Escolar Documentos
Profissional Documentos
Cultura Documentos
00/0
THEJOURNAL OF ~MMUNOLOCY Voi. 149. 2709-2714. No. 8.October 15. 1992
Copyrlght 0 1992 by The Amerlcan Assoclatlon of Immunologlsts Printed in U.S.A.
The potential of the immune system to inhibit or it is notclear that cancer is routinely eliminated by
stimulate tumor growth is a vivid example of the “immunosurveillance”as originally envisaged by Ehrlich
*two-edged sword” nature of immune responses. Our ( 1 ) and developed by Burnet (8).That is, a picture of the
results provide evidencethat thisdual capacity can immune system as serving a purely inhibitory role in
be attributed, in part, to thedual pathways of argi- cancer had to be rethought whenit was discovered during
nine metabolism exhibited by intratumor macro- the 1970’s that immunodeficient hosts did not exhibit an
phages. Specifically, i.p. tumor rejection in P815- increased incidence of cancer (with the minor exception
preimmunized miceis accompanied by an upshift in of lymphoid malignancies) (5, 9, 10). Also during this
intratumor macrophage arginine metabolism to the
nitric oxide (NO) synthase pathway that yields ci- period, Prehn and co-workers (11, 12) advancedthe the-
trulline and NO. A rapid and marked local increase ory that the immunesystem could actively stimulate
in IFN-y (both mRNA and protein) in preimmunized cancer. Although the “immunostimulation” theory has
mice during tumor rejection suggests that this cy- received little attention by most investigators in their
tokine plays a role in up-regulating nitric oxide pro- pursuit of anticancer defenses, sufficient evidence has
duction in vivo. Unliketumor rejection, progressive accumulated to conclude that cancergrowth can be stim-
i.p. P815 tumor growth in naive mice is associated ulated a s well as inhibited by the immune system (13-
with a marked decline in the production of citrul- 15).In particular, macrophages have been demonstrated
line/NO by intratumor macrophages. Examination toexhibit this dualcapacityunderdifferentcircum-
of macrophage arginine metabolism via arginase stances (16-21).
revealed a pattern opposite that of NO synthase. Recent results in this laboratory (22-24) and others
The local production of ornithine/urea markedly (25-28)havedemonstrated thatthe pathway macro-
increases during progressive tumor growth whereas phages use to metabolize arginine canpromote or inhibit
arginase activity decreases during tumor rejection. their functions, and thatof cells with which they com-
Inasmuch as nitric oxide inhibits tumor cell repli- municate. This knowledge combined with the aforemen-
cation whereas ornithine is the precursor of poly-
amines required for cell replication, these results tioned background in tumor immunology led us to pos-
are consistent with the conclusion that thepathway tulate that macrophage arginine metabolism at the site
macrophages use to metabolize arginine can influ- of atumor could influencewhethertumor growth is
ence the type of host immune responses against inhibited or stimulated. It was proposed that macrophage
cancer and otherconditions. arginine metabolism in the tumor bed via the NO3 syn-
thase pathway would favor tumor inhibition becauseNO
is tumoristatic/tumoricidal(26).If instead, NO production
Despite intensive and prolonged researchefforts in was down- regulated, tumor growth might be allowed or
tumor immunology dating back to the turn of the century even stimulatedbecausemacrophages are aprimary
when Ehrlich(1) and others (2) proposed first an antican- source of growth factors that promote proliferation of a
cer role for the immune system, there are still no con- variety of cells such as those involved in wound healing
sistently effective protocols for the immunotherapy of
and regeneration (29, 30). Further, if the predominant
human cancer. Clearly the immune system has the po-
pathway of arginine metabolism by intratumor macro-
tential to inhibit cancer. Indeed, several different potent
phages wasvia arginase (yieldingornithine and urea) (31)
antitumor mechanisms have been identified in animal
models (3.4, reviewed in 5-7).At the sametime, although rather than NO synthase then tumor growth might be
anticancer potential can be demonstrated experimentally further promoted because ornithine is the precursor of
polyamines that arerequired for cell replication (32, 33).
This report examines these hypotheses. A model was
Received for publication April 6, 1992.
Accepted for publication August3, 1992. used in which tumor growth or rejection occurs in the
The costs of publication of this article were defrayed in part by the peritoneum. This model facilitates analyses of the intra-
payment of page charges. This article must therefore be hereby marked tumor cellular infiltrate and local extracellular environ-
aduertfsement in accordance with 18 U.S.C. Section 1734 solely to indi-
cate this fact. ment (peritoneal fluid).It will be shown thattumor rejec-
This work was supported by National Institutes of Health Grant tion or growth is associated with local up regulation of
GM3224 and CA27523(RE). Certain of the results in this paper were
presented in preliminary form at the 2nd InternationalMeeting on Nitric the N O synthase or arginasepathway, respectively.
Oxide in London, October1991. These and other data suggest that intratumor macro-
‘Address correspondence and reprint requests to Charles D. Mills,
Department of Surgery, University of Minnesota, Box 120 University of Abbreviations used in this paper: NO, nitric oxide: PEC. peritoneal
Minnesota Hospitals and Clinics, Minneapolis,MN 55455. exudate cells.
2709
2710 MACROPHAGE
METABOLISM
ARGININE AND CANCER
801
100
W Immune 1200 1j
0 Naive 1000
L1 1,
Normal
60 Media
A
40
L
.-
c
0
FLgure 2. Changesinintratumorleukocyte 20
arginine metabolism during tumor rejection or
progressive tumorgrowth. PEC were collected
from groups of five immunized or naive mice 0
after P815 tumor implantation on the days indi- 3
cated. PEC were culturedat 1.5 x 10B/ml(0.2 ml) 1 3 5 7 1 5 7
in quadruplicate in 96-well microtiter plates for 30
20 h . Cell-free supernatant was collected and
stored at -80°C until analyzed. “Normal” = resi- 0
dentperitonealexudateleukocytes of normal
mice. “Media”= cell free media cultured for20 h.
20
Citrulline. ornithine, and urea were analyzedby
-5 2ooo
Iai
HPLC: nitrite (NO2)determined using the Griess
reagent. The same pattern of intratumor leuko-
cyte arglnine metabolism was observedIn three
nts. similar 2! 10
0
3
1 3 5 7 1 5 7
in the preceding section could be attributed to macro- onstrated elevated ornithine production as compared
phages, PEC were fractionated. Peritoneal exudate leu- with resident PEC from normal mice. Again, the majority
kocytes were incubated for 2 h at 37°C: leukocytes re- of leukocyte ornithine production from naive tumor bear-
maining adherent afterthorough washingare “adherent” ers or normal mice is found in the adherent fraction.
and the rest are “nonadherent.” Adherent cells comprised Although a percentage (approximately 50%)of the PEC
on average 54% of immune PEC and 45% of naive PEC. in naive mice at this time are tumor cells, P815 tumor
More than 93%of adherent immune or naive PEC were cells are nonadherent and produce little ornithine. Spe-
macrophages as assessed by morphology or nonspecific cifically, a culture supernatantof P815 tumor cells con-
tained 36 gM ornithine after 20 h incubation at 1.5 X
esterase staining. Themajority of remaining cells exhib-
106/ml (the maximum cell density if all PEC were tumor
ited lymphocyte-like morphology. It can be seen first in cells]. Together, these data indicate that macrophages
Figure 3A that, again, local NO production by peritoneal are primarily responsible for the increased local citrul-
exudate leukocytes from immunized mice is elevated 5 line/NO or ornithine/ureaproduction during tumorrejec-
days aftertumor implantation. More importantly, Figure tion orprogressive tumor growth,respectively.
3A shows that virtually all of the N O (measured a s NO2) NO synthase or arginase pathway predominates in
produced can be attributed to the adherent cell fraction. local extracellular tumor environment during rejection
In Figure 3B it can be seen that PEC recovered instead or growth, respectively. Direct examination of arginine
from naive mice 5 days after tumor implantation dem- metabolism at the site of tumor rejection or progressive
2712 MACROPHAGE ARGININE METABOLISM AND CANCER
301 A
W Immune
8o01
B
2iL
Figure 3. Evidence that macrophages are
primarily responsible for changes in intratu-
mor leukocyte arginine metabolism. PEC
were collected from groupsof five immune or
five naive mice 5 days after P815 tumorim- 10
plantation.Unfractionated PEC, adherent
PEC. or nonadherent PEC were cultured over-
night a s in Figure 2.
Non-AdherAdherent Un-Fract.
Non-Adher.
Adherent Un-Fract.
0 h
tumor growth (peritoneal fluid)revealed the same char- to be maximal 1 day after tumor implantation and to
acteristic differences observed with intratumor leuko- disappear by day 5. In support of these findings, Figure
cytes. That is, the local concentration of citrulline is 5C shows that leukocyte mRNA coding for IFN-7 is abun-
elevated in peritoneal fluid in immunized mice within 1 dant early after tumor challenge and rapidly declines
day of tumor implantation and remains somewhat ele- indicating a close correspondence between gene tran-
vated during tumor rejection (Fig. 4A).Inasmuch as ci- scription and translation/secretion. Finally, because lo-
trulline andNO are produced from argininein equimolar cal production of citrulline/NO remains elevated during
quantites by NO synthase (25), the concentration of this tumor rejection for at least1 wk, the data suggest that if
amino acid shouldreflect NO synthase activity. In IFN-y is responsible for increasedNO production in vivo,
marked contrast to these findings, it can be seen that then it is either required only transiently or that other
during progressive tumor growth, the intratumor citrul- signals play a role as well.
line concentration declines to levels significantly below In contrast to our knowledge of factors such as IFN-y
that found inperitoneal fluid from normal mice. Exami- that “activate” macrophages, less is known concerning
nation of arginine metabolism via arginase revealed pre- the signals that down-regulate local macrophage arginine
cisely the opposite pattern to that of N O synthase. Spe- metabolism to citrulline/NOand increase the production
cifically, there wasa progressive rise in thelocal concen- of ornithine/urea during progressive tumor growth. How-
tration of ornithine in peritoneal fluid during progressive ever, some informed speculation maybe useful.
tumor growth, but not during tumor rejection (Fig. 48). A tumor could regulate intratumor macrophage argi-
In contrast, thelocal concentration of ornithine is below nine metabolism directly or indirectly through other leu-
normal during specific tumor rejection. kocytes or another organ system. Regarding directregu-
Regulation of local arginine metabolism during tumorlation, tumors have been reported to produce a variety of
rejection or growth. Our results indicate that tumor re- products that could affect macrophage arginine metabo-
jection and progressive tumor growth are associated with lism. In particular, Nathan and co-workers(40, 41)have
characteristic and opposite shifts in macrophage argi- isolated a protein from several murine tumors that down-
nine metabolism. A s for the signals that regulate these regulates theNO synthase pathway in macrophages. One
changes in arginine metabolism, IFN-y has been reported tumor that secretes this protein (macrophage deactivat-
(36) to up-regulate the NO synthase pathway. Evidence ing factor) is the P815 mastocytoma used in our study.
consistent with the ability of IFN-y IFN to up-regulate Thus, it seems reasonable to postulate that such a factor
this pathway is shown in Figure 5A. It can be seen that plays a role in down-regulatingthe NO synthase pathway
the local extracellular concentration (peritoneal fluid) of during progressive P815 tumor growth. Another factor
IFN-y is markedly increased in immunized mice, but not that seems likely to play a regulatory role in intratumor
naive mice, after tumor implantation. Evidence that in- macrophage arginine metabolism is TGF-P. This cytokine
tratumor leukocytes are responsible forlocal the increase is produced by some tumors and has been shown to down-
in IFN-y is shown in Figure 5B where it can be seen that regulate NO production in macrophages in vitro (41).CSF
leukocytes recovered fromthe siteof tumor rejection, but includinggranulocyte-macrophage CSF arealso pro-
not from the site of tumor growth, secreted markedly duced by certain tumors (42) and these cytokines have
elevated levelsof IFN-7. Leukocyte secretion of IFN-r a n d been demonstrated to inhibit activation-associated func-
its concentration in peritoneal fluid were both observed tions of macrophages (43).Although CSF have not been
I
Immune
“i
A
0 Naive
Normal 60
FLgure 4. Changes in arginine metabolism in r
1i
the local extracellular environment during tumor
i
regression or progressive tumor growth. Perito-
neal fluid was collected and pooled from groups
of five immunizedor naive mice on the indicated =
days after P815 tumor Implantation. Citrulline
and ornithine concentrations were determined 0 20
using HPLC. A virtuallyidenticalpattern of
changes in citrulline and ornithine in peritoneal
fluid wasobserved in another experiment of the 3 5 0 5 9
same design. Normal peritoneal fluid represents
1 7 9 1 3
the mean & SD of five mice.