Human-Like Immune Responses in CD46

Transgenic Mice
Linda Johansson, Anne Rytkönen, Hong Wan, Peter
Bergman, Laura Plant, Birgitta Agerberth, Tomas Hökfelt
This information is current as and Ann-Beth Jonsson
of March 16, 2018.
J Immunol 2005; 175:433-440; ;
doi: 10.4049/jimmunol.175.1.433
http://www.jimmunol.org/content/175/1/433

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References This article cites 54 articles, 23 of which you can access for free at:
http://www.jimmunol.org/content/175/1/433.full#ref-list-1

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 The Journal of Immunology

Human-Like Immune Responses in CD46 Transgenic Mice1

Linda Johansson,2† Anne Rytkönen,2* Hong Wan,¶ Peter Bergman,‡ Laura Plant,¶
Birgitta Agerberth,‡ Tomas Hökfelt,§ and Ann-Beth Jonsson3¶
Neisseria meningitidis is a major cause of sepsis and/or meningitis. These bacteria normally cause disease only in humans, however,
mice expressing human CD46 are susceptible to meningococcal disease. To explain the sensitivity of CD46 transgenic mice to
meningococci, we evaluated early immune responses. Stimulation of TNF, IL-6, and IL-10 was stronger in CD46 transgenic mice
compared with nontransgenic mice, and resembled human responses. In CD46 transgenic mice, bacterial clearance in blood
started at later time points, and neutrophil numbers in blood were lower compared with nontransgenic mice. Further, elevated
levels of activated microglia cells and cyclooxygenase-2 were observed in brain of infected CD46 transgenic mice. Intraperitoneal
administration of meningococci lead to increased levels of macrophages only in the i.p. cavity of CD46 transgenic mice. Most of
the responses were impaired or absent using LPS-deficient meningococci, showing the importance of LPS in the early immune
response to meningococcal infection. Taken together, these data demonstrate that responses in mice expressing human CD46

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mimic human meningococcal disease in many aspects, and demonstrate novel important links between CD46 and the innate
immune system. The Journal of Immunology, 2005, 175: 433– 440.

N eisseria meningitidis (meningococcus) is a strict human
pathogen and a major cause of sepsis, septic shock, and
meningitis worldwide. These bacteria colonize the naso-
pharynx in 5–15% of healthy individuals for limited time periods
during nonepidemic conditions (1). Nasopharyngeal carriage of
these bacteria is normally entirely asymptomatic and only in a
small percentage of colonized people N. meningitidis reaches the
bloodstream, where it can cause meningococcemia and sepsis.
From the blood the bacteria cross the blood-brain barrier (BBB),4
reach the cerebrospinal fluid (CSF), and cause meningitis.
Attachment of pathogenic Neisseria to epithelial cells is medi-
ated by type IV pili and involves interaction with CD46 (2), a cell
surface complement regulator (3, 4) and receptor for several other
pathogens, e.g., adenovirus group B and D, human herpes virus 6,
measles virus, and Streptococcus pyogenes (5–11). Adherence and
colonization of host epithelial cells is followed by transcytosis and
rapid meningococcal multiplication in the bloodstream, upon
which three major cascade pathways are activated: the comple-
*Department of Infectious Diseases, Centre for Molecular Microbiology and Infec-
tion, Imperial College, London, United Kingdom; †Center for Infectious Medicine,
Department of Medicine, Karolinska University Hospital, and Departments of ‡Med-
ical Biochemistry and Biophysics and §Department of Neuroscience, Karolinska In-
stitutet, Stockholm, Sweden; and ¶Department of Medical Biochemistry and Micro-
biology, Uppsala University, Biomedical Center, Uppsala, Sweden
Received for publication October 4, 2004. Accepted for publication April 16, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Swedish Research Council (Dnr 108
46, 15302, 2887, 112 17, and 629-2002-6240), the Swedish Cancer Society, the
Swedish Society for Medicine, the Swedish Foundation for International Cooperation
in Research and Higher Education (STINT), the Magnus Bergvalls Foundation, Mari-
anne and Marcus Wallenberg’s Foundation, and Bristol-Meyers Squibb (Unrestricted
Neuroscience grant).
2
L.J. and A.R. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Ann-Beth Jonsson, Department of
Medical Biochemistry and Microbiology, Uppsala University, Biomedical Center, Box
582, SE-751 23, Uppsala, Sweden. E-mail address: Ann-Beth.Jonsson@imbim.uu.se
4
Abbreviations used in this paper: BBB, blood brain barrier; CSF, cerebrospinal
fluid; Iba1, ionized calcium-binding adaptor molecule 1; GFAP, glial fibrillary acidic
protein; COX-2, cyclooxygenase-2.
ment system, the coagulation and fibrinolysis pathway, and inflam-
matory responses (12).
Phagocytic leukocytes such as neutrophils and macrophages are
essential for innate immune responses against invading bacteria.
Interaction between bacteria and these host cells triggers potent
antimicrobial activity. Although phagocytes aim to destroy bacte-
ria, modulation of leukocyte apoptosis or cell death by bacteria has
developed as a mechanism of pathogenesis (13). Neutrophils are
short-lived cells produced in the bone marrow, circulating for 6 –9
h, and then migrating into tissue with a life span of an additional
3 days. Neutrophils leave the blood by interaction with integrins
and concentrate at the site of infection in response to chemotactic
factors (14). Bacteria are phagocytosed by neutrophils and exposed
to antibacterial substances. The mononuclear phagocyte system
includes cells derived from monocytes, e.g., macrophages and mi-
croglia in the brain. These cells are readily mobilized to sites of
infection for local activation, and macrophages are present at mu-
cosal surfaces and occur systemically. The CSF lacks cells capable
of initiating an effective immune response against invading patho-
gens. Meningeal macrophages act as phagocytic cells and facilitate
the influx of leukocytes at the BBB and hence play a crucial role
during clearance of bacteria entering the subarachnoidal space (15).
Neisserial LPS is responsible for the damage of human epithe-
lial and endothelial cells, and is an important activator of immune
responses upon infection (16). Morbidity and mortality of menin-
gococcal bacteremia is directly correlated with circulating menin-
gococcal LPS leading to activation of pro- and anti-inflammatory
mediators resulting in septic shock and, occasionally, death (17–
19). LPS binds to LPS-binding protein and eventually to CD14
expressed on monocytes, macrophages, and other host cells. Be-
cause CD14 lacks an intracellular signaling domain, interaction
between CD14, TLR 4, and MD-2 is necessary to mediate an in-
tracellular signal transduction. This signal results in production of
cytokines such as IL-1␤ and TNF (20 –21). Proinflammatory cy-
tokines are believed to elicit the systemic cascade reactions seen in
meningococcal disease such as multiorgan failure and death (22).
These cytokines play a crucial role in enhancing the bactericidal
capacity of phagocytosis, recruiting additional innate cell popula-
tions to the site of infection, and directing immune responses to the

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

434
invading microbe (23). LPS-deficient N. meningitidis may also in-
duce proinflammatory cytokine production. This immune response
is thought to involve bacterial components such as peptidoglycan,
lipoprotein, and CpG DNA (24).
A complete understanding of meningococcal disease requires an
animal model that mimics the human host. Several experimental
model systems using mice, rats, or rabbits have been evaluated
over the last decades, but these require either infant animals or
preinjection of inflammatory enhancers such as iron. Recently,
transgenic mice expressing human CD46 were demonstrated to be
susceptible to meningococcal disease (25). Crossing of the BBB
occurred in CD46 transgenic mice, but not in nontransgenic mice.
Additionally, CD46 transgenic mice showed 100% mortality 48 h
postchallenge, whereas nontransgenic mice survived.
In this study, immune responses in CD46 transgenic mice chal-
lenged with N. meningitidis were investigated with the aim to ex-
plain the rapid course of disease and the high mortality rate ob-
served in CD46 transgenic mice. In CD46 transgenic mice the
production of pro- and anti-inflammatory cytokines such as TNF,
IL-6, and IL-10, the ability of bacteria to cross the BBB, and the
stimulation of inflammatory responses in the brain tissue seem to
be major factors of the lethal outcome seen after meningococcal
challenge. The CD46 transgenic mouse model mimics human me-
ningococcal disease in many aspects and is therefore a very useful
tool in future vaccine trials against meningococcal infection.
Materials and Methods
Mouse strains
The hCD46Ge transgenic mouse line was generated as previously de-
scribed (26). CD46 in the mouse strain used (hCD46Ge) was detected in all
tested tissues (25–27). Using immunohistochemistry, CD46 was found on
epithelial cells, endothelial cells, glial cells, hepatocytes, in the glomerulus
(in kidney) and adrenal gland, as well as on B cells, T cells, neutrophils,
macrophages. The hCD46Ge transgenic mice breed normally but tend to
become obese with time (27). C57BL/6 and hCD46Ge mice were bred at
the animal facility. All mice were 5– 8 wk old when challenged with bac-
teria. All mouse procedures were performed in accordance with institu-
tional protocol guidelines under an approved protocol.
Bacterial strains and growth conditions
N. meningitidis FAM20 (P⫹, PilC1⫹, PilC2⫹) belongs to serogroup C (28,
29). The LPS-deficient FAM20 (P⫹, PilC1⫹, PilC2⫹, lpxA⫺), mutant has
previously been described (30). The lpxA gene encodes an enzyme respon-
sible for the first step of the lipid A biosynthesis pathway, adding the
O-linked 3-OH fatty acid to UDP-N-acetylglucosamine. The meningococ-
cal strains were grown on GC-agar containing Kellogg’s supplement (31)
at 37°C in 5% CO2 atmosphere and were passaged every 18 –20 h. The LPS
mutant (lpxA) was grown on GC-agar with 50 ␮g of kanamycin/ml.
Animal model
N. meningitidis was suspended in GC-liquid (1.5% w/v protease peptone
no 3 (BD Biosciences), 3 mM soluble starch (Sigma-Aldrich), 23 mM
K2HPO4 (Merck), 7 mM KH2PO4 (Merck), 50 mM NaCl (Merck)). Five-
to 8-wk-old mice were randomly redistributed in groups and injected i.p.
with 100 ␮l of bacterial suspension containing 108 bacteria or GC-liquid
alone. The actual number of wild-type bacteria in the challenge dose was
determined by viable count and equivalent amount of the mutant strain was
administrated. Blood samples were obtained from the tail at different time
points after bacterial challenge. The skin was disinfected with 70% ethanol
and a small incision was made with a sterile needle. A few microliters of
blood were collected in a heparinized capillary (Kebo). The blood was
diluted in PBS and serial dilutions were plated on GC-agar plates and
incubated overnight at 37°C in a 5% CO2 atmosphere. CFUs were counted
and bacteria were verified by gram staining, microscopy, and oxidase test.
Peritoneal wash
At different time points postchallenge with bacteria or GC-liquid animals
were sacrificed by cervical dislocation, and 5 ml of warm (37°C) sterile
PBS was injected i.p., and the peritoneal fluid was collected and stored on
ice. Recovered cells were pelleted by centrifugation at 300 ⫻ g for 10 min
MURINE IMMUNE RESPONSES TO MENINGOCOCCI
at 4°C. Cells were resuspended in 200 ␮l of 2% BSA (Sigma-Aldrich) in
PBS and incubated for 30 min at 4°C. Cells were centrifuged for 10 min
and resuspended in 100 ␮l of PBS. The cell number was determined using
a counting chamber.
Flow cytometry analysis
The fraction of macrophages and neutrophils in the population of perito-
neal wash cells was determined by flow cytometry using FITC-labeled rat
anti-mouse F4/80 (Serotec) and allophycocyanin-conjugated rat anti-
mouse Ly-6G (BD Biosciences) respectively. Propidium iodide staining
solution was used to define the living cells.
Blood smears
Blood was obtained from the tail vein. The skin of the tail was disinfected
with 70% ethanol, and a small incision was made at the tip of the tail with
sterile scissors. Blood smears were made, the slides were fixed with methanol,
and stained with Wright’s stain according to manufacturer’s recommendations
(Sigma-Aldrich). Samples were analyzed with light microscopy.
Immunoassays for cytokines and C5a in serum
Concentrations of murine IL-6, IL-10, and TNF were measured in serum of
N. meningitidis-challenged mice (108 bacteria/mouse) at different time
points postinjection by using sandwich ELISA according to manufacturer’s
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recommendations (Diaclone). For detection of C5a in mouse serum, a rat
anti-mouse C5a mAb I52-1486 (2 ␮g/ml) and biotinylated rat anti-mouse
C5a mAb I52-278 (1 ␮g/ml) from BD Biosciences were used, respectively,
as ELISA capture and detection Abs. A titration of pooled mouse serum
obtained from infected CD46 transgenic mice (24 h postchallenge) was
used to generate a standard curve. Undiluted pooled mouse serum was
arbitrarily assigned a concentration of 10 U/ml C5a.
Immunofluorescent microscopy
Mice were anesthetized and transcardially perfused with a 4% formalin
solution and rinsed as described previously (32). The brains were frozen,
cut at 14 ␮m in a cryostat, and processed for the indirect immunofluores-
cence technique (33) or the tyramide signal amplification method (34). The
astrocyte-marker glial fibrillary acidic protein (GFAP) was detected using
a rabbit anti-GFAP (1/100) from Sigma-Aldrich. An anti-rabbit secondary
Ab (1/40) from The Jackson Laboratory was used to visualize the signal
and the sections were analyzed in a Nikon Fluorescence microscope. As the
primary Ab for detecting COX-2 a rabbit anti-COX-2 (Cayman Chemical)
was used at a dilution of 1/2000. For detection of ionized calcium-binding
adaptor molecule 1 (Iba1) a rabbit Iba1 antiserum (1/2000) from Wako
Chemicals was used. The sections were then incubated with a secondary
swine anti-rabbit Ab conjugated with HRP and proceeded with reagents
from a commercial kit tyramide signal amplification (DuPont/NEN) in-
cluding FITC-conjugated streptavidin. The sections were analyzed in a
Nikon fluorescence microscope.
Statistics
Statistical significance was determined using the two tailed t test; values of
p ⬍ 0.05 were considered significant.
Results
CD46 transgenic mice are delayed in bacterial clearance
from blood
Survival of bacteria in the blood and subsequent crossing of the
BBB are considered critical for the development of meningococcal
disease. Intraperitoneal challenge of CD46 transgenic mice with N.
meningitidis is lethal, and bacterial crossing of the BBB occurs in
CD46 mice, but not in nontransgenic mice (25). In representative
experiments, 30% of the CD46 transgenic mice survived day 1
postchallenge, and none survived day 2, whereas all nontransgenic
mice survived the challenge. To explore possible variations in bac-
terial blood counts after i.p. challenge with N. meningitidis
FAM20, blood was collected at different time points during the
first 24 h, diluted, and spread on GCB-agar plates for determina-
tion of CFUs. At 1, 3, and 6 h postchallenge there were no sig-
nificant differences in CFU per milliliter between CD46 transgenic
mice and nontransgenic mice (Fig. 1), supporting the idea that
survival of nontransgenic mice most likely was not due to differ-
ences in bacterial numbers initially reaching the blood. However,
The Journal of Immunology 435

FIGURE 1. Bacterial counts (CFU/ml⫺1)

in blood of CD46 transgenic and nontrans-
genic mice challenged i.p. with N. meningi-
tidis. A, Mice were inoculated with 108 bac-
teria i.p. and blood samples were taken from
the tail vein at different time points postin-
oculation. Blood was diluted, spread on
GCB-agar plates, and bacterial colonies were
enumerated the following day. CD46 trans-
genic (Œ) and nontransgenic mice (‚) were
challenged with wild-type N. meningitidis
FAM20, or CD46 transgenic (f) and non-
transgenic mice (䡺) were inoculated with
LPS-deficient FAM20. ⴱ, Significant differ-
ence between CD46 transgenic and nontrans-
genic mice inoculated with FAM20 (p ⬍
0.05). The results represent mean CFU per

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milliliter of two separate experiments (eight
animals per group). B, Table shows mean
CFU per milliliter ⫾ SD corresponding to
values displayed in A.

10-fold more bacteria were found in blood of CD46 transgenic
mice compared with nontransgenic mice at 12 h postinoculation,
suggesting that bacterial survival and avoidance of clearance was
enhanced in CD46 transgenic mice.
LPS-deficient FAM20 (lpxA mutant) does not cause disease in
CD46 transgenic mice at equivalent doses (25). As shown in Fig.
1, bacterial numbers in blood of CD46 transgenic mice and non-
transgenic mice were 30-fold lower at 1 h postchallenge with LPS-
deficient bacteria compared with the wild-type strain. Further,
LPS-deficient meningococci were cleared 12 h postchallenge.
Increased TNF and IL-6 production in infected CD46
transgenic mice
Most death by meningococcal sepsis is not only due to the infec-
tion itself, but also from hypotension and organ failure character-
istic of septic shock. This is a manifestation of the uncontrolled
release of proinflammatory cytokines, such as TNF and IL-6. Host
TNF and IL-6 responses were analyzed by measuring cytokine pro-
duction in mouse serum by ELISA at different time points postinfec-
tion. As shown in Fig. 2A, TNF levels were raised after i.p. challenge
with FAM20. Stimulation was significantly higher in CD46 mice
compared with nontransgenic mice, and at 1 h postinfection TNF
levels ranged between 271 and 425 pg/ml in CD46 transgenic mice
and between 185 and 263 pg/ml in nontransgenic mice. In patients
with septic shock caused by meningococci TNF serum levels may
reach ⬃500 pg/ml (35), which is comparable to TNF levels detected
in CD46 transgenic mice challenged with N. meningitidis FAM20
(350 pg/ml). Infection of CD46 mice or nontransgenic mice with
LPS-deficient FAM20 did not stimulate the production of TNF in

FIGURE 2. Kinetics of serum cytokine levels in

CD46 transgenic and nontransgenic mice after i.p. chal-
lenge with N. meningitidis. Mice were injected with 108
N. meningitidis and serum levels of TNF (A), IL-6 (B),
and IL-10 (C and D) were determined by ELISA. A–C,
Serum levels of cytokines in mice inoculated with wild-
type N. meningitidis FAM20; D, IL-10 levels in mice
challenged with LPS-deficient FAM20. Results repre-
sent mean ⫾ SD of three experiments. CD46 transgenic
and nontransgenic mice were significantly different at
time points marked with asterisks (p ⬍ 0.05).
436 MURINE IMMUNE RESPONSES TO MENINGOCOCCI

serum. Cytokines could not be detected in mice before infection or in

mice injected with medium (data not shown).
IL-6 induction was strong at 3 and 6 h postchallenge in CD46
transgenic mice and then decreased rapidly (Fig. 2B). Nontrans-
genic mice showed significantly lower levels of IL-6. IL-6 was not
detected in noninfected mice, mice inoculated with media, or mice
inoculated with LPS-deficient meningococci (data not shown). In
patients with meningococcal disease the pattern of TNF and IL-6
response resembled those seen in CD46 transgenic mice with an
early peak and rapid decrease, and with the IL-6 peak occurring
shortly after TNF (36).

Increased IL-10 production in CD46 transgenic mice

IL-10 is an anti-inflammatory cytokine with suppressive effects on
the synthesis of proinflammatory cytokines and chemokines, such
as TNF, IL-1␤, IL-1␣, IL-2, IL-6, and IL-8. IL-10 injection has
been shown to increase survival rates in murine models of endo-
toxemia (37–39). High levels of IL-10 in sera have been reported
from patients with meningococcal septic shock and fatal cases had
IL-10 levels of ⬎1000 pg/ml (22). As shown in Fig. 2C, the IL-10

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serum concentration in CD46 transgenic mice challenged with
wild-type meningococci increased with time, and reached 1000
pg/ml at 24 h postchallenge, which is similar to levels measured in
patients with severe meningococcal disease. LPS-deficient bacteria
triggered detectable IL-10 production only at 1 and 3 h, but not at
later time points (Fig. 2D). Sera from noninfected mice or mice
injected with medium did not show detectable levels of IL-10 (data
not shown). These data indicate that IL-10 reaches human-like
high levels in CD46 mice, but not in nontransgenic mice. Further,
early IL-10 induction occurs in the absence of bacterial LPS, in
contrast to TNF and IL-6, which were not detected after challenge
with LPS-deficient bacteria.
Neutrophil and macrophage counts in blood after
meningococcal challenge
Mice were challenged i.p. and blood samples were collected at
different time points postchallenge. Neutrophils vs total number of
immune cells were determined by microscopical examination of
blood smears. As shown in Fig. 3A, the neutrophil levels were
stable over time in CD46 transgenic mice, but increased in non-
transgenic mice at 6, 12, and 24 h postchallenge. These levels were
significantly higher than neutrophil levels at earlier time points and
levels in nontransgenic mice injected with medium (control).
Transgenic and nontransgenic mice challenged with LPS-deficient
meningococci did not show increased levels of neutrophils com-
pared with control mice (Fig. 3B). Following the same procedure
as described above blood smears were also analyzed for presence
of monocytes. No significant differences were detected between
transgenic and nontransgenic mice challenged with wild-type
FAM20 or the lpxA mutant (data not shown). Taken together, these
data show that after meningococcal challenge, neutrophil counts in
CD46 transgenic mice are not increased as compared with non-
transgenic mice. Possible explanations for the impaired neutrophil
recruitment could be induction of neutrophil apoptosis/cell death,
or increased adherence of neutrophils to vascular endothelial
surfaces.
Serum C5a levels differ in CD46 transgenic and
nontransgenic mice
C5a is generated upon activation of the complement cascade. It has
both anaphylatoxin and chemotactic activity, and can trigger de-
granulation of granulocytes. Excessive production of C5a in hu-
mans correlates with increased cytokine release and severe sepsis
(40). Complement activation after meningococcal challenge was
FIGURE 3. Changes in blood neutrophil counts of CD46 transgenic and
nontransgenic mice following i.p. challenge with N. meningitidis. Mice
were inoculated with 108 N. meningitidis in medium or medium alone
(control). Blood samples were drawn from the tail vein and blood smears
were stained and analyzed by light microscopy. A, Alterations in blood
neutrophils in CD46 transgenic (f) and nontransgenic (u) mice infected
with wild-type N. meningitidis FAM20. B, Changes in neutrophil counts in
blood postinoculation with LPS-deficient FAM20. f, CD46 transgenic
mice; u, nontransgenic mice. Results in A and B show the mean neutrophil
change compared with mice injected with medium alone (control, 䡺). ⴱ,
Significant differences between nontransgenic mice and CD46 transgenic
mice or mice injected with medium (p ⬍ 0.05). Experiments were repeated
twice (eight mice per group).
determined by measuring C5a levels in mouse serum at different
times postinfection. CD46 transgenic mice contained higher basal
levels of C5a in serum compared with nontransgenic mice after
injection with medium or FAM20 (data not shown). The control
level of C5a was 9 U in CD46 transgenic mice, and 3 U in non-
transgenic mice, indicating that the presence of human CD46 in-
creased basal C5a levels in the mice. The high levels of C5a in
CD46 transgenic mice occurred at the same time as high bacterial
blood counts, indicating that in this system meningococci show
resistance to complement-mediated killing. As shown in Fig. 4,
serum C5a was significantly raised in both CD46 transgenic and
nontransgenic mice after i.p. challenge with FAM20, however,
with delayed kinetics in the nontransgenic mice. Production of C5a
in CD46 transgenic mice peaked at 3 h postchallenge and then
decreased over time. At 24 h postchallenge, no difference in serum
C5a was detected compared with control mice. In nontransgenic
mice, C5a was maximal at 12 h postchallenge. It is unlikely that
the correlation between high C5a levels resulted in the increased
neutrophil numbers in nontransgenic mice, because there was no
relationship between high C5a and neutrophil recruitment in CD46
transgenic mice. We cannot discount that the neutrophil recruit-
ment could be mediated by another chemotactic factor.
The Journal of Immunology 437

binding protein whose expression is restricted to macrophages/

microglia (42), which is further enhanced in activated microglia
(43). CD46 transgenic mice challenged i.p. with wild-type N. men-
ingitidis were perfused with fixative and the expression of Iba1
was investigated immunohistochemically at different time points
(3, 6, 12, and 24 h). Activated microglia were detected throughout
the brain tissue of challenged CD46 transgenic mice at all inves-
tigated time points (Fig. 5A; piriform cortex, C; striatum). Few
activated microglia cells were detected in naive transgenic mice
(Fig. 5B; piriform cortex, D; striatum).

FIGURE 4. C5a production in CD46 transgenic mice (f) and nontrans-

genic mice (u) after i.p. challenge with wild-type N. meningitidis FAM20.
Mice were injected with 108 wild-type N. meningitidis FAM20 or medium
alone (control, 䡺), and C5a levels in serum were analyzed by ELISA. The
control basal level of C5a was 9 U in CD46 transgenic mice, and 3 U in
nontransgenic mice. The figure shows the fold increase of C5a after bac-
terial challenge. ⴱ, Significant differences (p ⬍ 0.05) in C5a levels between
infected mice and mice inoculated with medium. Results represent mean ⫾
SD (6 –10 mice within a given group), from two independent mouse
Induction of cyclooxygenase-2 (COX-2) expression in brain of
CD46 mice
COX-2 is an enzyme responsible for the production of PG H2, the
first step in the prostanoid biosynthesis (44). TNF and IL-1␤ in-
duce COX-2 production (45). CD46 transgenic mice were chal-
lenged i.p. with wild-type N. meningitidis and perfused with fix-
ative at 3, 6, 12 and, 24 h postchallenge. Brains were removed,
frozen, and expression of COX-2 was investigated immunohisto-

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experiments.
Activation of microglia cells in CD46 transgenic mice
Bacteria cross the BBB in CD46 transgenic mice challenged i.p.
with N. meningitidis (25). It is likely that the presence of bacteria
in the meninges activate inflammatory responses in the brain. Mi-
croglia share many phenotypic and functional characteristics with
macrophages and provide an initial line of defense in the brain
against invading pathogens into the CNS (41). Iba1 is a calcium-
chemically. Three hours postinfection COX-2 expression was de-
tected in piriform cortex and expression peaked at 6 h (Fig. 5E).
COX-2 expression could not be detected at 12 and 24 h postin-
jection (data not shown). Noninfected CD46 transgenic mice did
not express detectable levels of COX-2 (Fig. 5F). Further, an ac-
tivation of astrocytes was detected by GFAP-staining in the in-
fected brains, but there was no difference between transgenic and
nontransgenic animals in this respect (data not shown). Because
bacteria did not reach the brain in nontransgenic mice, it is possible

FIGURE 5. Detection of activated microglia (A–D)

and COX-2 (E and F) induction in brain sections from
N. meningitidis-infected CD46 transgenic mice (A, C,
and E) and noninfected mice (B, D, and F). Following
i.p. injection with wild-type N. meningitidis FAM20,
CD46 transgenic mice were perfused with fixative at
different time points. Brain sections were stained for
microglia and COX-2 as described in Materials and
Methods. Microglia cells in the piriform cortex of
CD46 transgenic mice (A) and noninfected mice (B) 6 h
postchallenge. Microglia cells in striatum of CD46
transgenic mice (C) and noninfected mice (D) 6 h post-
challenge. E, COX-2-expressing cells in the piriform
cortex in CD46 transgenic mice 6 h postchallenge with
FAM20. F, COX-2 expression in noninfected CD46
mice. White bar, 50 ␮m.
438
FIGURE 6. Neutrophil and macrophage counts at
site of injection (i.p.) in CD46 transgenic and nontrans-
genic mice. Transgenic mice and nontransgenic mice
were injected with 108 meningococci in medium or me-
dium alone. The i.p. fluid was collected at 1 and 3 h
postchallenge, stained for live neutrophils and macro-
phages, and analyzed by flow cytometry. Neutrophil
counts in CD46 transgenic and nontransgenic mice
postinjection with wild-type N. meningitidis FAM20
and LPS-deficient FAM20, respectively, are shown in A
and B. Alterations of macrophage counts in mice chal-
lenged with FAM20 and changes in macrophage num-
bers in mice inoculated with LPS-deficient FAM20 are
shown in C and D. Data are normalized such that cell
counts from mice injected with medium alone (䡺, con-
trol) are represented as 100%. f, CD46 transgenic
mice; u, nontransgenic mice. ⴱ, Significant differences
between mice injected with medium (control) and mice
challenged with bacteria (p ⬍ 0.05). Results represent
mean ⫾ SD of three experiments.
that the activation of astrocytes arises from signaling mediated by
cytokines, immune cells, or LPS.
Neutrophil and macrophage levels in i.p. cavity
To investigate the primary neutrophil and monocyte recruitment at
the site of inoculation, CD46 transgenic mice and nontransgenic
mice were challenged i.p. with wild-type N. meningitidis FAM20.
The i.p. fluid was collected by peritoneal wash at 1 and 3 h post-
challenge, stained for live neutrophils and macrophages, and an-
alyzed by flow cytometry. Both CD46 transgenic mice and non-
transgenic mice demonstrated a decrease of neutrophil numbers
after 3 h postinoculation with wild-type N. meningitidis compared
with mice injected with medium alone (control) (Fig. 6A). On the
contrary, inoculation with LPS-deficient meningococci in CD46
transgenic mice resulted in a 4-fold increase of neutrophils i.p. at
3 h postchallenge (Fig. 6B). The corresponding low numbers of
LPS-deficient meningococci in the blood at this time suggest that
the bacteria remain within the peritoneal cavity, and recruit neu-
trophils to the site of increased infection. This increase was not
seen in nontransgenic mice. Taken together these data indicate that
wild-type meningococci trigger a decrease of viable neutrophils in
both transgenic and nontransgenic mice, and that LPS deficiency
leads to elevated neutrophil numbers in CD46 mice but not in
nontransgenic mice.
A significant influx of macrophages i.p. occurred at 1 and 3 h
postchallenge of N. meningitidis wild-type strain FAM20 in CD46
transgenic mice but not in nontransgenic or in control mice (Fig.
6C), indicating that CD46 plays an important role in inducing mac-
rophage responses. Challenge with the LPS-deficient meningo-
cocci did not induce an influx of macrophages i.p. in transgenic or
nontransgenic mice (Fig. 6D). These data argue that LPS is also
required to stimulate increased local macrophage levels.
Discussion
In the present study we demonstrate important differences in early
immune responses against meningococcal infection in transgenic
mice expressing human CD46 compared with nontransgenic mice.
MURINE IMMUNE RESPONSES TO MENINGOCOCCI
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Responses in CD46 transgenic mice mimic human meningococcal
disease in many aspects. Further, the data indicate novel important
functions of human CD46, and show additional links between
CD46 and innate immunity.
Mice expressing the human cell surface protein CD46 are sus-
ceptible to meningococcal disease, in contrast to nontransgenic
mice. Following i.p. challenge of transgenic CD46 mice and non-
transgenic mice with N. meningitidis, similar bacterial counts in
blood during the first hours postinjection were observed. To elu-
cidate the mechanisms for the lethal outcome after bacterial chal-
lenge of CD46 transgenic mice, we investigated cytokine re-
sponses during the first 24 h after infection. The cytokines of major
interest are the proinflammatory cytokines TNF and IL-6, and the
anti-inflammatory cytokine IL-10, all shown to be elevated during
meningococcal disease (36). Further, high levels of inflammatory
cytokines can be correlated with meningococcal disease severity.
TNF and IL-6 were significantly higher in CD46 transgenic mice
compared with nontransgenic mice with peak values occurring
during the first hours after infection. Interestingly, levels of proin-
flammatory cytokines at early time points in CD46 transgenic mice
show comparable levels to human patients with meningococcal
disease. Further, a similar pattern is observed in patients with me-
ningococcal disease with an early peak and rapid decrease of TNF
and IL-6, and with the IL-6 peak following the TNF peak (36). The
anti-inflammatory cytokine IL-10 concentration was higher in
CD46 mice at 3–24 h postchallenge. Previous studies have shown
that patients with lethal meningococcal disease have IL-10 levels
of ⬃1000 pg/ml (22). Similar values were detected in CD46 trans-
genic mice at 24 h after infection. The uncontrolled release of
cytokines in transgenic CD46 mice might explain the rapid course
of disease leading to death within 48 h. In patients with sepsis,
death is correlated with increased levels of cytokines that eventu-
ally lead to total organ failure rather than to the actual bacterial
load circulating in the bloodstream (46 – 48).
C5a is an important mediator which is involved in stimulating
mononuclear cells and the release of proinflammatory cytokines.
To investigate the possible contribution of complement activation
The Journal of Immunology
to sepsis in CD46 transgenic mice we measured the complement
activation marker C5a in serum from infected CD46 transgenic
and nontransgenic mice at different time points. A higher level of
complement activation was observed in CD46 transgenic mice
compared with nontransgenic mice, moreover, the maximum level
of activation was also observed at earlier time points postchallenge
in CD46 transgenic mice. These results are in accordance with the
TNF and IL-6 release in CD46 transgenic mice challenged with
wild-type meningococci. It is possible that C5a leads to early ac-
tivation of cells and mediates cytokine release, resulting in sepsis
in CD46 transgenic mice. There is accumulating evidence that neu-
trophils are a significant source of serum IL-6 during sepsis (40).
In the current study, a similar level of monocytes and lower levels
of neutrophils were observed in CD46 transgenic mice compared
with nontransgenic mice after challenge with wild-type FAM20.
Although a time correlation exists between serum C5a levels and
neutrophil infiltration in the blood in nontransgenic mice, it is un-
likely that the level of C5a induces the neutrophil response because
no link between neutrophil levels and serum C5a was observed in
CD46 transgenic mice.
In human meningococcal disease bacteria cross the BBB, reach
the CSF, and cause meningitis. In cells of the BBB, proinflamma-
tory cytokines have the ability to trigger transcription of different
genes, including COX-2 (49). We found that meningococcal in-
fection induced expression of COX-2 immunoreactivity in piri-
form cortex in CD46 transgenic mice. This response became evi-
dent by 3 h, and was most prominent at 6 h postchallenge. COX-2
expression could not be detected in challenged mice at 12 and 24 h
postinfection or in noninfected mice. In vivo, local increases in
COX-2 expression have been associated with inflammation, sei-
zures, and ischemia (50 –52).
In CD46 transgenic mice, activation of microglia occurred at all
tested time points postchallenge. Microglia rapidly respond to
CNS injury, yet the mechanisms leading to their activation and
inactivation remain poorly defined. It has been shown in mice that
microglia, which normally have small bodies with finely branched
appendages, are activated to proliferate and migrate to the site of
damage, and then drastically transform into expanded amoeboid
shapes with the ability to clear invading pathogens (53, 54). Mi-
croglia arise from macrophages outside the nervous system and are
unrelated to other cells of the nervous system. Activation of mi-
croglia has been regarded as a brain tissue reaction to cell death
and infection, the main purpose of which is to remove cellular
debris. Mounting evidence indicates that activated microglia, be-
sides removing cellular debris, may be actively involved in neu-
rodegenerative processes. In contrast, it is also suggested that mi-
croglia may exert neuroprotective effects, depending on the
situation.
After i.p. challenge with wild-type meningococci, both CD46
transgenic and nontransgenic mice demonstrated decreased neu-
trophil numbers in the i.p. cavity. Because the neutrophil numbers
did not differ between transgenic and nontransgenic mice after
challenge with wild-type bacteria, the reason for this specific neu-
trophil change is independent of CD46. Interestingly, LPS pre-
pared from Pseudomonas aeruginosa has been shown to decrease
neutrophils i.p. by TNF-induced apoptosis (55). In this study, chal-
lenge with LPS-deficient meningococci resulted in several-fold el-
evated neutrophil number in CD46 transgenic, but not in nontrans-
genic, mice indicating that CD46 plays an important role in the
initial recognition and response to LPS in N. meningitidis infec-
tion. As described previously (24, 56), LPS-deficient N. meningi-
tidis induces lower levels of serum cytokines than the wild-type
strain. Absence of high levels of TNF might lead to neutrophil
439
accumulation in the i.p. cavity of mice challenged i.p. with LPS-
deficient bacteria, rather than neutrophil apoptosis.
Macrophages, in contrast, were present in significantly higher
numbers in CD46 mice infected with meningococci compared with
nontransgenic mice and mice injected with medium alone. These
data argue that in CD46 transgenic mice, macrophages are stimu-
lated to migrate to the site of infection. Challenge with LPS-defi-
cient bacteria did not induce a comparable influx of macrophages
into the i.p. cavity.
By investigating immune responses to meningococcal infection
in transgenic CD46-expressing mice, we found similar patterns of
immune responses as compared with human meningococcal dis-
ease. The knowledge about these responses is important from a
therapeutic point of view, where new drugs targeted against these
factors could limit and impede the fulminant development of dis-
ease. The importance of an animal model mimicking human me-
ningococcal disease is also crucial to trial new vaccine candidates.
Acknowledgments
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We gratefully acknowledge Margareta Hagelin, Maj-Britt Alter, and
Katarina Åman for excellent technical assistance.
Disclosures
The authors have no financial conflict of interest.
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