0.38 (21%)
1.73 (33%)
288 (+113.3%)
32
0.62 (36.7%)
0.75(46%)
0.25(48%)
<0.005b
nr
<0.005b
<0.005b
<0.005b
<0.005b
Interferon beta-1b1
Interferon beta-1b
Annual relapse rate
Proportion of patients relapse-freec (%)
Annual rate of moderate and severe relapses
Number of hospital admissions due to MS
a
b
1.27
153
16
0.45
65
1.6 MIU
8 MIU
1.17 (8%)
180 (+18%)
21
0.32 (29%)
53 (18%)
0.84 (34%)
295 (+93%)
31
0.23 (49%)
37 (43%)
0.0001d
0.015d
0.007d
0.002d
0.046d
subset of patients on study for 104 weeks; ns not significant; nr not reported
12 MIU versus placebo; c over 2 years; d 8 MIU versus placebo
Table 2: Relapse-related outcomes in relapsing/remitting MS trials (continued)
Disability-related outcomes were assessed in all of the studies. In each study, progression was defined as an increase
of 1 EDSS point, confirmed after 3 or 6 months. Other disability-related outcomes were also assessed. These findings
are outlined in table 3; again, the implications of these findings are discussed in chapter two.
MRI outcomes were particularly important in all the trials. These are outlined in chapter six, together with the
implications of those findings for the mechanism of action of beta interferon.
Interferon beta-1a4
Outcome
Probability of onset of confirmed progression (%)
Placebo
P value
34.9
21.9
0.02
nr
Change in EDSS scorea

mean
0.61
0.02
improved (1 point) (%)
8.9
18.2
stable (%)
60.7
63.6
worse (1 point) (%)
30.3
18.2
0.02
Interferon beta-1a5
Interferon beta-1a
6 MIU
12 MIU
11.8
18.2 (+54%)
21.0 (+78%)
<0.05b
Change in IDSS per year
0.4
<0.05b
Change in EDSS (mean)
0.48
0.23
0.24
0.05b
Time to confirmed progressionc (months)

d
Interferon beta-1b1
Interferon beta-1be
Proportion of patients with confirmed progression (%)

Median time to progression (years)
1.6 MIU
8 MIU
46
47
35
nsf
4.18
3.49
4.79
nsf
subgroup of 111 patients (from 301) confirmed at 130 weeks; nr not reported; ns not significant
12 MIU versus placebo; c 25th percentile of progression; d IDSS = area under EDSS time curve
e 5-year data; f 8 MIU versus placebo
a
Table 3: Disability-related outcomes in the relapsing/remitting MS trials
TWO
Efficacy of Beta Interferon in Secondary Progressive MS

Only one trial of beta interferon in people with secondary progressive MS has been published.6 Further trials are
underway or are awaiting publication in this patient population. Unlike all but one trial in relapsing/remitting MS,
the primary outcome measure was disability-related. Table 4 outlines the design of the trial, and disability-related
outcomes are presented in table 5.
Duration
Treatment protocol
Frequency and route of administration
EDSS range
Primary outcome measures
Secondary clinical outcome measures
Tertiary outcome measures
MRI outcome measures
3 years, interim analysis after all subjects had completed

24 months treatment
8 MIU interferon beta-1b versus placebo
Every other day, sc
3.06.5
Time to confirmed 1 pointa progression of EDSS
Time taken to become wheelchair-bound
Annual relapse rate
Proportion of patients with confirmed progression
EDSS at endpoint
Proportion of patients with moderate or severe relapses
Disease activity
Disease burden
Other MR techniques
0.5 point between EDSS 6.07.0
Table 4: Design of the clinical trial of interferon beta-1b in secondary progressive MS
Additional points of interest include:

no effect of baseline EDSS score on the proportion of patients with confirmed progression
no effect of relapses prior to, or during, the study on the primary endpoint. However, relapses during the study
slightly increased the rate of progression in all patients.
Outcome
Placebo
Interferon beta-1b 8 MIU
P value
Delayed up to 12 months
<0.0031
38.9
0.0048
Delayed up to 9 months
<0.0133
24.6
16.7
0.0277
0.6
0.47
0.0299
Time to confirmed progression (40th percentile)

Proportion of patients with confirmed
progressiona (%)
49.7
Time taken to become wheelchair-bound

(EDSS 7.0)b
Proportion (%) of patients becoming
wheelchair-bound (EDSS 7.0)b
EDSS change from baseline
a
lost to follow-up = not progressed; unconfirmed

b
Table 5: Disability-related outcomes in the clinical trial of interferon beta-1b in secondary progressive MS
Relapse-related outcomes are described in table 6 overleaf. Due consideration is given to the behaviour of
secondary progressive MS some patients do not experience overt clinical relapses, and in those that do, relapse
rate typically is much less than in relapsing/remitting MS.
MRI outcome measures are discussed in chapter six. The outcomes of studies using the newer MR techniques,
including magnetisation transfer imaging and atrophy measurements, have not yet been published.
A second trial of beta interferon in secondary progressive MS has recently been completed. Initial reports suggest
that interferon beta-1a at doses of 6 MIU and 12 MIU three times a week failed to show a significant treatment
effect on the primary outcome measure of time to onset of confirmed progression.7 On secondary outcome
measures related to relapses, the high dose showed significant treatment effects. MRI disease activity was also
reduced, and a doseresponse effect was seen. However, the full implications of these findings must await
publication of the study results.
THREE
Outcome
Placebo
P value
overall
0.64
0.44 (31.3%)
0.0002
with prior relapsesab
0.77
0.56 (27.2%)
without prior relapsesab
0.33
0.19 (42.4%)
403
644
0.003
0.5
0.33 (34%)
0.001
Proportion of patients with moderate or

severe relapses (%)
53.1
43.6
0.0083
Proportion of patients receiving steroid therapy (%)
67.9
53.6
<0.0001
Proportion of patients hospitalised for MS (%)
42.2
33.6
Annual relapse rate
Annual rate of moderate and severe
relapsesb
prior relapses = within 2 years prior to study; b data on file
Table 6: Relapse-related outcomes in the clinical trial of interferon beta-1b in secondary progressive MS
Summary
Three preparations of beta interferon have proven to be effective in relapsing/remitting MS. The benefits are
reasonably consistent, with disease activity being reduced by 1834% depending on the trial and dosing arm. At
similar weekly doses, comparable clinical benefit was observed. Treatment benefit is also apparent on relapserelated outcomes including hospitalisations and steroid use. All the preparations reduced the number of people with
progression of disability, significantly so in two trials.
The effectiveness of beta interferon in secondary progressive MS has the support of a single published trial, although
these results are convincing. Both the time to confirmed progression and the time taken to become wheelchairbound were significantly delayed after only 1 year of treatment. Both completed trials confirm that the effects of
beta interferon on disease activity in secondary progressive MS reflect the observations in relapsing/remitting disease.
Beta interferon is, therefore, an effective treatment for people with relapsing/remitting MS, and there is convincing
data from one published study that interferon beta-1b also offers clinically meaningful benefit in people with
secondary progressive MS.
References
FOUR
1.
The IFN Multiple Sclerosis Study Group. Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a
multicenter, randomized, double-blind, placebo-controlled trial. Neurology 1993; 43: 655661.
2.
Paty DW, Li DK, University of British Columbia MS/MRI Study Group and the IFN Multiple Sclerosis Study Group. Interferon beta-1b is
effective in relapsing-remitting multiple sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial.
Neurology 1993; 43: 662667.
3.
The IFN Multiple Sclerosis Study Group and The University of British Columbia MS/MRI Analysis Group. Interferon beta-1b in the treatment
of multiple sclerosis: Final outcome of the randomized controlled trial. Neurology 1995; 45: 12771285.
4.
Jacobs LD, Cookfair DL, Rudick RA, et al. Intramuscular interferon beta-1a for disease progression in relapsing multiple sclerosis. Ann Neurol
1996; 39: 285294.
5.
PRISMS Study Group. Randomised double-blind placebo-controlled study of interferon beta-1a in relapsing/remitting multiple sclerosis. Lancet
1998; 352: 14981504.
6.
European Study Group on interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon
beta-1b in treatment of secondary progressive multiple sclerosis. Lancet 1998; 352: 14911497.
7.
Paty DW, the SPECTRIMS Study Group. Secondary progressive efficacy clinical trial of recombinant interferon beta-1a in MS (Abstract).
Presented at the 9th ENS meeting, Milan, Italy, 1999.
CHAPTER TWO
Implications of Beta Interferon Clinical

Trial Results
The results of the clinical trials in relapsing/remitting and secondary progressive multiple sclerosis (MS) clearly
demonstrate that beta interferon is beneficial for people with these forms of the disease. Careful consideration of the
trial results, however, reveals features that may have important implications for the mechanism of action of beta
interferon in MS. This chapter will consider a number of the features and will speculate on their influence on our
understanding of the mechanism of action of beta interferon.
Implications from Relapsing/Remitting MS Trials

The predominant features of relapsing/remitting MS are the random, sometimes frequent, bouts of inflammation,
demyelination and other pathological processes that result in clinically apparent signs. Magnetic resonance imaging
(MRI) also shows foci of inflammation in the brain that appear and resolve with a frequency greater than that of
clinically apparent signs. Changes in disability are less pronounced, but accumulation of MRI-determined burden of
disease is a relentless process. Thus, there are several, apparently disparate, aspects to the clinical picture of
relapsing/remitting MS.
The main trials of beta interferon in this form of MS15 are reasonably consistent in that they show:
approximately one third reduction in relapse rate at higher doses
minor doseresponse effects on most outcome measures (figure 1)
rapid onset of effect within 1 year for relapse rate and within a few weeks for MRI disease activity
disproportionately large effects on inflammation as measured by MRI disease activity
slowing in the accumulation of MRI burden of disease
a tendency to reduce the number of patients with observed progression of disability.
The doseresponse effect is observed

clinically (chapter one), and can also be
seen when the activity of beta interferon
is assessed using biological response
markers (chapter four). Increasing the
dose brings greater benefits in those trials
with two dose arms, but it is also clear
that there may be a plateau effect at
approximately 3035% reduction in
relapse rate. In the PRISMS study, the
doubling of the dose did not achieve a
proportionate reduction in the relapse
rate. In fact, increasing the dose tends to
increase the incidence of side-effects,
hinting that greater doses will adversely
affect tolerability with little clinical gain.
12 MIU 3x/wk
sc
IFN1b1,2
OWIMS5
MSRCG3,5
PRISMS4
40
35
Reduction in relapse rate* (%)
6 MIU 3x/wk
sc
8 MIU eod
sc
33
33
18.0
28.0
37
30
12 MIU
once/wk
sc
25
20
1.6 MIU eod

sc
19
6 MIU
once/wk
im
15
15
10
10
5
0
6 MIU once/wk
sc
0
5.6
* 1-year data
P<0.001 versus placebo
6.0
6.0
12.0
36.0
Weekly beta interferon dose (MIU)
The observations that severe relapses

Figure 1: Doseresponse to beta interferon in several trials
were less frequent, and that the clinical
effect on severe relapses was greater than on relapses overall in the interferon beta-1b trial, suggest that one effect of
beta interferon is to reduce the aggressiveness of ongoing inflammatory events. This is in addition to reducing the
number of such events.
None of the trial reports indicate whether beta interferon reduces the duration of relapses. This outcome is difficult
to measure, but it has implications for the mechanism of action of beta interferon. If relapse duration is unchanged,
it would suggest that once the inflammatory event has started, beta interferon does not enhance the downregulatory
FIVE
mechanisms involved in resolving the lesion. If relapses are shortened by treatment, this would suggest that
resolution of lesions is accelerated, and that beta interferon acts on both of these aspects.
An important observation from trials involving high doses of beta interferon is that the clinical benefit begins within
12 months of starting treatment indeed, an indication of the clinical benefit of beta interferon in the interferon
beta-1b trial was apparent after 2 months. Time to maximum clinical effect may be a further doseresponse effect,
implying some cumulative effect to treatment. However, the marked reduction in relapse rate after 1 year with 1.6
MIU interferon beta-1b every other day, in contrast with the lesser effect of 6 MIU interferon beta-1a once a week,
may suggest an alternative explanation that beta interferon levels need to be maintained above baseline
throughout the week to offer a treatment effect at these relatively low doses. Also of note is that, in the interferon
beta-1b trial, the magnitude of the reduction in relapse rate was sustained for up to 5 years, suggesting a long-term
benefit. What remains to be answered is whether clinical benefit persists after treatment has stopped, and if so, for
how long?
In all of the large trials, MRI parameters were measured, and the results largely supported the clinical findings.25,6
Essentially, both disease activity and the accumulation of disease burden were reduced by appropriately dosed beta
interferon. These findings are discussed in greater detail in chapter six.
Each of the beta interferon trials showed that fewer treated patients accumulated permanent neurological deficit.
Even today, the findings, especially at the lower end of the EDSS, remain controversial given the relative insensitivity
of the assessment scales and clinical uncertainty of minor changes in disability. Nevertheless, since all the trials hint
at such a benefit, and given that relapse rate and disability progression are weakly correlated, the suggestion is that
beta interferon may act directly on certain aspects of the pathology underlying disease progression, and can slow
progression of MS in the early stages of the disease. In more advanced, secondary progressive MS, the trial of
interferon beta-1b provided robust evidence to show a slowing down in the progression of disability.7
Observation
Implications
Doseresponse effect
Plateau effect
Reduction in relapse severity
No reported reduction in relapse duration
Rapid onset of activity
Sustained clinical effect
correlation between relapses
Poor
and progression of disability
High, tolerable doses are probably essential to obtain maximum benefit

Beta interferon does not address all relapse-related pathology
Beta interferon may downgrade the severity of inflammation in active lesions
Beta interferon may not accelerate the resolution of ongoing
inflammatory lesions
Beta interferon acts directly on an early step in pathology
MS pathology remains sensitive to sustained beta interferon treatment
Suggestion of disparate but linked pathology underlying each clinical aspect
Table 7: Beta interferon mechanism of action: implications from the relapsing/remitting MS studies
Implications from the Secondary Progressive MS Trial

To date, only one trial report of beta interferon in secondary progressive MS has been published.7 The findings have
important implications for the underlying pathology of secondary progressive MS, the mechanism of action of beta
interferon, and for the treatment of all people with this type of MS. A second trial has been completed, and the
preliminary findings presented,8 but an assessment of the implications of these findings for the mechanism of action
of MS must await publication of the trial report. The results of this study will not be discussed in this chapter.
The most important clinical finding of the interferon beta-1b trial7 was a marked delay in the time it took to develop
confirmed disability progression. This supports the suggestion from relapsing/remitting studies that beta interferon
can beneficially act on the pathology that underlies this aspect of the disease. From the mechanism of action
perspective, the most important finding was that this beneficial effect was observed regardless of the occurrence of
relapses during, or in the 2 years prior to, the study. This suggests that the pathology underlying disability is distinct
from the pathology that causes relapses, although the slightly more rapid progression of patients treated or not
SIX
with relapses does indicate some overlap. Thus the mechanism of action of beta interferon is likely to be sufficiently
diverse to target both pathologies.
Other clinical outcomes include the clinical effect of beta interferon on relapse rate (which was consistent with that
observed in relapsing/remitting MS an approximate 30% reduction in relapses), a dramatic reduction in MRI
disease activity, and a reduction in MRI disease burden that contrasted with an annual increase in the placebo
group.
Observation
Marked effect on disability progression

on relapses consistent
Effect
with relapsing/remitting MS trials
on progression
Benefit
independent of relapses
Implications
Confirmation that beta interferon can slow the progression of the underlying pathology
Consistent relapse-related pathology between the disease types
Disparate but overlapping pathologies responsible for relapses and progression of
disability
Table 8: Beta interferon mechanism of action: implications from the secondary progressive MS trial
Summary
Outcomes of large clinical trials of beta interferon all point to the same fact: this agent is effective in
relapsing/remitting MS and one agent is proven to be effective in secondary progressive MS. They also suggest that the
pathology of MS is complex, and that beta interferon has positive effects on various components of this diverse
pathology, either by targeting one common aspect, or by targeting several diverse features. The mechanism of action
is also likely to be consistent between the two stages of the disease and not influenced by pre-existing pathology.
These, and other interpretations of the trial outcomes, may prove to be useful in elucidating the most important
effects of this agent in MS.
References
1.
The IFN Multiple Sclerosis Study Group. Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a
multicenter, randomized, double-blind, placebo-controlled trial. Neurology 1993; 43: 655661.
2.
3.
1996; 39: 285294.
4.
1998; 352: 14981504.
5.
Freedman MS, for the OWIMS Study Group. Dose-dependent clinical and magnetic resonance imaging efficacy of interferon beta-1a (Rebif) in
multiple sclerosis. Ann Neurol 1998; 44: 992. Abstract 9.
6.
Paty DW, Li DK, University of British Columbia MS/MRI Study Group and the IFN Multiple Sclerosis Study Group. Interferon beta-1b is
effective in relapsing-remitting multiple sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial.
Neurology 1993; 43: 662667.
7.
European Study Group on interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon beta1b in treatment of secondary progressive multiple sclerosis. Lancet 1998; 352: 14911497.
8.
Paty DW, the SPECTRIMS Study Group. Secondary progressive efficacy clinical trial of recombinant interferon beta-1a in MS (Abstract).
Presented at the 9th ENS meeting, Milan, Italy, 1999.
Further Reading
New treatments for multiple sclerosis a review of clinical trials. Proceedings of the MS Forum Symposium, Istanbul, Turkey, 1997. Worthing:
PPS Europe, 1998.
SEVEN
CHAPTER THREE
Multiple Sclerosis Immunopathology and

Immunomodulation
Although the factors that induce multiple sclerosis (MS) or trigger relapses remain obscure, the pathological processes
involved in causing demyelination are generally well understood. What are less well understood are the mechanisms
that translate demyelination into sustained neurological deficit. Research continues to clarify the fine details of these
processes, and also tries to identify potential avenues by which these pathological processes could be modulated to
bring about clinical benefit. This chapter will summarise our current understanding both of the immunopathology of
MS and of the potential therapeutic approaches.
The Inflammatory Basis of MS

It is now generally agreed that the initial step in an MS episode is the activation of T cells specific for myelin
antigens. Many hypotheses have been put forward to explain this activation of autoreactive T cells, including
bystander activation, superantigen stimulation and cross-reaction based on molecular mimicry, but as yet none can
be considered to be definitive. Nevertheless, activated autoreactive T cells can be found within the circulation
(figure 2),1 where they may, in certain individuals, subsequently generate an autoimmune response.
Systemic
circulation
Loss of peripheral
tolerance
Bloodbrain
barrier
Central nervous
system
Recruitment of cells
Myelin injury
Chemokines
Microglia
T cell
Astrocyte
Activation by
superantigens, molecular
mimicry, or unknown
mechanisms
Activation of
endothelium
Cytokines
Conduction block
These inflammatory episodes are selfActivated

Autoreactive
Antigen-presenting cell
autoreactive T cell
T cell
limiting. The activation of proinflammatory cells also results in
Figure 2: Pathogenesis of MS. Reproduced with permission from Rudick et al. New Engl J Med 1997.1
stimulation of immunomodulatory
pathways, including the production of
inhibitory cytokines, which suppress the activity of autoreactive T cells, macrophages and other accessory cells. This
results in a resolution of the inflammation within several days to a few weeks.
The Pathological Consequences of Inflammation

The active inflammatory lesion can have several pathological consequences. The first, and probably the one with
minimal long-term consequences, is direct conduction block of nerve signals by pro-inflammatory cytokines
(figure 3).2 This can rapidly induce neurological dysfunction. However, the increasing concentrations of
immunomodulatory cytokines, and the consequent reduction of inflammatory mediators towards the end of the
inflammatory episode, relieve the conduction block and restore function to otherwise undamaged axons.
EIGHT
1999 Massachusetts Medical Society. All rights reserved
Activated T cells all share the ability

to cross the bloodbrain barrier.
This is normal behaviour, and
does
not
usually
incur
any
consequences. However, autoreactive
cells that subsequently encounter
their target antigen in the appropriate
context within the central nervous
system (CNS) will respond by
producing cytokines and chemoattractants that will stimulate an
inflammatory
response.
Clonal
expansion of the autoreactive T cells,
and the migration of accessory cells
into the inflamed region, enhance the
inflammatory process and lead to
tissue injury.
CONDUCTION
BLOCK
RECOVERY
The second and most widely recognised

consequence
of
inflammation
is
demyelination (figure 4).3 Loss of the
insulating myelin sheath decreases the
efficiency of action potential conduction.
This leads to reduced conduction velocity
and, in severe cases, conduction block.4
This also has less obvious but equally
important implications for the health of
the axon itself. Loss of myelin destabilises
the molecular structure of the axonal
cytoskeleton, possibly predisposing the
nerve cell to further injury.5
NORMAL
DEMYELINATION
ADAPTATION
REMYELINATION
Axonal recovery following demyelination

is a two-stage process. The first stage is
axonal adaptation, which takes place
over
several
days
and
involves
redistribution of sodium channels, a
Figure 3: Conduction block by inflammatory
process that may be under the control of Figure 4: Demyelination, axonal recovery and
cytokines
remyelination
astrocytes. Within a week, the sustained
but slow transmission of action potentials begins across the demyelinated segment, restoring some neurological
function. However, allied to this is electrical instability. Ectopic impulses may be generated, and these can travel in
either direction along the naked axon. Such ectopic
Tissue sample
Lesions
Transected axons/mm3
impulses may lead to tingling or pain sensations. The
analysed
(mean SE)
second stage of recovery is remyelination, mediated by
Active lesions
5
11,236 2275
surviving oligodendrocytes or maturing oligodendrocyte
Chronic active lesions
13
precursors within the brain parenchyma. Remyelinated
edge
3138 688
axons typically show shortened internodes and thin myelin.
core
875 246
This process re-establishes stable conduction of action
Non-lesion
white
matter
11
17 2.8
potentials at speeds approaching normal, leading, over a
Control white matter
5
0.7 0.7
period of some weeks, to partial or complete restoration of
neurological function.
Table 9: Distribution and extent of axonal transection in MS lesions
DEMYELINATION and TRANSECTION NEURONAL DEATH
1999 Lippincott Williams & Wilkins
NORMAL
Figure 5: Demyelination and axonal transection
The loss and subsequent restoration of myelin and neural function

provides a good model for the relapsing/remitting nature of early MS.
However, it is not a useful explanation of the progressive disease. The
final direct pathological consequence axonal transection (figure 5) may
provide a good explanation of the progressive worsening of MS.
In a recent study, active and chronic MS lesions were examined for
pathological axonal changes using immunohistochemistry.6 The first
important observation was extensive pathological changes to the axonal
neurofilament in MS lesions. In acutely active lesions, these injured axons
were identified throughout the inflamed area, whereas in chronic lesions
they tended to be located at the margins, where inflammation was
ongoing. The second observation, confirmed by three-dimensional
confocal microscopy, was the presence of terminal ovoids marking
transected axons. These ovoids were located in areas of active
demyelination. Table 9 shows the number of transected axons in different
types of lesion. These findings demonstrate that axonal transection is an
ongoing process even at the earliest stage of MS. Axonal loss may be a
major contributing factor to permanent deficit and cerebral atrophy,
which are both features of progressive MS.3
NINE
NORMAL
Potential Targets for Immunomodulation

There are many potential targets for immunomodulatory activity in the immunopathological process underlying MS.
These include:
activation of pro-inflammatory autoreactive T cells

reactivation of autoreactive T cells by antigen-presenting cells (APCs) within the CNS
production of pro-inflammatory cytokines within the CNS
conduction blockade by pro-inflammatory cytokines
activation of bloodbrain barrier endothelium and bloodbrain barrier transit by activated autoreactive T cells
recruitment of accessory cells
myelin injury
axonal damage, transection and loss.
In addition to these processes, the innate immunomodulatory mechanisms that self-limit the inflammatory response
may be enhanced or supplemented.
Central to any immune response is the interaction between the T cell and the APC. This is a complex event that
includes antigen recognition, accessory molecule signalling, adhesion molecule interaction, and cytokine production
and reception (figure 6).7 Any of these may impact on the outcome of the event and drive both the APC and the
T cell along a variety of outcome pathways that will influence the character of the ensuing immune response. Beta
interferon may sufficiently influence the expression of any of these molecules to modify the outcome of this
interaction. For example, it may bias the commitment of nave T cells towards the Th1 or Th2 phenotypes (chapter
eight), or it may lead to the presentation of autoantigen in a non-inflammatory context.
Cytokines
ANTIGEN-PRESENTING CELL
Cytokine
receptors
LFA-3
B7-1
B7-2
CD40
CD4
CD28
CTLA-4
CD40-L
I-CAM-1
I-CAM-2
MHC
CD2
CD3
LFA-1
Cytokine
receptors
T CELL
Cytokines
Costimulation
Integrin
T cell/MHC
complex
Adhesion
Ig Super-family member
Figure 6: The T cell:APC interaction
In one study, levels of soluble VCAM-1 (a membrane ligand for VLA-4 that can be shed into the circulation,
probably from endothelial cells) were rapidly increased in people starting beta interferon treatment.10 VLA-4 is
believed to be important in the mechanism by which inflammatory cells migrate into active lesions. Declining VLA-4
levels, and increasing soluble VCAM-1 levels, suggest a reduced potential for T cells to localise within inflammatory
lesions.11 In a second study, the ability of leucocytes to cross the bloodbrain barrier was reduced by beta interferon
treatment. This correlated with a reduction in the expression of matrix metalloproteinase-9 and the ability of this
enzyme to degrade fibronectin.12
TEN
VCAM
TCR
VLA-4
1997 Oxford University Press. Reproduced with permission
A major feature of the inflammatory

process is the recruitment of
accessory cells into the lesion;
without this influx, disease is very
limited. Studies using gadoliniumenhanced
magnetic
resonance
imaging (which reveals loss of
bloodbrain barrier integrity and
inflammation)
show
that
beta
interferon treatment almost immediately halts the development of new
lesions (chapter six).8 This antiinflammatory activity is confirmed by
a decline in leucocyte count within
the cerebrospinal fluid of treated
patients.9 How this occurs is not fully
understood, but it may involve
redirection of leucocyte trafficking,
which would imply an influence of
beta interferon on specific adhesion
molecules including the integrins.
Thus, beta interferon may have many potential immunomodulatory effects in MS, and several of those protecting
bloodbrain barrier integrity, altering cellular trafficking and reducing the capacity of cells to cross the bloodbrain
barrier have been demonstrated. Many more are under investigation.
Summary
The inflammatory mechanism that induces demyelination may be responsible for more of the pathological
consequences of MS than previously thought. Three possible outcomes can be envisaged conduction block,
demyelination and recovery, or axonal transection and there is good evidence that axonal transection occurs early
in the disease. Thus, MS may now be considered a neurodegenerative disease, different from others in that the
early, normally clinically silent, progression is illuminated by clinically apparent signs of ongoing inflammation.
Beta interferon has many effects on the ongoing inflammatory process. Perhaps the most important are protecting
bloodbrain barrier integrity, altering inflammatory cell homing to the CNS, and reducing the ability of inflammatory
cells to enter the CNS. Early treatment with beta interferon, with the aim of reducing the early accumulation of
permanent neurological damage, and searching for ways to potentiate the protective effects of beta interferon, may
offer even more effective ways to reduce the effects of this disease.
References
1. Rudick RA, Cohen JA, Weinstock-Guttman B, et al. Management of multiple sclerosis. New Engl J Med 1997; 337: 16041611.
2. Bornstein MB, Crain SM. Functional studies of cultured human brain tissues as related to demyelinative disorders. Science 1965; 148:
12421244.
3. Trapp BD, Ransohoff RM, Fisher E, Rudick RA. Neurodegeneration in multiple sclerosis: Relationship to neurological disability. Neuroscientist
1999; 5: 4857.
4. Waxman SG. Pathophysiology of demyelinated and remyelinated axons. In: Cook SD (ed). Handbook of multiple sclerosis. 2nd Edn. New
York: Marcel Dekker, 1996, 257294.
5. Kirkpatrick LL, Brady ST. Modulation of the axonal microtubule cytoskeleton by myelinating Schwann cells. J Neurosci 1994; 14: 74407450.
6. Trapp BD, Peterson J, Ransohoff RM, et al. Axonal transection in the lesions of multiple sclerosis. New Engl J Med 1998; 338: 278285.
7. Hohlfeld R. Biotechnological agents for the immunotherapy of multiple sclerosis: Principles, problems and perspectives. Brain 1997; 120:
865916.
8. Calabresi PA, Stone LA, Bash CN, et al. Interferon beta results in immediate reduction of contrast-enhanced MRI lesions in multiple sclerosis
patients followed by weekly MRI. Neurology 1997; 48: 14461448.
9. Rudick R, Cookfair D, Simonian N, et al. Cerebrospinal fluid abnormalities in a phase III trial of Avonex (IFN beta-1a) for relapsing multiple
sclerosis. J Neuroimmunol 1999; 93: 814.
10. Calabresi PA, Tranquill LR, Dambrosia JM, et al. Increases in soluble VCAM-1 correlate with a decrease in MRI lesions in multiple sclerosis
treated with interferon beta-1b. Ann Neurol 1997; 41: 669674.
11. Calabresi PA, Pelfrey CM, Tranquill LR, et al. VLA-4 expression on peripheral blood lymphocytes is downregulated after treatment of multiple
sclerosis with interferon beta. Neurology 1997; 49: 11111116.
12. Stve O, Dooley NP, Uhm JH, et al. Interferon beta-1b decreases the migration of T lymphocytes in vitro: Effects on matrix metalloproteinase-9.
Ann Neurol 1996; 40: 853863.
ELEVEN
CHAPTER FOUR
Biological Markers of Beta Interferon

Activity
Beta interferon reduces multiple sclerosis (MS) disease activity following subcutaneous or intramuscular
administration. One of the challenges of identifying the mechanism of action is that of relating the clinical benefit in
the central nervous system to the peripheral administration. Even immediately following administration, it is difficult
to detect beta interferon in the circulation with sufficient reliability. Thus, traditional assessments of the kinetics of
treatment have not been forthcoming.
An alternative approach to assessing the kinetics of beta interferon is to study its effects on biological response
markers (BRMs), levels of which respond to beta interferon administration. This chapter will review beta interferon
BRMs and discuss the implications for treatment and the mechanism of action of beta interferon.
What are Biological Response Markers?

Beta interferon has a wide
range of effects on the body.
One of these is to prepare both
the immune system and normal
body tissue to combat viral
infection (table 10). As part of
this process, the expression of a
range of cell-surface molecules
and soluble mediators, and
production of metabolites, is
altered. Expression of some of
these substances is highly
sensitive to the presence of beta
interferon, and their expression
may increase or decline
dramatically. Among those
molecules with a short half-life,
rapid expression and high peak
levels are those that are
considered to be the best
markers.
Process
Effect
Activation
Process
Tissue damage
Major histocompatibility complex (MHC)

class I expression
MHC class II expression
Lymphotoxin production by Th1 cells

Tumour necrosis factor alpha production
by macrophages
Macrophage cytolytic activity
B7.2 expression on monocytes
Suppressor cell function
Release of mitogen and oxygen

intermediates by macrophages
B7.1 expression on B cells
CD14 expression on macrophages
Recruitment
Circulating lymphocyte numbers
Lymphocyte entry to lymph nodes
Recovery
IL-10 production by macrophages
Expansion
Interleukin (IL)-2 production by Th1 cells
Transforming growth factor beta-1 production

by peripheral blood mononuclear cells
b
IL-2 receptor expression
Prostaglandin E2 release from macrophages
T cell proliferation
Lymphocyte exit from lymph nodes

Endothelial cell adhesion
molecule expression
Gamma interferon production
Antibody synthesis
Cytotoxic T cell function
Cytotoxic natural killer cell function
Cytostasis
restored to normal in people with MS; in vitro; increased; decreased; variable

b
A number of BRMs have been

used in studies of beta Table 10: The myriad effects of beta interferon
interferon (table 11). Because of
the diverse influence of beta interferon on the immune system, and the uncertainty surrounding the precise
mechanism of action of this agent in MS, the BRMs cannot be considered surrogate markers of efficacy in this
disease. Nevertheless, they can be useful in providing insight into the many characteristics of this agent.
Biological response marker
Source and function
Cellular 2',5'-oligoadenylate synthase
Interferon-induced enzyme that degrades viral RNA
Serum neopterin
A metabolite produced by interferon-activated macrophages
Serum 2-microglobulin
A component of MHC class I antigens, upregulated by interferons
IL-10
An immunomodulatory cytokine
Serum human MxA protein
An inhibitor of viral replication
Serum soluble IL-2 receptor
Soluble antagonist of the pro-inflammatory IL-2
IL-12 receptor
Cellular response to IL-12
Table 11: Some beta interferon biological response markers
Other
Trafficking
TWELVE
Effect
Insights from Biological Response Marker Studies

BRM studies have demonstrated a doseresponse to beta interferon. In one study of a
single administration of interferon beta-1b or
interferon beta-1a at a range of doses and
routes of administration, the response of three
BRMs appeared to be related to dose.1 Data
for human MxA are given in table 12. This
study also showed that there was no apparent
difference in the biological activity of the two
beta interferons at the approved dose and
route of administration.
Treatment
MIU
Human MxA
AUCc
Cmax
(ng/ml SD)
Interferon beta-1a, im
54.9 32.1
3942 2856
86.6 13.8a
6232 2406a
6b
107.0 17.5
9246 2993
106.3 26.5
8947 2652
12
159.6 11.5
14507 3614
49.2 27.7
2715 2154
96.6 20.7
8971 3656
102.8 17.6
9925 3728
130.0 11.3
12316 3154
12
134.8 10.1
12775 2679
81.1 17.3
5177 2797
85.0 34.7
6046 3232
8b
111.8 13.5
9424 3962
12
137.1 37.1
12157 3934
16
126.5 12.4
11423 1500
Interferon beta-1a, sc
Another aspect relating to dose is the duration

of biological response to beta interferon. In a
study comparing the biological response to
interferon beta-1a and interferon beta-1b
using approved doses and regimens, it was
demonstrated that once-a-week dosing was
inadequate to maintain most BRMs above
baseline throughout the week, whereas
frequent dosing sustained the BRM response
(figure 7).2
(ng.k/ml SD)
Interferon beta-1b, sc
a n = 4 (otherwise, n = 5); b clinically utilised dose and route of administration;

AUC area under the curve; c relative to baseline
Table 12: Doseresponse of the biological response marker, human MxA, to beta interferon
The influence of the route of administration on

the biological activity of beta interferon has been extensively debated. Several studies have reported contradictory
findings (table 13).1,35 Nevertheless, the available evidence favours the view that subcutaneous and intramuscular
routes of administration are equivalent in terms of their influence on beta interferon biological activity.
*#
#
*#
3
2
1
*#
#
#
*#
#
0
Median change in MxA

(ng/ml)
C
200
*#
#
150
#
#
100
#
#
#
*#
#
#
*#
50
*#
0
0
24
48
72
96
120
*#
*#
*#
# *#
*#
#
#
*#
0
Interferon beta-1a
Interferon beta-1b
2
#
*#
*#
72
96
144 168
24
48
120
Drug administration
Time on Study (hours)
Time on Study (hours)
*P<0.05, interferon beta-1b versus interferon beta-1a; #P<0.04 versus baseline
144 168
Reproduced with permission
#
#
Median change in 2microglobulin

(g/ml)
*#
Median change in IL-10

(pg/ml)
Median change in Neopterin

(ng/ml)
A 5
Figure 7: Regular dosing is required to sustain biological response to beta interferon
THIRTEEN
Study
Findings
Strzebecher et al1
Doseresponse effect; interferon beta-1a 6 MIU im, interferon beta-1a 6 MIU sc and interferon beta-1b
8 MIU sc were equivalent
Salmon et al3
Extent and duration of the biological response of 2,5-oligoadenylate synthase to interferon beta-1a
6 MIU sc or im was independent of route of administration
Alam et al4
Interferon beta-1a 6 MIU, formulated for and administered im, gave a significantly greater biological
response than interferon beta-1a 6 MIU, formulated for and administered sc
Munafo et al5
Interferon beta-1a 6 MIU formulated for sc and administered sc and im was equivalent to interferon
beta-1a 6 MIU that had been formulated for and administered im
Table 13: Biological response marker study findings
The Biological Response to Treatment

In addition to the specific BRMs used to assess the kinetics of beta interferon treatment, there are many other
biological effects seen as a direct consequence of beta interferon treatment. Among these are the clinical outcomes
changes in relapse rate and MRI activity in particular but also effects on cytokines, adhesion molecules and other
parameters of the immune system, as well as side-effects, injection-site reactions and neutralising antibody.
In people receiving long-term beta interferon treatment, there is evidence to support an influence on gamma
interferon and gamma interferon-secreting cells; an increase in interleukin (IL)-10 production, with a possible
correlation with clinical response; a reduction in tumour necrosis factor alpha production; and other changes. These
all point towards a shifting of the immune response towards the Th2 phenotype (chapter eight), although it is not yet
clear whether the change in IL-10 production is a feature of T cells or monocytes.
Several groups have examined the change in IL-10 production in people treated with beta interferon. Kinetic studies
after the first and fifth injection of interferon beta-1a induced significant elevations of serum IL-10 within 48 hours.6
In the same study, levels of IL-10 in the cerebrospinal fluid were increased in people with a favourable clinical
outcome at 2 years, compared with those that continued to show progression of disease. Also reported in this study
was an immediate increase in the production of IL-4 but a slightly reduced gamma interferon production by
peripheral blood mononuclear cells. The IL-10 observations are important since this cytokine is key in promoting
and mediating Th2 responses, and levels appear to be correlated with clinical outcome in MS.
Others have reported different findings. In one series, IL-10 production in people starting treatment increased
markedly, but returned towards baseline within a few months.7 There were differences between individuals in terms
of the IL-10 response, and some evidence to suggest an association between high IL-10 production and improved
clinical outcome. Overall, there is evidence that MS disease activity is related to elevated IL-12 and reduced IL-10
levels, and that beta interferon acts by normalising levels of both cytokines and shifting the immune system towards
the Th2 phenotype (chapter eight). However, there are many outstanding questions to be resolved.
Evidence for a shift towards the Th2 phenotype comes from reports of increased production of autoantibodies. This
is a well-recognised consequence of alpha interferon treatment, and some studies suggest that this is also true for
beta interferon.8 Autoantibodies also tend to be more frequent in autoimmune diseases regardless of treatment. It
remains to be determined whether development of these autoantibodies is a common event and if they will lead to
clinically manifest disease in some treated individuals. Isolated case reports suggest autoantibody-mediated
alterations to liver and thyroid function.
Neutralising antibodies to beta interferon are a recognised phenomenon in people receiving this treatment.
Depending on the assay and the threshold criteria used to assess neutralising antibody titres, between 10% and 30%
of people will, at some time, develop neutralising antibody. However, it is not yet clear whether different preparations
are more or less antigenic, nor is the influence of altering route or frequency of administration understood.
These antibodies may have in vivo consequences. In a study of people receiving interferon beta-1a once-weekly by
subcutaneous injection, the presence of neutralising antibody was correlated with a reduced response of neopterin
FOURTEEN
and 2-microglobulin. There was also a trend towards reduced treatment benefit on MRI outcomes.9 In the North
American trial of interferon beta-1b in relapsing/remitting MS, presence of neutralising antibody suggested reduced
clinical efficacy in cross-sectional analyses for relapse reduction and MRI activity.10 However, when neutralising
antibody titres were assessed longitudinally, many patients reverted to an antibody-negative status. Longitudinal
analysis found only an indication of attenuated treatment effect on relapse rate. No significant correlation between
antibody status and progression or MRI outcomes at the 8 MIU dose were found.11 It is interesting to note that many
individuals who develop antibody revert to, and remain, neutralising antibody-negative. In one study that followed
individuals treated with interferon beta-1b for over 8 years, neutralising antibody disappeared in the majority of
patients.12
Summary
BRMs might be a useful tool to monitor the kinetics of beta interferon treatment. They support the view that
intramuscular and subcutaneous routes of administration are equivalent, and have been used to demonstrate a
doseresponse effect and a sustained effect only with frequent dosing. The biological response to treatment also
suggests that IL-10 levels may be increased at least over the short term and that this may bias the immune system
towards a Th2 phenotype. Evidence of increasing autoantibody levels supports this view. Neutralising antibody is
also a biological reaction that may, over the short term, influence clinical efficacy of beta interferon, but long-term
follow-up suggests that the majority of treated individuals revert to antibody-negative status.
References
1. Strzebecher S, Maibauer R, Heuner A, et al. Pharmacodynamic comparison of single doses of interferon beta-1a and interferon beta-1b in
healthy volunteers. J Interferon Cytokine Res 1999; 19: in press.
2. Williams GJ, Witt PL. Comparative study of the pharmacodynamic and pharmacologic effects of Betaseron and AVONEX. J Interferon
Cytokine Res 1998; 18: 967975.
3. Salmon P, Le Cotonnec J-Y, Galazka A, et al. Pharmacokinetics and pharmacodynamics of recombinant human interferon-beta in healthy male
volunteers. J Interferon Cytokine Res 1996; 16: 759764.
4. Alam J, Goelz S, Rioux P, et al. Comparative pharmacokinetics and pharmacodynamics of two recombinant human interferon beta-1a products
administered intramuscularly in healthy male and female volunteers. Pharmaceutical Res 1997; 14: 546549.
5. Munafo A, Lugan-Trinchard I, Nguyen TXQ, Buraglio M. Comparative pharmacokinetics and pharmacodynamics of recombinant human
interferon beta-1a after intramuscular and subcutaneous administration. Eur J Neurol 1998; 5: 187193.
6. Rudick RA, Ransohoff RM, Lee J-C, et al. In vivo effects of interferon beta-1a on immunosuppressive cytokines in multiple sclerosis. Neurology
1998; 50: 12941300.
7. Rep MHG, Schrijver HM, van Lopik T, et al. Interferon-beta treatment enhances CD95 and IL-10 expression but reduces interferon-gamma
producing T cells in MS patients. J Neuroimmunol 1999; 96: 92100.
8. Durelli L, Ferrero B, Oggero A, et al. Autoimmune events during interferon beta-1b treatment for multiple sclerosis. J Neurol Sci 1999; 162:
7483.
9. Rudick RA, Simonian NA, Alam JA, et al. Incidence and significance of neutralising antibodies to interferon beta-1a in multiple sclerosis.
Neurology 1998; 50: 12661272.
10. The IFN MS Study Group, the University of British Columbia MS/MRI Analysis Group. Neutralising antibodies during treatment of multiple
sclerosis with interferon beta-1b: Experience during the first three years. Neurology 1996; 47: 889894.
11. Petkau J, White R. Neutralising antibodies and the efficacy of interferon beta-1b in relapsing-remitting multiple sclerosis (Abstract). Mult Scler
1997; 3: 402.
12. Rice GP, Pazner B, Oger J, et al. The evolution of neutralising antibodies in multiple sclerosis patients treated with interferon beta-1b.
Neurology 1999; 52: 12771279.
FIFTEEN
CHAPTER FIVE
Kinetics of Beta Interferon Treatment

Beta interferon has many effects within the body. As an immune modulator, it is capable of inducing the expression of
proteins in both cells of the immune system and normal tissue cells as part of the overall programme to control
infection. Of the many events that are influenced by beta interferon either directly, or indirectly as a consequence of
the activity of induced proteins only a small proportion are likely to be responsible for the clinical benefit obtained by
beta interferon treatment in multiple sclerosis (MS). This chapter will examine key features of the response to beta
interferon and highlight the clues that could tell us more about the mechanism of action of this agent.
How does beta interferon exert its clinical effect?

The complexity of the bodys response to beta interferon enables the expression of many proteins that may
themselves have either a direct or an indirect impact on the disease. By carefully studying the cascade of events
following beta interferon administration, some clues may be found that tell us which of these effects are due
directly to beta interferon, or are due to one of its induced products.
A
Positive effect on disease
Negative effect on disease
A...R Consequences*
Beta interferon
F
Time (hours)
N
O
Q
+
R
Time (weeks)
*such as elevated cytokine production, reduced cell activity, etc.
Clinical Observations with Beta Interferon Treatment

In almost all trials of beta interferon, the observed clinical effect has been measurable within weeks of commencing
treatment. However, it is not immediate on all outcome measures. Reductions in relapse rate tend to become
apparent from the second month of treatment, reaching a sustained maximum treatment effect from the third
month. This suggests that the clinically beneficial effect is not immediate either because a period of sustained
treatment is required to reduce the onset of relapses, or because relapses initiated before treatment was started
continue to cause clinical disease.
This is in contrast with the observed effects on gadolinium-enhanced magnetic resonance imaging (MRI) scans,
which reveal a rapid decline in the total number of active, enhancing lesions. MRI findings generally reveal a
maximal effect within 2 months of starting treatment and may be much faster.1
Flu-like Symptoms
Another clear consequence of beta interferon treatment is the occurrence of flu-like symptoms. These symptoms
occur following administration of alpha, beta or gamma interferon. Severity appears dose-dependent and inversely
related to body size. Flu-like symptoms also show kinetics:
in the short term, with symptoms appearing within 46 hours and fading after a further 48 hours
in the longer term, where the incidence of flu-like symptoms declines from over 56% of patients experiencing
these symptoms to approximately 10% of patients by 3 months of treatment, although they may recur at any
time.
SIXTEEN
The short-term kinetics of flu-like symptoms related to beta interferon are similar to those of alpha and gamma
interferon. They suggest a direct, or almost direct, stimulation by beta interferon of endogenous pyrogens such as
interleukin (IL)-1, IL-6 and tumour necrosis factor alpha (TNF). It is interesting to note that beta interferon can
induce gamma interferon and that this can induce IL-6 and TNF. It is also of note that neopterin, a useful biological
response marker, responds far more sensitively to gamma interferon than to beta interferon, and that levels of
HLA-DR an antigen of the major histocompatibility complex are elevated on circulating monocytes 12 weeks
after starting treatment. This is another gamma interferon-mediated response.
In the longer term, the decline in beta interferon-related side-effects reflects changes in the levels of gamma
interferon-secreting cells in the circulation. Immediately following initiation of beta interferon treatment, levels of
circulating gamma interferon-secreting cells increase in approximately 60% of patients.2 Longer-term follow-up
shows that the number of these circulating cells had returned to normal within 3 months. The longer-term profile of
flu-like reactions to alpha interferon reflects that of beta interferon; however, in patients receiving gamma interferon
there is no decline in the frequency of symptoms over time.
Injection-site Reactions
Injection-site reactions are also typical in people injecting beta interferon via the subcutaneous route. Again, they
have kinetics that can provide clues to their aetiology. Onset of these reactions occurs 1224 hours after injection,
peaks within 12 weeks and they resolve after 34 weeks. The erythema is largely due to vasodilatation, and may be
a consequence of a depot effect of administered beta interferon.
Histology of these lesions suggests that cellular infiltration begins within 18 hours of beta interferon administration.
Recruited cells include CD8+ T cells, activated macrophages and a limited number of natural killer cells. In terms of
the inducing agent, vasodilatation occurs in response to nitric oxide, which is itself produced in response to either
gamma interferon, TNF or lipopolysaccharide. CD8+ cells are preferentially activated by alpha and beta interferon,
and macrophages mature and express HLA-DR in response to gamma interferon.
Implications for the Mechanism of Action

Beta interferon administration has many effects. Some are mediated directly, such as the preferential recruitment of
CD8+ T cells and expression of 2-microglobulin, and some are mediated indirectly. Many of the clinical
consequences of beta interferon administration, such as flu-like symptoms or injection-site reactions, and molecular
changes such as neopterin production, macrophage maturation, HLA-DR expression, and pyrogen production, may
be due to the increased expression of gamma interferon, which itself is directly enhanced by beta interferon.
Perhaps part of the mechanism of action of beta interferon in MS consists of a normalisation of gamma interferon
responses an effect that becomes fully established after approximately 3 months.
The effects of beta interferon on CD8+ T cells could have far-reaching consequences. Proliferation of CD8+ T cells,
in part, reflects the antiviral activities of a subset of these cells in a response that supplements the innate antiviral
properties of beta interferon. However, CD8+ CD28 T cells are regulatory in nature under the appropriate
circumstances, they can produce immunosuppressive molecules such as transforming growth factor beta and the
recruitment of these cells may also enhance immune suppression.
In animal models, CD8+ T cells are implicated in the prevention of relapses in experimental allergic
encephalomyelitis, but not in recovery from a relapse; CD8 knockout mice typically exhibit relapsing and remitting
disease, whereas normal mice generally experience monophasic disease.3 In people with MS, CD8+ T cell function
appears to be disturbed, and this may reflect one aspect of the immune dysfunction responsible for MS.4 In vitro and
clinical evidence suggests that beta interferon normalises CD8+ T cell function, and this may enhance protection
against onset of new relapses.
SEVENTEEN
How beta interferon may exert its clinical effect possible effects
The consequences and kinetics of events following beta interferon administration suggest several that may
contribute to the clinical benefit of the agent.
Induce CD8+ T cells
Beta interferon
Induce MHC class I

Induce IL-10, IL-4
+ Normalise CD8+ suppressor activity

?
Increase viral surveillance
+ Divert immune responses

towards a Th2 phenotype
+ Antagonise gamma interferon
Induce gamma interferon
Induce neopterin
Induce pyrogens and fever
+ Normalise gamma interferon

production
Downregulate adhesion
molecules
+ Positive effect on disease
Negative effect on disease
+ Reduce inflammatory cell

homing to the CNS
? Uncertain effect on disease
Summary
The biological response to beta interferon is diverse and can be revealed not only by changes in molecular biological
response markers, but also by clinical effects including MRI outcomes and side-effects of treatment. These all suggest
a cascade of events following beta interferon administration, several of which may contribute to the clinical benefit
of the agent. Among these events are some that are likely to be mediated by gamma interferon, including some of
the less desirable outcomes, and some directly mediated by beta interferon, such as restoration of poor suppressor
T cell function. These events provide solid evidence for the activity of beta interferon, and suggest important routes
by which beta interferon may exert clinical benefit.
References
1.
Calabresi PA, Stone LA, Bash CN, et al. Interferon beta results in immediate reduction of contrast-enhanced MRI lesions in multiple sclerosis
patients followed by weekly MRI. Neurology 1997; 48: 14461448.
2.
Dayal AS, Jensen MA, Lledo A, Arnason BGW. Interferon-gamma-secreting cells in multiple sclerosis patients treated with interferon beta-1b.
Neurology 1995; 45: 21732177.
3.
Arnason BGW, Dayal A, Qu Z-X, et al. Mechanisms of action of beta interferon in multiple sclerosis. Semin Immunopathol 1996; 18: 125148.
4.
Antel JP, Arnason BGW, Medof ME. Suppressor cell function in multiple sclerosis: Correlation with clinical disease activity. Ann Neurol 1979;
5: 338342.
EIGHTEEN
CHAPTER SIX
Clinical Significance of Magnetic

Resonance Imaging Findings with
Beta Interferon
Magnetic resonance imaging (MRI) is an immensely valuable technique that can objectively assess changes in the
central nervous system (CNS). In multiple sclerosis (MS), it is a powerful tool that allows quantification of disease
activity, disease burden and pathological and biochemical changes as the disease progresses. It is of greatest
importance as a tool for use in clinical trials as an efficacy outcome measure, but it may also provide useful insights
into the mechanism of action of beta interferon in MS.
MRI in the Beta Interferon Trials

In the clinical trials of the three beta interferon preparations, MRI proved to be very informative. All the trials
assessed both changes in T2-weighted burden of disease and disease activity according to the number of new, active
or enlarging lesions. These two techniques assess different aspects of MS pathology, much as EDSS and relapse rate
assess different clinical aspects. Each can provide information relating to the mechanism of action of beta interferon.
Several trials also used gadolinium (Gd)-enhancement as a measure of disease activity. This technique very likely
enables bloodbrain barrier disruption to be visualised.
Despite the variety of protocols used, the MRI findings are reasonably consistent between the various trials (tables
14ad).16 MRI-determined disease activity is suppressed by 6090%. This depends on a number of factors,
including the dose used and the assessment technique, and contrasts with the modest 3035% reduction in clinical
relapse rate. Also notable is the rapid onset of effect, which typically reaches maximum within 3 months. These
observations suggest that the pathological processes visualised by MRI and those responsible for clinical relapses
overlap, but that they are not identical. This may simply be due to the inability of MRI to elucidate all relapseassociated pathology. However, it does indicate that different aspects of the mechanism of action of beta interferon
are being observed using clinical and MRI assessments.
Measure
Placebo
n
Mean SD

n
Mean SD
P value
Gd-enhancing lesion number

baseline
132
2.32 0.37
141
3.17 0.62
ns
at 1 year
123
1.59 0.31
134
1.04 0.28
0.02
at 2 years
82
1.65 0.48
83
0.80 0.22
0.05
baseline
132
219.0 36.2
140
255.0 45.1
ns
at 1 year
123
96.5 21.2
134
70.0 24.9
0.02
at 2 years
82
122.4 48.5
82
74.1 38.3
0.03
Median (range)
Median (range)
Gd-enhancing lesion volume (mm3)
T2 lesion volume (mm3)

baseline
change at 1 year
12,075 (nr)
113
455
120
(765019,035)
baseline
change at 2 years
1410
(988530,645)
0.01
152
ns
(10,45517,655)
13,620 (nr)
80
9238 (nr)
78
10,210 (nr)
ns
628
ns
(563030,430)
ns not significant; nr not reported
Table 14a: MRI outcomes of the beta interferon trials interferon beta-1a3,4
NINETEEN
MRI burden of disease continues to increase in untreated individuals at approximately 5% per year. This is indicative
of the accumulation of residual pathology. However, there was a very poor correlation between this accumulation
and the increase in clinically apparent neurological impairment again indicating a disparity between clinical and
MRI parameters. This was further emphasised by the almost complete suppression of MRI disease burden
accumulation in the interferon beta-1b trial over 5 years, and the failure to demonstrate convincingly a slowing of
disease progression in this trial. Thus, until the pathological consequences of MRI disease burden are more clearly
understood, the implications of the treatment effect of beta interferon on this parameter are unclear.
Measure
Placebo
T2 lesion burden (% median change over 2 years)
Interferon beta-1a
6 MIU
12 MIU
P Value
1.2
3.8
<0.0001b
67a
78a
<0.0001b,c
22
T2 active lesion number (%)

a
versus placebo; b 12 MIU versus placebo; c 12 MIU versus 6 MIU (P = 0.0003)
Table 14b: MRI outcomes of the beta interferon trials interferon beta-1a5
Measure
Placebo
Active scans (change from placebo)

Active lesions/year
New lesions/year
T2 lesion areab
baseline (median/mm2)
change at 1 year (%)
change at 2 years (%)
a
Interferon beta-1b
1.6 MIU
8 MIU
P valuea
29.4%
3.0
2.0
11.8% (60%)
1.0 (66%)
0.5 (75%)
5.9% (80%)
0.5 (83%)
0.5 (75%)
0.006
0.009
0.002
1503
6.7
11.9
21.0
18.7
30.2
1086
5.7
12.4
6.1
11.7
10.6
1525
4.9
5.6
3.8
0.8
3.6
ns
0.0012
0.0015
0.0002
0.0055
0.0363
8 MIU versus placebo; b in 217 patients with 4- or 5-year scans; ns not significant
Table 14c: MRI outcomes of the beta interferon trials interferon beta-1b (relapsing/remitting MS)1,2
Although most of the trials

included people with
New lesions (months 06)
10.4
3.6 (65%)
<0.0001
relapsing/remitting
New lesions (months 1824)
7.0
1.6 (78%)
0.0008
disease, the one published
T2 lesion volume
trial in people with
baseline (median/mm3)
23,820
21,600
ns
change at 1 year (%)
1.8
4.9
secondary progressive MS
2.2
6.8
<0.0001
reported
similar
MRI
10.8
5.2
findings. Disease activity
over the first 6 months of
ns not significant
the study, and between
Table 14d: MRI outcomes of the beta interferon trials interferon beta-1b (secondary progressive MS)6
months 18 and 24, was
substantially reduced, and progression of disease burden was comparable with that in the relapsing/remitting studies
despite a substantially greater baseline disease burden.
Measure
Placebo
P value
Overall, there are a number of implications that can be drawn from these findings:
newly active MRI lesions are a consistent feature of untreated people, reinforcing the view that MS is an ongoing,
rather than an episodic, inflammatory disease
beta interferon has a marked treatment effect on disease activity
in people receiving no treatment, a steady accumulation of disease burden of 510% per year is observed, reflecting
gross damage within the brain. This finding appears to be true in both mild and more severe disease cases
beta interferon can slow, halt or slightly reverse accumulation of disease burden, and this can be sustained over
several years. However, the bulk of existing burden remains, suggesting that much of the observed disease
burden is permanent or escapes the effect of beta interferon.
TWENTY
Further MRI Studies with Beta Interferon

One of the challenges of large clinical trials is that the MRI outcome measures are normally assessed in a crosssectional fashion. Longitudinal assessment of MRI parameters can provide detailed insight into the behaviour of MS
over time. As part of a natural history study, one group with MS was monitored from early in the disease every
month using several MRI techniques.7,8 These studies show that the number of Gd-enhanced lesions correlating with
bloodbrain barrier disruption can vary quite dramatically from month to month within individuals. There is a good
correlation between bursts of MRI activity and clinically apparent relapses. However, each individual had a
characteristic level of disease activity when averaged over several months. By contrast, the level of disease activity
between individuals could vary considerably. Similarly, total T2-weighted disease burden was shown to vary from
month-to-month, but overall, it increased with time. Both the intra- and inter-individual variations observed in these
serial studies may explain why the correlations between disease burden and EDSS scores in cross-sectional studies
tend to be weak.
The treatment effect of beta interferon on individuals assessed in this way is marked. In the group of 14 individuals
studied by Stone et al, mean total contrast-enhancing lesion number in the 7 months prior to starting interferon
beta-1b was 3.46 0.83, and during the 6 months following treatment was 0.48 0.16, representing an 86%
reduction in disease activity.8 In longer-term follow-up, suppression of disease activity remains substantial, but there
is some variability after 23 years (figure 8).9 Similarly, T2 lesion load shows initial reductions that are followed by
increased variability and a trend towards increase by 3 years. This variability may be due to a number of factors.
NIH baseline vs treatment interferon beta-1b trial (32 RRMS Pts)

10
Total lesions
Interferon beta-1b
BWMLL
9
Mean total lesions/Pt/Mo
7
6
5
4
3
2
Bulk white matter lesion load (cm3)
Part of the variability in these observations

derives from individual responses to
treatment. Some individuals respond
immediately and completely to interferon
beta-1b, while others continue to develop a
reduced number of new enhancing lesions.
Over time, some individuals fail on
treatment, and they present with increasing
numbers of enhancing lesions that can
approach baseline levels. In most cases, this
is largely correlated with the appearance of
neutralising antibodies. However, the
presence of neutralising antibodies does not
necessarily imply a return of MRI lesions
some individuals with particularly high
neutralising antibody titres continue to show
an almost complete absence of enhancing
lesions. In addition to treatment failure, a
small proportion of individuals simply fail to
respond to beta interferon treatment with a
reduction in MRI disease activity.
1
0
8 4 2 0 2
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Month
Figure 8: Mean disease activity and burden in 32 patients over 3 years
One interesting observation from serial studies is that cessation of beta interferon treatment in individuals responding
to treatment does not necessarily lead to an immediate rebound in disease activity or continued accumulation of
disease burden. This may have implications both for the clinical management of people on treatment and for the
mechanism of action of beta interferon.
The advent of newer MRI techniques is also adding to the information on the dynamics of disease activity in
individuals. Magnetisation transfer ratio imaging provides a good marker of permanent damage resulting from
inflammatory lesions, while atrophy measurement is providing some evidence that beta interferon can slow the
decline in brain volume.
TWENTY ONE
Summary
MRI provides an objective tool to assess both disease activity and accumulation of disease burden in people with
MS. It is apparent that MS is an ongoing, active disease, even in the early stages. Lesions are characterised by a loss
of bloodbrain barrier integrity and subsequent inflammation. There is also a notable heterogeneity in MRI activity
within the population. Treatment with beta interferon can almost completely suppress the appearance of new
inflammatory lesions within a matter of a few weeks, and there is some evidence to suggest that the benefit of
treatment can be sustained for some weeks after treatment itself has ceased. However, some individuals fail on
treatment, and this may be due to the presence of neutralising antibody. Newer MRI techniques will provide greater
pathological specificity, which, it is hoped, will be able to shed more light on the mechanism of action of beta
interferon.
References
1.
Paty DW, Li DKB, the University of British Columbia MS/MRI Analysis Group. Interferon beta-1b is effective in relapsing-remitting multiple
sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial. Neurology 1993; 43: 662667.
2.
The IFN MS Study Group, the University of British Columbia MS/MRI Analysis Group. Interferon beta-1b in the treatment of multiple
sclerosis: Final outcome of the randomized controlled trial. Neurology 1995; 45: 12771285.
3.
1996; 39: 285294.
4.
Simon JH, Jacobs LD, Campion M, et al. Magnetic resonance studies of intramuscular interferon beta-1a for relapsing multiple sclerosis. Ann
Neurol 1998; 43: 7987.
5.
1998; 352: 14981504.
6.
European Study Group on Interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon beta1b in treatment of secondary progressive multiple sclerosis. Lancet 1998; 352: 14911497.
7.
Harris JO, Frank JA, Patronas N, et al. Serial gadolinium-enhanced magnetic resonance imaging scans in patients with early, relapsing-remitting
multiple sclerosis: Implications for clinical trials and natural history. Ann Neurol 1991; 29: 548555.
8.
Stone LA, Frank JA, Albert PS, et al. The effect of interferon beta on bloodbrain barrier disruptions demonstrated by contrast-enhanced
magnetic resonance imaging in relapsing/remitting multiple sclerosis. Ann Neurol 1995; 37: 611619.
9.
Frank JA, McFarland HF. Personal communication.
Further Reading
Imaging in Multiple Sclerosis. Proceedings of the MS Forum Modern Management Workshop, 1997, Aylesbury, UK. Worthing: PPS Europe, 1997.
New Treatments for Multiple Sclerosis A Review of Clinical Trials. Proceedings of the MS Forum Symposium, 1997, Istanbul, Turkey. Worthing:
PPS Europe, 1998.
TWENTY TWO
CHAPTER SEVEN
Beta Interferon-mediated
Intracellular Signalling
At the most fundamental level, the mechanism of action of beta interferon will depend on the intracellular signalling
pathway that translates the presence of the molecule at the cell surface into a pattern of gene expression within the
cell nucleus. The specific response of the beta interferon receptor, overlap between intracellular signalling pathways
of beta interferon and other mediators, and the resultant pattern of gene expression, will all contribute to our
understanding of the biological activity of this agent. This chapter will consider all these aspects of intracellular
signalling in response to beta interferon, and suggest possible mechanisms of action.
Intracellular Signalling The Model Pathway

Over recent years, a prototype model has been
developed to describe the signalling pathway that
conveys a chemical signal the presence of a
cytokine, a hormone or other signalling molecule
or a receptor-mediated intercellular signal at the
surface of the cell into an altered pattern of gene
expression within the cell nucleus. The following
events form the basis of this signalling pathway
(figure 9):
binding of a chemical messenger or receptor
ligand to a specific receptor at the cell surface,
resulting in a signal
transduction of the signal across the cell
membrane, either directly or via an accessory
molecule
phosphorylation of a specific intracellular
protein by the receptor or accessory molecule
transport of the phosphorylated intracellular
signalling protein, or transduction of the signal
via additional molecules, to the nucleus
interaction between the phosphorylated
signalling protein and transcription control
elements, leading to modified gene expression.
Chemical messenger
Receptor
Cell membrane
Phosphate
group
Signal transduction
Phosphorylation of
signalling molecule
P
CYTOPLASM
Direct
transport
Signal transduction
NUCLEUS
Direct
signalling
Regulatory protein
Indirect
signalling
Transcription
DNA
Figure 9: The model intracellular signalling pathway
In reality, there are many different pathways by which a signal can be transduced to the cell nucleus, and many of
these pathways overlap and interact. This allows for great flexibility in the cellular response to different stimuli, but
great complexity when attempting to understand the specific responses to a single stimulus.
Beta Interferon Intracellular Signalling

Only in the last few years has the signalling pathway used by the interferons been identified, and work continues to
address many outstanding questions. Cloning of the human genes for the type I (alpha and beta) and type II (gamma)
interferon receptors enabled researchers to show that none of the receptor chains contained intrinsic tyrosine kinase
activity within their intracellular domains an essential function of signal transducers at the cell surface. This implies
that, in order to activate an intracellular signalling pathway, an accessory molecule with this activity must be
associated with these receptor chains.1
The Janus family of tyrosine kinases (JAK) was first discovered using genetic screening techniques that identified
genes with tyrosine kinase catalytic domains.2 Further analysis revealed a family of four mammalian JAK kinases with
common features (see box: The JAK family of tyrosine kinases). It was found that members of the JAKs bound
TWENTY THREE
specifically to cytokine receptor chains and were

able to activate the next step in the transduction
pathway.
The JAK family of tyrosine kinases

The Janus (JAK) family of tyrosine kinases are so-called because they share two
kinase-like domains. One is active, the other is probably enzymatically inactive
but it may play a role in the interaction with some cytokine receptors. There are
four mammalian JAKs JAK1, JAK2, JAK3 and Tyk2. JAKs are activated by many
cytokines; however, there is some degree of specificity in the JAK that is
activated by a specific cytokine receptor, and this may play some role in
controlling the response to the cytokine.
This next step in signal transduction involves a

family of signal transducers and activators of
transcription (STATs) see box: The STAT family of
signal transducers. STATs are selectively activated by
specific JAKs and also by certain cytokine receptors
that carry intrinsic tyrosine kinase activity. The roles
of STAT1 and STAT2 as important signal transducers
for the interferons have been elucidated.35
Pseudokinase
Kinase
C
JH7
JH6
JH5
JH4
JH3
JH2
JH1
JH JAK homology
The STAT family of signal transducers
How do These Components

Fit Together?
The STAT family of transcription factors is encoded from seven unique Stat
genes. However, differential processing of the mRNA allows for the generation
of more than seven end proteins. As with the JAKs, different cytokine receptors
will induce the activation of different STATs. STAT homo- or heterodimers travel
to the nucleus where, with or without interacting with additional control
proteins, they can regulate gene transcription.
The type I interferon receptor binds all the alpha

interferon sub-types and beta interferon. The type II
interferon receptor binds gamma interferon only.
Both are believed to be heterodimers. However,
whereas both type I receptor chains bind interferon,
only one type II receptor chain has a binding site.
The signal transduction pathway for type I
interferons is as follows:
Tyr
Ser
C
DNA-binding
domain
SH Src homology
SH3-like
SH2
Transactivation
domain
Tyr tyrosine phosphorylation site

Ser serine phosphorylation site
the two receptor chains, IFN R1 and IFN R2,

are both constitutively associated with members of the JAK family IFN R1 with Tyk2; IFN R2 with JAK1
binding of type I interferon to the receptor chains causes them to aggregate, allowing Tyk2 and JAK1 to activate
each other
ligand binding also causes the intracytoplasmic receptor domain to become receptive to STAT binding
bound STATs become activated by the receptor-associated JAKs
activated STATs form homo- or heterodimers that move into the cell nucleus where, either alone or in
association with other proteins, they bind to gene promotors and induce, or suppress, expression (figure 10).6
Diverse JAK/STAT
Signalling
Interferon /
JAK1
Tyk2
P
Tyk2
JAK1
P
ST
AT
3
P P
STAT3
STAT2
STAT1
P
Although the JAK/STAT pathway was

identified initially by its role in
interferon signalling, it is increasingly
recognised that many cytokines utilise
this pathway.7 This raises an
important question: how are these
diverse signals transduced to the cell
nucleus using this one system, and
why are the various outcomes of
activators still specific for a given
signal?
STAT2
STAT1
STAT1
STAT5
p48
STAT4
P
ST
AT
3
P
P
STAT1
GAS
ST
AT
3
ST
AT
3
GAS
Figure 10: JAK/STAT signalling and type I interferons
TWENTY FOUR
STAT1
P
STAT1
GAS
ST
AT
1
STA
T2 P p48
ISRE
ISGF3
Specificity in the JAK/STAT pathway is

achieved at two levels. First, of the
four JAK family members and the
seven STAT members, each cytokine receptor may

only bind certain JAKs and can activate only certain
STATs (table 15). In addition, different STATs will
only recognise, and bind to, particular DNA
response elements (the supplementary transcription
control factors) and DNA promotor sequences,
thereby limiting the genes that they may activate.
However, this pathway does answer one important
question regarding cytokines. The complex signal
pathway may go some way to explaining why the
biological activity of many cytokines overlap, and
also why biological activity of one cytokine can be
modified by costimulation of target cells using a
second cytokine or more.
It is also becoming clear that the JAK/STAT pathway
is not active in isolation. Some cytokine and growth
factor receptors do not need JAKs to activate STATs.
JAKs are also able to activate signal transduction
elements in other pathways. Similarly, STATs may
become phosphorylated by serine/threonine kinases,
and this may modulate the ability of tyrosine kinaseactivated STATs to modify gene expression.
Differential Alpha/Beta
Interferon Signalling
Ligand
Activation of JAK family kinases

JAK1
JAK2
JAK3
IFN type I (,)
IFN type II ()
IL-2c
IL-3b
IL-4e
IL-5b
IL-6a
IL-7c
IL-9c
IL-10
IL-11a
IL-12
Tyk2
STATs
1,2,3,5a/b
1,3,5a/b
+
3,5a/b
5a/b
6
5a/b
1,3
1,3
1
+
1,3
3,4
IL-13c
IL-15c
EGF
PDGF
1,3
CSF-1
1,3,5a/b
GH
G-CSF
5a/b
1,3,5a/b
1,3,5a/b
1,3,5a/b
GM-CSFb
TPO
5a/b
+
1,3,5a/b
Angiotensin II
PRL
LIFa
1,3
CNTFa
1,3
OSMa
1,3
CT-1a
1,3
EPO
1
5a/b
5a/b
The JAK/STAT pathway enables many cytokine

OB-Rd
+
1,3
receptors to send signals to the cell nucleus. Each
a
b
+ activity; via gp 130 receptor chain; via beta receptor chain;
cytokine receptor can induce relatively specific
c via gamma receptor chain; d via leptin
responses depending on the JAKs and STATs
involved in the pathway. However, alpha and beta Table 15: The specificity of cytokine receptors for JAKs and STATs
interferons induce distinct biological responses
through the same receptor,8 raising the question of the mechanism underlying this differential signalling.
Both type I interferon receptor chains (IFN R1 and IFN R2) can bind alpha and beta interferons. The prevailing
model describes the binding of type I interferon to the two receptor chains in a sandwich orientation, since the
binding surfaces of type I interferons are found on opposing sides of the molecule. Using a study involving the sitedirected mutation of the interferon receptor chains, three models for binding of alpha and beta interferon to the
receptor complex were identified (figure 11):9
alpha interferon binds to a single IFN R1/IFN R2 complex
beta interferon binds to a single IFN R1/IFN R2 complex, but interacts with IFN R2 in a way distinct from
alpha interferon
beta interferon binds to two IFN R2 chains, cross-linking two receptor complexes.
These different binding modalities may be associated with specific patterns of JAK and STAT activation and,
consequently, result in biological response patterns unique to the individual interferon. In mice, there has been little
progress in identifying the differential signalling between the type I interferons, but the tools are now in place to
allow these investigations to begin. Findings in the human system, and initial indications from Tyk2-knockout mice,
suggest that Tyk2 plays a role in this differential signalling pathway, but the significance of this has yet to be
explored.
TWENTY FIVE
IFN R1
IFN R1
IFN R2
IFN
IFN R2
IFN R2
IFN
IFN
IFN
IFN
Cell
membrane
JAK1
P
Tyk2
JAK1
P
Tyk2
JAK1
Cytoplasm
P
Tyk2
JAK1
P
Tyk2
JAK1
P
Tyk2
JAK1
P
Tyk2
Figure 11: Differential alpha and beta interferon receptor binding
Summary
The mechanism by which the binding of beta interferon to its receptor induces changes in the expression of genes
within a cell is now well understood, although there are many fine details that await elucidation. However, the
JAK/STAT pathway that is critical to this signalling pathway is not exclusive to beta interferon. Indeed, it appears that
many cytokines exploit the same JAK/STAT pathway. Diversity in the JAK and STAT components, and their
behaviour once activated, provides some specificity, but allows for a substantial degree of interaction between
different cytokine signalling pathways. This may provide the basis for the diverse responses of cells to a given
cytokine in different environments.
One question has been addressed the differential signalling obtained by alpha and beta interferons via the same
receptor complex. Beta interferon can bind to the receptor in two ways one is structurally very similar to the
binding mode of alpha interferon, whereas the second involves the cross-linkage of two separate receptor
complexes. Can the clinical benefit of beta interferon be attributed to either one of these binding modes? If so, it
opens the possibility for more selective treatments. If not, perhaps this explains why alpha interferon may have some
small clinical benefit in this disease.
References
1.
Mller M, Briscoe J, Ibelgaufts H. Signalling through cytokine class II receptors. In: Heldin CH, Purton M (eds). Signal Transduction. London:
Chapman & Hall, 1996.
2.
Wilks AF. Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction. Proc Natl Acad Sci USA 1989; 86:
16031607.
3.
Fu XY, Schindler C, Improta T, et al. The proteins of ISGF-3, the interferon alpha-induced transcriptional activator, define a gene family
involved in signal transduction. Proc Natl Acad Sci USA 1992; 89: 78407843.
4.
Schindler C, Fu XY, Improta T, et al. Proteins of transcription factor ISGF-3: One gene encodes the 91- and 84-kDa ISGF-3 proteins that are
activated by interferon alpha. Proc Natl Acad Sci USA 1992; 89: 78367839.
5.
Schindler C, Shuai K, Prezioso VR, Darnell JE Jr. Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor.
Science 1992; 257: 809813.
6.
Briscoe J, Kohlhuber F, Mller M. JAKs and STATs branch out. Trends Cell Biol 1996; 6: 336340.
7.
Mller M, Ibelgaufts H, Kerr IM. Interferon response pathways a paradigm for cytokine signalling. J Viral Hepatitis 1994; 1: 87103.
8.
Rani MRS, Foster GR, Leung S, et al. Characterisation of -R1, a gene that is selectively induced by interferon beta compared with interferon
alpha. J Biol Chem 1996; 271: 2287822884.
9.
Lewerenz M, Mogensen KE, Uze G. Shared receptor components but distinct complexes for alpha and beta interferons. J Mol Biol 1998; 282:
585599.
TWENTY SIX
CHAPTER EIGHT
Effect of Beta Interferon on

Immune Regulation
Current models of multiple sclerosis (MS) involve the immune system in many aspects of the aetiology and
pathology. Beta interferon may act by influencing several of the underlying immunological processes, or it may have
a more fundamental effect. In MS, the immune system is thought to have a bias towards an autoimmune phenotype.
The concept that beta interferon may alter this immune bias and exert its clinical benefit in this way will be explored
in this chapter.
Autoimmune Bias in Multiple Sclerosis

It is well understood that T helper cells can be subdivided into two main groups depending on the patterns of their
cytokine production. These two cell phenotypes regulate the type of immune response and can cross-regulate each
other. Th1 cells are pro-inflammatory, with a corresponding cytokine production profile (table 16), whereas Th2
cells produce a distinct set of cytokines that promote allergic diseases and an antibody-mediated immune response.
Overactive Th1 cells are believed to promote inflammatory-type autoimmune diseases, including MS. As a
consequence, methods that shift the differentiation of T cells towards the Th2 phenotype are actively being sought
as potential treatments for MS.
Th1 and Th2 cells are mutually antagonistic. The cytokines
produced by each population both stimulate further
recruitment of nave T cells to the same population and
suppress recruitment to the other. Other mechanisms to
reinforce this polarisation of differentiation include cytokine
receptor and accessory molecule expression, corticosteroidmediated immune modulation, and modifiers of intracellular
signalling pathways.
Th1 cytokines
Th2 cytokines
Interleukin (IL)-2
Gamma interferona
IL-4b
IL-5
Lymphotoxin
IL-6
IL-10c
IL-13
a important pro-Th1 cytokine; b important pro-Th2

cytokine; c principal anti-inflammatory mediator
Table 16: The cytokine profiles of Th1 and Th2 cells
Role of Interleukins 10 and 12 in MS

Interleukin (IL)-10 and IL-12 are critical to the differentiation of nave T cells along the Th1 and Th2 phenotypic
pathways. IL-10 is an 18 kD protein produced by a variety of cells, including CD4+ and CD8+ T cells and activated
B cells. IL-12 is a heterodimer of 35 kD and 40 kD subunits produced by activated B cells, macrophages and other
antigen-presenting cells. The 35 kD subunit (p35) is expressed by many cells but cannot be secreted alone. In the
cells incapable of producing the 40 kD subunit (p40), the function of p35 is not known. IL-12p40 can be secreted
alone, and it binds the IL-12 receptor with lower affinity than the heterodimer. It cannot induce receptor signalling
but may block binding of, and signalling induced by, the heterodimer.
103
102
IL-10
****
****
IL-12p35
***
****
IL-12p40
TNF
****
*
IFN
101
100
101
**
***
C RR SP
C RR SP
C RR SP
C RR SP
C Control; RR Relapsing/remitting MS; SP Secondary progressive MS;
TNF tumour necrosis factor alpha; IFN gamma interferon
*P<0.07; **P<0.02; ***P<0.005; ****P<0.0001
C RR SP
104
Geometric mean
(mRNA in fg st.eq. x 108)
It has been proposed that

any change in the bias of
the immune system would
be determined by changes
in the relative expression of
these cytokines. A small
study involving people with
relapsing/remitting
and
secondary progressive MS
was
carried
out
to
determine whether there
was any apparent change in
the expression of these
cytokines and in a range of
other cytokines known to be
Figure 12: Cytokine mRNA expression in mononuclear cells of people with MS
TWENTY SEVEN
involved in the inflammatory response. The study also asked

whether the two disease types varied in this respect.1 Blood
samples from eight people with relapsing/remitting MS and five
people with secondary progressive MS were obtained monthly
and analysed for the expression of cytokine mRNA by the
peripheral blood mononuclear cells (PBMCs).
IL-12p35
Relapse
300
Cytokine mRNA in standard equivalents (fg x 106)
200
100
0
For each cytokine, a longitudinal analysis of all samples was

used to assess differences in cytokine expression by people with
MS. Neither tumour necrosis factor alpha (TNF) nor gamma
interferon expression were significantly changed in people with
MS compared with controls (figure 12). However, substantial
declines in IL-10 expression were observed in people with MS,
which was greatest in those with secondary progressive disease.
Expression of the two IL-12 subunits was also substantially
altered.
IL-12p40
40
P<0.05
20
0
IL-10
150
100
The same study correlated cytokine expression with clinical and

MRI disease activity. Again, TNF and gamma interferon
showed little change. Both IL-10 and IL-12p35 tended to
increase following relapse, but the most striking observation was
an increase in IL-12p40 prior to relapse (figure 13).
50
0
+2
+6
Weeks before/after relapse
Figure 13: Cytokine mRNA and relapses in people with MS
Magnetic resonance imaging (MRI) data are less informative,

with only three significant associations between cytokine changes and MRI lesions (figure 14). In both
relapsing/remitting and secondary progressive individuals, IL-12p35 is elevated following MRI lesions, and in
relapsing/remitting patients IL-10 is elevated at the time of the lesions. These data suggest possible differences in the
control of disease activity by IL-10 in relapsing/remitting and secondary progressive disease.
In people with secondary progressive MS,

IL-10 remains low, but IL-12p40 mRNA is
high when MRI activity develops.
Secondary progressive MS
IL-12p35
100
100
P<0.05
P<0.05
50
0
50
0
IL-12p40
IL-12p40
300
300
200
200
100
100
0
IL-10
IL-10
100
100
P<0.01
50
0
50
1
0
+1
Months before/after active lesions
1
0
+1
Months before/after active lesions
Figure 14: Cytokine mRNA and appearance of active MRI lesions in people with MS
On the basis of these, and other observations, it is clear that IL-10 and IL-12 play an important role in regulating
disease activity in MS, but there may be differences between relapsing/remitting and secondary progressive disease.
TWENTY EIGHT
have low IL-10 and transforming growth

factor beta levels prior to relapse but
have high TNF mRNA. IL-12p40
mRNA levels also increase
express increased levels of IL-10 mRNA
and circulating IL-10 in association with
remission. Transforming growth factor
beta levels increase but there is a
decline in TNF mRNA.
Relapsing/remitting MS
IL-12p35
Cytokine mRNA in standard equivalents (fg x 106)
Other studies have, to a certain extent,

shown similar changes in the expression of
IL-10 and other cytokines.2,3 Together,
these data show that people with relapsing/
remitting MS:
Modifying the IL-10/IL-12 Network

Given that IL-10 and IL-12 appear to have an important role in MS, it is tempting to find methods to accelerate the
changes normally observed during remission, with the goal of beneficially modifying disease activity. One approach
is administration of corticosteroids around the time of a relapse. Several studies have suggested that administration
of hydrocortisone enhances plasma IL-10 levels,4,5 and administration of methylprednisolone to people with MS
increases both IL-10 mRNA and serum IL-10 levels.6 As shown in figure 15, this agent may rapidly reverse the IL-10
and IL-12 changes associated with relapses.7 Further studies also indicate that glucocorticosteroids may exert their
beneficial effect by influencing the IL-10/IL-12 system.8 These observations raise the question of the consequences
of administering beta interferon concurrently with steroids for the IL-10/IL-12 balance.
104
MethylRelapse prednisolone
103
Cytokine/-actin mRNA
Another important consideration is the

regulation of receptor expression. The
IL-12 receptor consists of two chains, 1
and 2, which can be differentially
expressed, and which can modify the
response to IL-12. Nave T cells express
the 1 IL-12 receptor chain, which has a
low affinity for IL-12 and is not involved in
intracellular signalling. Exposing these cells
to IL-4 stimulates the expression of the
IL-4 receptor, which reinforces the IL-4
signal. In mice, the IL-4 receptor induces
intracellular signalling via STAT6, leading
to the cell committing to the Th2
phenotype. This is enhanced by IL-1.
Conversely, in humans, alpha and beta
interferons stimulate expression of both 1
and 2 IL-12 receptor chains on nave
T cells. The 2 chain induces STAT4
activation, which, with the addition of
IL-18, commits the cell to the Th1
phenotype. IL-4 inhibits this commitment.9
Ratio IL-10:IL-12p40
IL-12p40
IL-10
102
101
100
101
102
103
1
MRI lesions
1
Months
10
Figure 15: Methylprednisolone reverses changes in IL-10 and IL-12 that are associated
with relapses
Given the observation that beta interferon stimulates commitment towards pro-inflammatory Th1 cells, how can it
be beneficial in MS? Data from recent studies suggest that beta interferon can also increase IL-10 and IL-4 mRNA
expression in PBMCs. After 2 years of treatment with beta interferon, IL-10 levels in the cerebrospinal fluid were
significantly increased,10 pointing to a long-term influence on the commitment of T cells within the central nervous
system.
Summary
The role of IL-10 and IL-12 in moderating disease activity in MS appears to be reasonably clear IL-10 is
suppressive while IL-12 seems to promote disease activity. By influencing the balance of these two cytokines, for
example by using glucocorticosteroids such as methylprednisolone, the inflammatory component of MS can be
beneficially altered. However, the influence of beta interferon is less clear. In humans, it appears that beta interferon
drives T cells towards the Th1 phenotype, yet it also enhances IL-10 expression over the long term. Are there
unrecognised interactions between beta interferon and the IL-10/IL-12 network, or are these effects consistent with
the kinetics of a number of different responses? This question remains to be addressed. Nevertheless, it is clear that
strategies to increase the proportion of IL-10 relative to IL-12 appear to offer benefit to people with MS.
TWENTY NINE
References
1. van Boxel-Dezaire AHH, Hoff SCJ, van Oosten BW, et al. Decreased IL-10 and increased IL-12p40 mRNA are associated with disease activity
and characterize different disease stages in multiple sclerosis. Ann Neurol 1999; 45: 695703.
2. Rieckmann P, Albrecht M, Kitze B, et al. Cytokine mRNA levels in mononuclear blood cells from patients with multiple sclerosis. Neurology
1994; 44: 15231526.
3. Rieckmann P, Albrecht M, Kitze B, et al. Tumour necrosis factor alpha messenger RNA expression in patients with relapsing/remitting multiple
sclerosis is associated with disease activity. Ann Neurol 1995; 37: 8288.
4. van der Poll T, Barber AE, Coyle SM, Lowry SF. Hypercortisolaemia increased plasma interleukin-10 concentrations during human
endotoxemia a clinical research center study. J Clin Endocrinol Metab 1996; 81: 36043606.
5. Dandona P, Aljada A, Gang G, Mohanty P. Increase in plasma interleukin-10 following hydrocortisone injection. J Clin Endocrinol Metab 1999;
84: 11411144.
6. Gayo A, Mozo L, Suarez A, et al. Glucocorticosteroids increase IL-10 expression in multiple sclerosis patients with acute relapse.
J Neuroimmunol 1998; 85: 122130.
7. van Boxel-Dezaire AHH. Unpublished observations.
8. Visser J, van Boxel-Dezaire A, Methorst D, et al. Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro. Blood
1998; 91: 42554264.
9. Rogge L, Barberis-Maino L, Biffi M, et al. Selective expression of an IL-12 receptor component by human T helper 1 cells. J Exp Med 1997;
185: 825831.
10. Rudick RA, Ransohoff RM, Lee JC, et al. In vivo effects of interferon beta-1a on immunosuppressive cytokines in multiple sclerosis. Neurology
1998; 50: 12941300.
THIRTY
CHAPTER NINE

Mediators of Inflammation
In many respects, experimental allergic encephalomyelitis (EAE) has provided considerable insight into multiple
sclerosis (MS). Beta interferon is just one of many cytokines that can influence an immune response. The cytokine
network represents the complex interaction of these molecules, and it is important to understand how the cytokine
network can drive the immune response towards, or away from, an inflammatory response, and how beta interferon
may influence the cytokine network and the subsequent immune response. This chapter will consider the role of
inflammatory cytokines in the mouse EAE model, and will discuss the implications for MS.
The Inflammatory Model of EAE

In common with MS, the processes involved in inducing inflammation in the central nervous system of an EAE
mouse are:
crossing of the bloodbrain barrier by an activated, antigen-specific T cell, and exposure to the specific antigen
within the central nervous system
recruitment of additional inflammatory cells monocytes and macrophages, B cells, additional T cells and
natural killer cells mediated by chemokines and helped by elevated expression of adhesion molecules on
blood vessel endothelial cells
tissue damage including demyelination, oligodendrocyte injury and death, axonal damage and gliosis.
There is also evidence that a perpetuation of the inflammatory response occurs, whereby the availability of
additional antigens in the context of the inflammatory environment causes the initially focused immune response to
diversify. This mechanism would facilitate epitope spreading.
In mice, the Th1/Th2 model of T helper cell differentiation is well understood. As in humans, Th1 cells produce
many pro-inflammatory cytokines, including gamma interferon and tumour necrosis factor alpha (TNF),
lymphotoxin alpha (LT) and lymphotoxin beta (LT) the three members of the LT/TNF family. On crossing the
bloodbrain barrier and recognising their target antigen, these cells produce cytokines that stimulate changes in
nearby cells the upregulation of surface antigens, and cytokine and chemokine production that ultimately result
in the initiation of an inflammatory response.
The lymphotoxins are particularly important in this
TNF
process. They share many effects, but each can
Receptor
p55, p75
induce specific responses (table 17).1 Among the
Cytotoxicity
+
most interesting effects are those not normally
associated with inflammation lymph node
Lymph node development
development and splenic organisation. In addition,

Spleen organisation
+
expression of MAdCAM and VCAM, adhesion
NF-B activation
+
molecules characteristic of high endothelial venules
ICAM, VCAM induction
+
in lymphoid tissue, is upregulated on vascular
MAdCAM induction
+
endothelium. Together, these observations suggest
that part of the inflammatory response is the Table 17: Characteristics of the LT/TNF family
development of an environment permissive to the
perpetuation and diversification of the immune response.
LT
LT
p55, p75
LTR
The influence of the LT/TNF family and their receptors in the severity of disease in EAE has been extensively studied
using myelin oligodendrocyte glycoprotein (MOG)-induced EAE in knockout mouse strains. Although there were
differences among the reported outcomes, some features were consistent:
LT is crucial for disease since LT-knockouts failed to develop EAE or any signs of inflammation or
demyelination
THIRTY ONE
TNF may have some role in the initial crossing of the bloodbrain barrier by the infiltrating T cells, but
otherwise there is little difference between normal and knockout mice in terms of the nature or severity of the
disease
the p55 TNF receptor makes an important contribution to the severity of disease; mice lacking the receptor only
develop mild disease
by contrast, animals lacking the p75 TNF receptor develop extremely severe disease, suggesting that this receptor
has a protective effect.
The roles of many cytokines in EAE, derived from studies using gene-targeted mice, have recently been
summarised.2 Interestingly, gamma interferon and its receptor do not appear to be crucial for MOG-induced EAE.
Interleukin (IL)-6, however, is essential for disease since mice deficient in the IL-6 gene are more susceptible to EAE.
Mice deficient in IL-4 are not consistently more susceptible, suggesting a minimal or non-existent role for this
cytokine.
The Effects of Beta Interferon

In common with the human system, beta interferon in the mouse acts on a number of the inflammatory mechanisms
involved in EAE pathology. Antigen presentation is affected through altered expression of major histocompatibility
complex (MHC) class I and II molecules, and overall T cell activation is suppressed due to a downregulation of IL-2
receptor expression. Th1-type cytokines are downregulated, whereas the Th2-type cytokines are upregulated (table 18).
Data from human studies confirm that beta interferon induces
IL-4 and IL-10 parameters.3 In mice, passive transfer of EAE using
cells stimulated in vitro to proliferate in the absence of beta
interferon tends to result in severe disease, and the cells produce
high levels of gamma interferon and lower levels of IL-10 and IL-4.
By contrast, stimulating these cells in the presence of beta
interferon shifts the cells towards expression of IL-4 and IL-10,
reduces production of gamma interferon, and leads to milder
disease. This experiment confirms that immune deviation, rather
than any other form of tolerance induction, is involved.
Weber et al recently demonstrated that both interferon beta-1a
and interferon beta-1b inhibit the production of LT from human
T cell lines reactive to myelin basic protein, suggesting that a
further mechanism of action of beta interferon is through the
downregulation of a crucial pro-inflammatory cytokine.4
Interestingly, there was no observed effect on IL-4, but both
agents induced IL-10 production.
Effect
MHC class I
MHC class II
IL-2 receptor
++
+/
Th1 cytokines
gamma interferon
TNF
LT
IL-12
Th2 cytokines
IL-4
IL-10
transforming growth factor beta
+++
+++
+++
+++
Inflammatory events
adhesion molecules
recruitment into CNS
matrix metalloproteinase-9
macrophage activation
B cell differentiation
T cell replication
natural killer cells
The ability of inflammatory cells to cross the bloodbrain barrier is

also compromised by beta interferon. Besides a reduction in
chemokine production and adhesion molecule expression on Table 18: Effects of beta interferon on immune
endothelial cells respectively reducing the attractiveness of the parameters of EAE and MS
inflammatory lesion and the ability of the cells to adhere to the
endothelium these cells are also less capable of penetrating the bloodbrain barrier. The ability of beta interferontreated activated T cells to degrade fibronectin, a critical process required for cells to cross the bloodbrain barrier,
appears to be linked to a reduction in the expression of matrix metalloproteinase-9.5
THIRTY TWO
Summary
EAE pathology depends critically on inflammatory processes. Among these are production of inflammatory cytokines
and recruitment of effector cells to the lesion, but an additional feature is the modification of endothelial cells. Part
of the inflammatory response therefore appears to be the development of new lymphoid-like regions that could
promote diversification of the inflammatory response. Principal among the cytokines responsible for these changes
are members of the LT/TNF family.
Beta interferon has a marked anti-inflammatory effect, and can downregulate many of the inflammatory features
and molecules typical of EAE lesions. If these findings can be translated directly to MS, then this strongly suggests that
the mechanism of action of beta interferon in MS is to downregulate inflammation. However, questions remain
whether a direct translation of these findings can be made, and whether beta interferon has other activities not
directly related to inflammation.
References
1.
Sacca R, Cuff CA, Ruddle NH. Mediators of inflammation. Curr Op Immunol 1997; 9: 851857.
2.
Hjelmstrom P, Juedes A, Ruddle NH. Cytokines and antibodies in myelin oligodendrocyte glycoprotein-induced experimental allergic
encephalomyelitis. Res Immunol 1998; 149: 794804.
3.
Rudick RA, Ransohoff RM, Lee J-C, et al. In vivo effects of interferon beta-1a on immunosuppressive cytokines in multiple sclerosis. Neurology
1998; 50: 12941300.
4.
Weber F, Janovskaja J, Polak T, et al. Effect of interferon beta on human myelin basic protein-specific T-cell lines: Comparison of interferon
beta-1a and interferon beta-1b. Neurology 1999; 52: 10691071.
5.
Stve O, Dooley NP, Uhm JH, et al. Interferon beta-1b decreases the migration of T lymphocytes in vitro: Effects on matrix metalloproteinase-9.
Ann Neurol 1996; 40: 853863.
Further Reading
Rieckmann P. The effects of interferon-beta on cytokines and immune responses. In: Reder AT (ed). Interferon Therapy of Multiple Sclerosis. New
York: Dekker, 1997, 161191.
THIRTY THREE
CHAPTER TEN

Immune Activation
The variety of molecular signals involved in the communication between antigen-presenting cells (APCs) and nave
T cells was highlighted in chapter three. The antigen-specific recognition event, involving the T cell receptor, the
major histocompatibility complex (MHC) and the antigen fragment itself, forms only part of the overall message.
Increasingly, accessory molecules are being recognised as important contributors to this message. The particular role
of interactions between CD28 and B7 and between CD40 and its ligand, on T cell responses, and the impact of beta
interferon on those interactions, will be explored in this chapter.
The Importance of Accessory Molecules

Accessory molecules can greatly
influence the outcome of the
Cytotoxic T cell
T cell:APC interaction, and also
Ag
interactions between T cells and other
Melanoma
MHC I
TCR
cells such as tumour cells. A
particularly clear illustration of this
can be found from a model using
CD28
melanoma cells. Melanoma cells
expressing antigen fragments via
MHC class I molecules are recognised
by nave, antigen-specific cytotoxic
Cytotoxic T cell
T cells. However, if the melanoma
Ag
cells do not express the accessory
MHC I TCR
Engineered
molecule B7, the nave T cell will not
melanoma
respond.
By
engineering
the
CD28
B7
melanoma to express B7, the T cell
IL-2
can be induced to activate, proliferate
and kill the melanoma (figure 16).
Thus, the presence or absence of the Figure 16: B7 is critical to an active immune response
CD28:B7 signal plays a critical role in
determining the outcome of the antigen-specific recognition event for nave T cells.
No reaction
Activated, proliferating
cytotoxic T cells
The CD28:B7 Signalling Pathway

The B7 molecule is expressed in two forms, B7.1 and B7.2, on a variety of APCs including monocytes, macrophages,
B cells and a small population of T cells. B7.2 is expressed constitutively at a low level, but expression of B7.1
increases rapidly at the onset of inflammation. The B7 ligand, CD28, is expressed on T cells and stimulates these
cells towards activation. However, within 48 hours of this response, a second B7 ligand, CTLA-4, is expressed at high
levels by these activated T cells. CTLA-4 is an inhibitory molecule, and its interaction with B7 provides an inhibitory
signal to the T cell. It is suggested that initial activation of nave T cells via CD28 and B7.2 induces inflammation and
expression of B7.1. Within 48 hours, expression of CTLA-4 increases, causing a downregulation of the developing
inflammatory response. Thus CD28:B7-induced inflammatory responses are self-limiting.
CD28:B7 in Multiple Sclerosis

In multiple sclerosis (MS), there is strong evidence that B7.1 is substantially overexpressed on lymphocytes, in
particular B cells, and is also more abundant on monocytes compared with controls. Elevated B7.1 expression on
B cells leads to an exaggerated antigen-specific immune response. In contrast, when macrophages or dendritic cells
present antigen and overexpress B7.1, there is an increase in generalised immune activation. B7.2 is also found to
be overexpressed in MS, although to a much smaller degree.
THIRTY FOUR
Treatment with beta interferon has a marked effect on B7 expression on the circulating cells. Within a month of
starting beta interferon treatment, B7.1 expression on lymphocytes is greatly reduced, and remains low for at least
36 months. Levels of B7.2 show a non-significant, transient decline.1 This effect can be attributable specifically to
the response of B cells. In contrast to the reduction of B7 expression on B cells, B7.1 and B7.2 expression on
monocytes increases in response to the interferons. These observations indicate a distinct treatment effect of beta
interferon on the two limbs of the immune system the highly specific antigen presentation of the B cell, and the
relatively non-specific antigen presentation of the monocyte/macrophage.
In the central nervous system of people with MS, B7.1 expression is found on lymphocytes surrounding veins, and
B7.2 is expressed by macrophages within the inflammatory lesions. Microglia, which normally do not express B7,
carry B7.1 in MS brain, and these cells can activate T cells in vitro. In normal brain, levels of both markers are very
low. These findings suggest that the central nervous system of people with MS is at an elevated state of activation
and is probably capable of locally amplifying immune responses.2,3
Overall, elevated expression of B7 in MS, particularly on B cells, results in enhanced costimulation and a B celldriven activation of T cells. Smaller amounts of antigen are needed to induce an activation response, and tolerisation
to antigen may be less likely. Beta interferon treatment normalises B7 expression and downregulates the overall
immune responsiveness in the blood.
CD40:CD40 Ligand Signalling

The role of the accessory molecules
CD40 and CD40 ligand in MS has
been explored.4 In the normal
situation, the antigen-specific recognition event involving these accessory
molecules leads to activation of both
the T cell and the APC, increased
CD40 expression on the APC and
expression of the CD40 ligand on the
T cell. This event also induces
interleukin (IL)-12 secretion, which in
turn increases expression of the IL-12
receptor and this leads to increased
gamma
interferon
production.
This can then increase B7 expression
on monocytes, B cells, and T cells,
thus enhancing immune activation
(figure 17).
T CELL
ANTIGEN-PRESENTING CELL
T cell receptor
CD40 ligand
2
expression
IL-12 receptor
Ag/MHC
CD40
IL-12
secretion
Increased
IFN
secretion
Figure 17: The CD40:CD40 ligand response
In MS, this pattern of activation remains intact, but at each step the response is enhanced. Thus, the CD40:CD40
ligand recognition event is an important target for immune modulation. It is unclear whether beta interferon acts
directly on this interaction, but it can act on downstream events IL-12, IL-12 receptor, and gamma interferon
expression mediated by the CD40:CD40 ligand interaction are all downregulated by beta interferon.
Other Accessory Molecule Interactions

A range of other accessory molecules interact during the antigen-specific recognition event, and they may have an
influence on MS. Already mentioned is the interaction of B7 with CTLA-4. This inhibitory interaction suppresses
further proliferation of activated T cells. Without this control mechanism in place, overproliferation of T cells leads to
grossly enlarged lymphoid organs and a poorly regulated immune system.5 Again, it is not clear whether beta
interferon influences the expression of CTLA-4.
THIRTY FIVE
Accessory molecule
Effect of beta interferon
Signalling molecules
B7.1, B7.2
Increases B7 expression on monocytes, reduces B7 expression on B cells
CD28
Unknown
CTLA-4
Unknown
CD40, CD40 ligand
Reduces the expression of IL-12, IL-12 receptor and gamma interferon mediated by this interaction
Adhesion molecules
ICAM-1
Minimal effect on resting endothelial cells and astrocytes, slight increase of cytokine-induced
expression in human endothelial cells
Induction on melanocytes and CNS tumours
LFA-1
Unknown
VLA-4
Reduced on monocytes
LFA-3
No change
VCAM
Minimal effect on endothelial cells. However, increased in serum when gadolinium-enhancing MRI
lesions are present, and following beta interferon therapy, possibly from shedding by cerebral
endothelial cells
E-selectin
Minimal effect on endothelial cells
Table 19: Effect of beta interferon on accessory molecules
Adhesion molecules may also play a role in immune activation in MS. Although conventionally not considered to be
signalling molecules, the interaction between accessory molecules, and the level of accessory molecule expression,
can alter the overall strength of antigen-specific and accessory molecule binding. This in turn alters the subsequent
immune responses. There are many adhesion molecules that are of interest in MS. Table 19 shows the effects of
beta interferon on these signalling and adhesion molecules.
Summary
Although the antigen-specific interaction between the T cell and the APC is vital to ensure a response, the accessory
molecules that are also involved in this interaction shape the response. The most important interactions are
B7:CD28, which stimulates an inflammatory response, B7:CTLA-4, which acts later to slow the development of the
inflammatory response, and the CD40:CD40 ligand interaction, which induces IL-12 and gamma interferon
secretion, both of these being pivotal to the expansion of the inflammatory response. In MS, there is overexpression
of B7 and an enhanced excitability of the CD40 response. These both suggest that the immune system is in a
heightened inflammatory state. Adhesion molecules also play a role in the T cell:APC interaction. Even though they
do not transduce signals directly, adhesion molecules can influence the binding affinity of the interaction and modify
immune responses.
Beta interferon acts beneficially on each of these interactions to suppress the potential for an inflammatory response.
Can these effects explain the clinical benefit of beta interferon? It is likely that they represent one of numerous
clinically relevant biological effects.
References
1.
Gen K, Dona DL, Reder AT. Increased CD80+ B cells in active multiple sclerosis and reversal by interferon beta-1b therapy. J Clin Invest
1997; 99: 26642671.
2.
Williams K, Ulvestad E, Antel JP. B7/BB-1 antigen expression on adult human microglia studied in vitro and in situ. Eur J Immunol 1994; 24:
30313037.
3.
Dangond F, Windhagen A, Groves CJ, Hafler DA. Constitutive expression of costimulatory molecules by human microglia and its relevance to
CNS autoimmunity. J Neuroimmunol 1997; 76: 132138.
4.
Balashov KE, Smith DR, Khoury SJ, et al. Increased interleukin 12 production in progressive multiple sclerosis: Induction by activated CD4+
T cells via CD40 ligand. Proc Natl Acad Sci USA 1997; 94: 599603.
5.
Waterhouse P, Penninger JM, Timms E, et al. Lymphoproliferative disorders with early lethality in mice deficient in CTLA-4. Science 1995;
270: 985988.
THIRTY SIX
CHAPTER ELEVEN
Effect of Beta Interferon on the Natural

History of Multiple Sclerosis
Beta interferon has been proven to be an effective treatment for relapsing/remitting and secondary progressive
multiple sclerosis (MS) in several well-designed, double-blind trials. However, these trials have only shown efficacy
for in most cases 2 years. In only one trial were controlled data available for up to 5 years, but by this time less
than half of the original patients remained in the study. There is no evidence that clinical benefit is limited to a short
period indeed the studies of interferon beta-1b suggests that it should offer clinical benefit from mild to severe
disease but there is also little evidence that clinical benefit persists beyond 23 years. Will long-term benefit reflect
short-term findings? How could the disease course be altered? How could these changes be monitored over the long
term? This chapter will consider these questions and speculate on the possible answers.
Will Long-term Benefit Reflect Short-term Findings?
Median change from baseline (%)
2.0
Interferon beta-1b 1.6 MIU
1.6
Placebo
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Baseline
Year 1
Year 2
25
Interferon beta-1b 1.6 MIU

Placebo
10
5
0
5
1 Year
2 Years
Year 4
Year 5
No significant effect was

observed on the rate of
progression of disability but
the power of the trial to detect
an effect was low. However,
the
Kaplan-Meier
curve
showing risk of probability of
progression
showed
a
consistent tendency over the 5
years of study towards a
slowing of progression. Fewer
people overall progressed in
the high-dose treatment group
compared with placebo.
15
10
Year 3
Figure 18: Relapse rates in the relapsing/remitting interferon beta-1b trial. Declining relapse rates in
the placebo group represent the influences of regression to the mean and natural history of MS
30
20
1.8
Relapse rate
The trial of interferon beta-1b in

relapsing/remitting MS, originally
designed for 2 years with a further
1 year continuation period, was able
to acquire blinded data for up to
5 years in some patients. For each
year of data the patient cohorts
declined in size mainly due to
optional
participation
in
the
continuation study and the loss to
follow-up of some patients. Within
the limits of the incomplete follow-up
the magnitude of the clinical effect
on relapse rate continued at
approximately 30% over the 5 years
(figure 18). The burden of T2 lesions
on MRI scans also continued to show
a substantial difference between the
treated and untreated group (figure
19).1
3 Years
4 Years
5 Years
Several points about the

clinical outcome of this trial
permit speculation that clinical benefit persists. The magnitude of relapse rate reduction showed little sign of
dwindling with time. The MRI burden of disease data show a sustained difference between treated and placebo
Figure 19: MRI burden of disease in the relapsing/remitting interferon beta-1b trial
THIRTY SEVEN
80
Placebo
Sulphasalazine
60
40
P = 0.098
20
0
0
2
Years to sustained progression
Figure 20: Disability progression in the sulphasalazine trial in progressive MS patients
Naturally, there are considerable concerns

with extrapolating this data into the long term. The most important object lesson was the clinical trial of
sulphasalazine in active relapsing/remitting or progressive MS. After 18 months of treatment, progression of disability
in the progressive subgroup was significantly delayed by treatment. However, continuing the study to 3 years
revealed that this treatment effect had been lost (figure 20).3
How Could the Disease Course be Modified?
Several
groups
have
collected
comprehensive long-term natural history
DSS 10
data, using both clinical and MRI
80
parameters, and they suggest some
interesting points. From a study of over
1000 people with MS, an actuarial
60
analysis of time to landmark stages of
disability was compiled (figure 21).4 This
DSS 8
40
suggested that within 15 years of
diagnosis, 50% of people with MS will
reach EDSS 6.0, and 10% will reach
20
EDSS 8.0. However, this analysis did not
DSS 3
DSS 6
take into account the baseline
0
characteristics of the individuals or the
0
10
20
30
40
50
60
influence of these characteristics on
Time (years)
outcome. A follow-up study of the same
Figure 21: An actuarial analysis of avoiding disability from onset of MS
cohort, however, did consider the
predictive value of the early clinical
course.5 In these analyses, the number of relapses within the first 2 years of prospective follow-up (figure 22), the
first interattack interval and the time to reach EDSS 3.0 greatly influenced the median time to reach EDSS 6.0. In a
further analysis of the data, several factors predictive of a favourable prognosis were identified (table 20).6 The link
100
Patients (%)
With all the caveats in mind concerning the extrapolation of short-term results into the long term, are there any
clues that may suggest how the disease course may be changed over the long term by effective treatment? An
alternative way to consider this question is to ask how differences in relapse rate, MRI disease activity and burden of
disease, and progression of disability in the early years of disease, influence the subsequent course of the disease in
people not receiving immunomodulatory treatments. For treatments that can reduce these parameters, it is plausible
that the difference between the natural course of disease with baseline parameters corresponding to the pre-treated
and treated clinical status of patients will represent the long-term treatment effect.
THIRTY EIGHT
Also important is the evidence from the

secondary progressive interferon beta-1b trial
that shows the same magnitude of effect on
relapses, reductions of MRI disease activity and
disease burden consistent with those from the
relapsing/remitting MS trials, and a significant
slowing of the accumulation of disability.2
Clinical effect of interferon beta-1b is apparent
within the range of disability severity up to
EDSS 67, although clinical effect in more
advanced disease remains an unknown.
100
Cumulative probability of progressing
patients, and the data on progression of

disability shows a separation of the KaplanMeier curves consistent with sustained effect
over 5 years.
80
Patients (%)
between relapses per se and progression

is not fully defined. Nevertheless, the
predictive value of relapse rate early in
the disease for time to reach EDSS 6.0 in
this cohort was particularly marked. In
individuals with five or more relapses,
median time to EDSS 6.0 was
approximately 67 years. In people with
only one relapse, median time to EDSS
6.0 approached 18 years a difference
of at least a decade, and a clinically
meaningful difference in outcome for the
two groups of subjects.
100
60
40
>
5 relapses
20
1 relapse
24 relapses
0
0
10
20
30
40
50
Time from onset of MS (years)

Figure 22: Time to reach EDSS 6.0 depends on the number of relapses early in disease
Variable
Favourable
Neutral
Unfavourable
Gender
Female
Male
Age at onset
Younger
Older
Initial symptoms
Optic neuritis,
insidious pyramidal
Attack frequencya
Low
High
Longer
Shorter
Low
High
First interattack
intervala
Disability status at 5 yearsa

a
Modifiable with beta interferon treatment?
Table 20: Factors indicative of a favourable prognosis
Sensory, acute
pyramidal, brain stem
Cerebellar
Despite the evidence that early disease

characteristics can influence subsequent
disease course, it is not possible to say
whether modifying the disease course
using beta interferon will have
comparable long-term effects. These
analyses are inadequate to resolve the
long-term clinical implications of a 30%
decline in relapse rate, and there may be
aspects of the disease that beta
interferon does not affect which have an
influence on the disease course.
Nevertheless, it is tempting to speculate
that reducing relapse rate and slowing
disease progression will have long-term
benefits.
How Could Clinical Effects be Monitored Over the

Long Term?
The most important clinical development tool is the double-blind, randomized clinical trial. However, such trials
have many features that would make their long-term use to monitor the clinical benefit of beta interferon
problematic. The limitations of these trials are outlined in table 21.
One possible approach to long-term monitoring of treatment effect is to observe the clinical effect of the drug in
the patient population once it has been approved for clinical use. Simply observing the disease course in these
individuals will allow investigators to develop a model for the natural history of the disease under treatment. By
comparing this against the natural history of the disease without treatment, a reasonable assessment of the clinical
benefit of the drug over the long term can be made. For such an approach to be practical, however, there are
three main requirements. First, reliable, comprehensive long-term natural history data for the specific disease must
be available. Such data already exist for MS. Second, an infrastructure must be in place to monitor the clinical
course of people receiving treatment. Third, resources to support a long-term monitoring programme must be
available. These include financial resource and political will. However, in MS such an approach could offer many
benefits, including practicability. Indeed in an era where therapies have been proven to be effective, the standard
paradigm of the randomised controlled clinical trial will need to be reconsidered if longer range goals of
assessment are to be met.
This approach faces many challenges, and is one of several options for assessing the long-term clinical benefit of beta
interferon in MS. However, current trials are inadequate to address this question. Until a means is identified of
assessing treatment effect over the long term, this will remain an open question concerning the existing treatments.
THIRTY NINE
Summary
The course of MS far exceeds the duration of
clinical trials that assess the efficacy of beta
interferon. Therefore, the question of longterm clinical effect remains. Controlled clinical
data show a sustained treatment effect for up
to 5 years, but there are very few data to
support the use of beta interferon any longer.
Neither are there data that suggest the efficacy
of beta interferon is limited to 5 years.
It is tempting to consider how baseline disease
factors predict long-term disease outcome, and
whether modifying these factors using beta
interferon could alter the disease course. Three
factors relapse rate, interattack interval, and
time to EDSS 3.0 predict future outcome,
and two of these three factors were influenced
favourably during the short-term clinical trials.
Feature
Limitations
Placebo group
Not ethical when a clinically effective treatment is

available
or
Problems maintaining the placebo group patients

defect to treatment
Alternative treatment
Cost two treatments, size of the patient cohorts,

administration
Only possible when there are effective alternative
treatments
Duration
Patient sample size
Blinding
Expense
Restrictive design
Control group behaviour
Tend to be short 23 years in MS

To show adequate power in comparative trials,
large patient cohorts are necessary
Ensuring blinding can be difficult
Drug costs, administration, coordination, etc
Trials are prospectively designed to test efficacy on
specific outcomes, with a power to show an effect
based on assumptions about the possible
outcome
Lack of power to stratify patients
Reliance on normal behaviour i.e. in line with
the natural history of the disease
Enrolment criteria to control for variable behaviour
ensures trial outcomes are only certain to be true
for that cohort
Loss to follow-up
Drop-outs reduce the power of trials and occur
because of:
The only true method of addressing the
perceived inefficacy
question is to perform a clinical trial. However,
drug-specific side-effects
existing trial protocols are inadequate. One
loss to follow-up
approach would use existing natural history
Surrogate markers
Reliability and validity
data as a control group and would monitor the
Predictive of the long term?
behaviour of a cohort receiving treatment as
Statistical power
Underpowered studies lack ability to show a
part of normal clinical management. Other
clinical effect
approaches may be equally valid. However,
Overpowered studies suggest statistically significant
effect with little or no clinical relevance
until data are available, it will remain a matter
of clinical judgement whether treatment with Table 21: Limitations of randomized, double-blind, clinical trials
beta interferon offers the long-term clinical
benefit that is desired.
References
FORTY
1.
2.
European Study Group on interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon
beta-1b in treatment of secondary progressive multiple sclerosis. Lancet 1998; 352: 14911497.
3.
Noseworthy JH, OBrien P, Erickson BJ, et al. The Mayo ClinicCanadian cooperative trial of sulfasalazine in active multiple sclerosis.
Neurology 1998; 51: 13421352.
4.
Weinshenker BG, Bass B, Rice GPA, et al. The natural history of multiple sclerosis: A geographically based study. 1. Clinical course and
disability. Brain 1989; 112: 133146.
5.
Weinshenker BG, Bass B, Rice GPA, et al. The natural history of multiple sclerosis: A geographically based study. 2. The predictive value of
the early clinical course. Brain 1989; 112: 14191428.
6.
Weinshenker BG, Rice GPA, Noseworthy JH, et al. The natural history of multiple sclerosis: A geographically based study. 3. Multivariate
analysis of predictive factors and models of outcome. Brain 1991; 114: 10451057.
Concluding Remarks
How does beta interferon work in multiple sclerosis (MS)? This important question remains to be answered fully, but
there is now good evidence to provide a partial explanation. It is clear from a number of clinical trials that the effects
of beta interferon are reproducible and they appear to be consistent in relapsing/remitting and at least for
interferon beta-1b in secondary progressive MS. These studies provide some clues both to the underlying
pathology of MS, and to the way the treatment works. Similar treatment effects were observed in both types of MS
indicating an independence of the level of underlying pathology, and the interferon beta-1b secondary progressive
trial showed an effect on progression in the absence of relapses. This points to an effect on a common pathology
upstream of both relapses and progression of disability, or an effect on diverse pathological aspects of the disease.
Recent findings cast the progression of disability in MS in a new light. Axonal loss is a feature of early MS, and is
likely to contribute to cerebral atrophy and accumulating disability. Axonal loss is simply the most severe
consequence of the inflammatory activity in the brain, and follows conduction block and demyelination. Thus,
methods to reduce the impact of inflammation during early disease are likely to have significant long-term benefits.
However, it remains to be seen whether disability in MS is related to other pathological processes.
Beta interferon itself is difficult to detect once administered. However, the kinetics of treatment can be monitored
using biological response markers. These clearly show the importance of sustained treatment to maintain biological
effect, and they confirm the doseresponse relationship observed in the trials. Several of these biological response
markers provide insights into the possible effects of treatment. Most important, perhaps, are changes in interleukin
(IL)-10 and IL-12, which play a role in suppressing or promoting inflammation, respectively. However, many effects
both biochemical or clinical suggest that gamma interferon is induced by beta interferon treatment. The timings
of these effects also indicates a cascade of responses, any of which could have a positive or indeed negative
effect on the disease process.
Inflammation is clearly an important process in the pathology of MS, and this can be visualised objectively using
magnetic resonance imaging (MRI) techniques. Findings from the large clinical trials of beta interferon all indicate
that the appearance of new inflammatory lesions is greatly reduced by treatment. In addition, bloodbrain barrier
integrity is largely preserved, and the accumulation of persistent damage is slowed. However, damage can still
accumulate in the absence of inflammatory lesions, suggesting that acute inflammation does not explain all the
pathology that is underway in the MS brain.
One of the keys to understanding the effects of beta interferon on the immune system is the detailed intracellular
signalling and gene expression that occurs in response to binding at the cell surface. While much work remains to be
done to elucidate this system, it is clear that beta interferon is one of many cytokines and immune modulators that
use a common signalling pathway. Thus, beta interferon acts on, and can be influenced by, many other factors and
this leads to particularly complex cell behaviours.
Largely, beta interferon has anti-inflammatory properties. Many studies have shown that it antagonises the effects of
gamma interferon yet in humans it stimulates gamma interferon production. It also reduces expression of IL-12
and enhances IL-10 expression. These two cytokines are important in determining the overall bias of the immune
response towards inflammatory or humoral responses. Other anti-inflammatory effects include suppression of the
activities of the lymphotoxin family, which in turn appears to reduce the diversification of the immune response to
antigens of the central nervous system. It is also notable that beta interferon can reduce the ability of activated
immune cells to cross the intact bloodbrain barrier, an effect hinted at by MRI findings.
Beta interferon can influence the outcome of immune cell activation by modifying the response of antigenpresenting cells and T cells to accessory molecule signals. In MS, expression of B7 is enhanced, placing the immune
system at a heightened state of readiness to become activated. Beta interferon normalises this situation, and also
suppresses the CD40-based mechanism for the induction of the pro-inflammatory IL-12. It also has an effect on
adhesion molecule expression that may serve further to drive the immune system away from an overtly proinflammatory, activated state.
Overall, therefore, beta interferon has myriad effects on the immune system, and many of those effects may be
beneficial in MS (table 22). Other effects may have no effect whatsoever on MS, and still more may be deleterious.
FORTY ONE
Action
Consequences
Reduce central nervous

system (CNS) inflammation
Reduce intensity of the inflammatory insult to axons,

decreasing the risk of severe demyelination, axonal
transection and axonal loss
Decline in cerebrospinal
leucocyte count
Reduced CNS inflammation
Increase in soluble VCAM-1
Blocks VCAM-1:VLA-4 binding and inflammatory cell

homing to the CNS
Downregulation of
metalloproteinase-9
Preserve bloodbrain barrier integrity by preventing

crossing by activated inflammatory cells
Enhance interleukin (IL)-10

production
Promote Th2-type T cell recruitment
Enhance IL-4
production
Promote Th2-type T cell responses
However, the clinical trials have

shown that the balance of all these
effects is favourable. Nevertheless,
there still remain some questions,
including whether the effects of
beta interferon can be enhanced,
and which effects of beta
interferon are most beneficial in
the treatment of MS?
Of greatest interest to the person

with MS, however, is whether beta
interferon treatment is going to
Downregulate IL-2 receptor
Reduce potential for T cell activation
expression on T cells
offer sustained benefits, and
Increase production of
Controversial. Suggests bias in the immune system
indeed
whether
short-term
autoantibodies
towards a humoral Th2-type T cell response
benefits
will
have
greater
longEnhance suppressor cell function Restores a suppressor activity deficit in
people with MS
term consequences. Interestingly,
Flu-like symptoms, injection-site
Suggests temporary induction of gamma interferon
relapse rate and interattack
oedema
and endogenous pyrogen production
interval seem to predict future
Preferential recruitment of
Enhance local T cell-mediated suppression
outcome,
and
both
were
CD8 T cells into MS lesions
favourably altered in clinical trials.
Downregulate adhesion
Reduce homing signals, alter activation signals
molecule expression
However, these questions cannot
Downregulate IL-12 production
Suppress Th1-type T cell recruitment and activation
yet be answered fully from clinical
Enhance IL-12 receptor
Increase sensitivity to IL-12
trial data, although there are
expression on T cells
approaches that may be taken to
Reduce chemokine
Reduce attractiveness of inflammatory lesions for
obtain these data. It remains a
production
circulating inflammatory cells
clinical judgement whether the
Enhance transforming growth
Promote Th2-type T cell responses
factor beta production
sustained effects observed to
Downregulate B7 expression
Reduce (pro-inflammatory) T cell activation
5 years with this treatment will
Normalise CD40-induced
Reduce Th1-type T cell recruitment
continue into the longer term, and
IL-12 production
whether the benefits will accrue
Table 22: Possible mechanisms of action of beta interferon in MS
with time. Certainly, if beta
interferon acts beneficially on all the pathological aspects of MS, and the natural history of MS can be altered
correspondingly, then this will reinforce the view that beta interferon is a significant milestone in the
treatment of MS.
Further Reading
Yong VW, Chabot S, Stve O, Williams G. Interferon beta in the treatment of multiple sclerosis: Mechanisms of action. Neurology 1998; 51:
682689.
Arnason BGW, Dayal A, Qu ZX, et al. Mechanism of action of interferon beta in multiple sclerosis. Semin Immunopathol 1996; 18: 125148.
FORTY TWO

Mechanism of Action and Clinical Effects of Beta-Interferon in MS (Venice Workshop 1999)

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Proceedings of the MS Forum

Modern Management Workshop

The MS Forum is supported by an unrestricted educational grant from

The MS Forum is supported by an unrestricted educational grant from

The MS Forum is supported by an unrestricted educational grant from

Proceedings of the MS Forum

Professor Michel Clanet

Professor George Ebers

Professor Cesare Fieschi

Professor Ioannis Milonas

Professor Mathias Mller

Leiden, The Netherlands

Professor Michel Clanet

Professor George Ebers

Professor Cesare Fieschi

Professor Ioannis Milonas

Professor Mathias Mller

Leiden, The Netherlands

Professor Michel Clanet

Professor George Ebers

Professor Cesare Fieschi

Professor Ioannis Milonas

Professor Mathias Mller

Leiden, The Netherlands

The Clinical Evidence for Beta Interferon in

Implications of Beta Interferon Clinical Trial Results

Multiple Sclerosis Immunopathology and

Biological Markers of Beta Interferon Activity

Kinetics of Beta Interferon Treatment

Clinical Significance of Magnetic Resonance Imaging

Beta Interferon-mediated Intracellular Signalling

Effect of Beta Interferon on Immune Regulation

Effect of Beta Interferon on Mediators of Inflammation

Effect of Beta Interferon on Immune Activation

Effect of Beta Interferon on the Natural History of

The Clinical Evidence for Beta Interferon

Efficacy of Beta Interferon in Relapsing/Remitting MS

IFN MS Study Group13

2 years plus 1-year extensiona

8 MIU versus 1.6 MIU

6 MIU versus placebo

12 MIU versus 6 MIU

Frequency and route

Every other day, sc

Three times a week, sc

Primary outcome measures

Secondary outcome measures

Time to first relapse

Time to first relapse

MRI outcome measures

rolling recruitment enabled 5-year data to be obtained in some patients

Table 1: Design of three clinical trials of beta interferon in relapsing/remitting MS

Interferon beta-1a 6 MIU

Annual relapse rate

Median time to first relapse (days)

Proportion of patients relapse-freea (%)

Table 2: Relapse-related outcomes in relapsing/remitting MS trials (continued overleaf)

Table 2: Relapse-related outcomes in relapsing/remitting MS trials (continued)

Interferon beta-1a 6 MIU

Change in EDSS scorea

improved (1 point) (%)

worse (1 point) (%)

Change in IDSS per year

Change in EDSS (mean)