Department of Microbiology and Immunology, University of Kentucky, College of Medicine, Lexington
Correspondence: Dr. Donald Cohen, Department of Microbiology and Immunology, University of Kentucky, College of Medicine, 800 Rose St., Lexington, KY 40536-0084. E-mail: dcohen{at}pop.uky.edu
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and IL-6 was significantly reduced in both
alveolar and peritoneal macrophages from infected versus uninfected
mice. IL-10 hyporesponsiveness was not related to direct infection by
the retrovirus, because bone marrow-derived macrophages infected in
vitro with LP-BM5 were as responsive to IL-10 as were uninfected bone
marrow-derived macrophages. TNF- appeared to contribute to
development of IL-10 hyporesponsiveness, because exposure of normal
macrophages to TNF- but not interferon-
reduced macrophage
responsiveness to IL-10. Reverse transcriptase-PCR and flow cytometry
demonstrated normal expression of the and ß chains of the IL-10
receptor in macrophages from infected mice, suggesting that IL-10
hyporesponsiveness is not related to a change in receptor expression.
The potential role of reduced IL-10 responsiveness in the chronicity of
inflammation in this and other diseases is discussed.
Key Words: cytokine • inflammation • retroviruses • TNF-
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, IL-1, IL-6, and IL-12 and the expression of major
histocompatibility complex class II and costimulatory molecules, such
as B7 and CD40 [1
]. The reduction of proinflammatory
cytokine synthesis by IL-10 has been shown to correlate with enhanced
survival in animal models of septic shock and immune complex alveolitis
[5
6
7
8
9
]. Given the protective responses induced by IL-10
administration in such animal models, the use of IL-10 as a possible
therapeutic tool has been suggested for several clinical inflammatory
conditions, such as chronic autoimmune diseases, septic shock, and
adult respiratory distress syndromes [10
11
12
13
].
Synthesis of IL-10 usually occurs as a consequence of acute and chronic
inflammatory responses, and neutralization of IL-10 often exacerbates
inflammatory lesions. However, a number of examples exist in which
IL-10 is expressed during chronic inflammatory states in which the
immunosuppressive effects of IL-10 appear to be minimal. In this
regard, coexpression of interferon (IFN) and/or TNF- with IL-10
has been shown to occur during autoimmune diseases, such as rheumatoid
arthritis [14
], systemic lupus erythematosus (SLE)
[15
], and multiple sclerosis [16
], and in
inflammatory conditions such as allograft rejection
[17
]. Simultaneous expression of IFN-, TNF-, and
IL-10 has also been reported to occur during chronic infections by HIV,
Borrelia, and Plasmodium species
[18
19
20
21
]. Suggestions have been made by some
investigators that coexpression of IL-10 with cytokines such as IFN-
and TNF- may indicate that the level of IL-10 in these conditions is
not sufficient to completely suppress the inflammatory response.
However, a corollary of this argument is that the in vivo expression or
responsiveness of IL-10 receptors might be altered such that even high
levels of IL-10 are incapable of down-regulating inflammation. We
previously showed that C57Bl/6 mice infected with the LP-BM5 retrovirus
develop a chronic progressive interstitial pneumonitis in which mRNAs
for IFN-, TNF-, IL-1, and IL-10 are coexpressed during the
chronic phase of infection [22
]. We demonstrate in this
report that the elevated expression of proinflammatory cytokines in the
face of chronic expression of IL-10 is caused in part by a decrease in
the responsiveness of macrophages to IL-10 and that hyporesponsiveness
is not caused by a change in expression of the IL-10 receptor.
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Viral infection
LP-BM5 virus is a mixture of murine leukemia viruses containing
the disease-causing retrovirus Bm5 and an ecotropic helper virus and
mink cell focus-forming virus that are constitutively produced by
chronically infected SC-1 cells [23
]. Chronically
infected SC-1 cells (clone 6) were obtained from H. C Morse III
[National Cancer Institute, National Institutes of Health (NIH),
Bethesda, MD], and stocks were prepared as previously described
[22
]. For infection, mice were inoculated
intraperitoneally with 1 mL of LP-BM5 virus stock, which contains an
effective dose sufficient to cause a 50% increase in the spleen weight
in 1–2 weeks and immunodeficiency by 8 weeks [22
]. All
experiments in the current studies used mice infected for 10–12 weeks.
All experiments were performed at least three times unless otherwise
indicated.
Macrophage isolation and treatment in vitro
Alveolar cells were collected from normal mice by lavage of
lungs with 10 mL of phosphate-buffered saline-EDTA. Cells from four
mice per group were pooled, washed, and brought to a concentration of
106 cells/mL in RPMI 1640 medium containing 5% fetal calf
serum (FCS). Normal peritoneal cells were collected by lavage of the
peritoneum with 5 mL of phosphate-buffered saline. Isolated cells from
four mice per group were pooled, washed, and brought to a concentration
of 106 cells/mL in RPMI medium containing 5% FCS. All
cells were cultured in 24-well plates at 106 cells per well
for 2 h at 37°C in 5% CO2.
Nonadherent cells were removed by rinsing plates twice with medium. Adherent cells were >90% macrophages as determined by flow cytometry with F4/80 antibody. The adherent cells were incubated with IL-10 (5 ng/mL) for 2 h at 37°C after which lipopolysaccharide (LPS) from Escherichia coli O111:B4 (5µg/mL) (Difco, Inc., Detroit, MI) was added, and plates were cultured for an additional 24 h. Supernatants were collected and stored at -20°C.
Quantification of cytokine protein
Cell culture supernatants were analyzed for cytokine content by
ELISA as previously described [22
], using antibodies and
standards purchased from PharMingen, Inc. (San Diego, CA). For the
analysis of IL-6, the following reagents were used at concentrations
recommended by the manufacturer: capture antibody, purified rat
anti-mouse IL-6 (PharMingen catalog no. 18071D); detection antibody,
biotin rat anti-mouse IL-6 (PharMingen catalog no. 18082); standard,
recombinant mouse IL-6 (PharMingen catalog no. 19251V). For the
analysis of TNF-, the following reagents were used at concentrations
recommended by the manufacturer: capture antibody, purified anti-mouse
TNF- (PharMingen catalog no. 18131D); detection antibody, biotin rat
anti-mouse TNF- (PharMingen catalog no. 18352D); standard,
recombinant mouse TNF- (PharMingen catalog no. 19321T).
Generation and infection of bone marrow-derived macrophages
Femurs from normal C57Bl/6 mice were flushed with RPMI 1640
medium containing 10% FCS. Single-cell suspensions of bone marrow
cells were cultured in 100-mm-diameter tissue culture plates in RPMI
1640 containing 10% FCS and 50% culture supernatant from the LadMac
cell line, which is transfected with the murine macrophage-colony
stimulating factor (M-CSF) gene and constitutively produces M-CSF
[24
]. This cell line was generously provided by W.
Walker, St. Jude Children’s Hospital, Memphis, TN. Plates containing
bone marrow cells were cultured at 37°C in 5% CO2 to
allow macrophage development. Twenty-four hours after culture, 10 mL of
LP-BM5 virus-containing supernatant or 10 mL of medium (control) were
added to the cultures. Plates were incubated an additional 6 days
before analysis to allow complete macrophage development and virus
infection to occur. Infection with LP-BM5 retrovirus was verified by
reverse transcriptase (RT)-PCR for expression of RNA for the
disease-causing BM5 virus as described below.
RT-PCR for analysis of cytokines, cytokine receptor, and viral RNA
For analysis of IFN-, IL-10, and IL-10R mRNA expression,
total RNA was extracted from lung lymphoid cells (IFN- and IL-10) or
lung and peritoneal macrophages (IL-10R mRNA) and were pooled from four
mice per group. Infected mice were used at 10–12 weeks postinfection
unless otherwise indicated. Normal controls were age matched to the
infected mice. For analysis of viral RNA, total RNA was extracted from
cultured bone marrow-derived murine macrophages. One microgram of total
RNA was reverse transcribed into cDNA with the Promega Reverse
Transcription System (Promega Corp., Madison, WI) and then amplified
with Taq polymerase for 30 cycles in a Perkin-Elmer thermal
cycler (denaturation at 94°C for 1 min, primer annealing at 55°C
for 2 min, and primer extension at 72°C for 2 min). Primers were
synthesized based on analysis of the DNA sequence in GenBank, using the
on-line primer selection program from the Virtual Genome Center,
University of Minnesota, Minneapolis (http://alces.med.umn.edu/raw
primer.html) or from previously published sequences. Primers for the
disease-causing BM5 retrovirus were as previously published
[25
]. Sequences of these primers are indicated below.
: forward, 5'-TACTGCCACGGCACAGTCATTGAA-3'; reverse,
5'-GCAGCGACTCCTTTTCCGCTTCTT-3' (405 bp) : forward, 5'-GCTGCCTTCAGACTCTTC-3'; reverse,
5'-AACCCCTCTGTGATCGGA-3' (508 bp)
Preliminary studies have indicated that 30 is the number of
cycles that is subsaturating for IFN-, IL-10, IL-10R chains, and
BM5. ß-actin RT-PCR was performed for 25 cycles. PCR products were
separated by electrophoresis on 2% agarose gels and were visualized by
ethidium bromide staining on a GelPrint 2000i imaging system
(BioPhotonics Corp., Ann Arbor, MI). The PCR product images presented
are negative images of otherwise unaltered band images. Integrated
optical densities of negative-image bands were obtained using NIH Image
software.
Flow-cytometric analysis of IL-10 receptor expression
Surface expression of IL-10 receptors was determined by the
binding of fluorescein isothiocyanate (FITC)-labeled
recombinant human IL-10 (R&D Systems, Minneapolis, MN), according to
the manufacturer’s instructions. Briefly, normal and infected alveolar
and peritoneal cells were preincubated with anti-FcR to block Fc
receptor binding and then incubated with RPE-Cy5-labeled F4/80
(Serotec, Inc., Raleigh, NC) and FITC-labeled IL-10. As a negative
control, groups of cells were stained with F4/80 Ab and FITC-labeled
IL-10 which had first been neutralized by preincubation with anti-IL-10
antibody. Flow cytometry was performed with a FACSCalibur cytometer
(Becton-Dickinson, San Jose, CA). Cells were gated on
F4/80+ macrophages and then analyzed for the capacity to
bind FITC-labeled IL-10 or neutralized FITC-labeled IL-10.
Statistics
Significant differences between groups were analyzed using the
SigmaPlot software program (SSPS Science, Chicago, IL). Data
were analyzed using Student’s t-test or one-way analysis of
variance (ANOVA) as indicated in the figure legends. Multiple
comparisons versus a control group were compared using the Bonferroni
test.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-10 has been shown to occur early after infection and to
persist throughout the infectious process [27
]. In the
current study, analysis of cytokine mRNA expression in the lungs of
normal and infected mice demonstrated that beyond 8 weeks
postinfection, both IFN- and IL-10 were expressed simultaneously
(Fig. 1
). IFN- expression was observed by 2 weeks after infection and
peaked between weeks 4 and 8. In contrast, significant levels of IL-10
were not observed until 8 weeks after infection, and high levels of
expression were maintained through 16 weeks of infection. As expected,
expression of IL-10 was associated with reduced expression of IFN-
at 12 and 16 weeks after infection, indicating the known inhibitory
effect of IL-10 on IFN- expression [28
]. However, we
consistently observed that the expression of IFN- never returned to
normal levels in spite of the strong expression of IL-10 in lung
tissue. Moreover, we have shown that severe lung inflammation can
persist through 16 weeks of infection in LP-BM5-infected mice
[22
], suggesting that persistent expression of IL-10
failed to control the inflammatory response in the lungs of infected
mice.
 View larger version (17K): [in a new window] |
Figure 1. Kinetics of cytokine expression in lungs during LP-BM5 infection. RNA
was extracted from isolated interstitial lymphoid cells, which were
collected and pooled from four mice per group at the indicated time
after infection. RNA was analyzed by RT-PCR for expression of IFN-
and IL-10. Data are expressed as integrated optical density of the PCR
products. Data are representative of two separate experiments.
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and persistent
pulmonary inflammation are associated with a change in the cellular
response to IL-10, alveolar macrophages were isolated from normal and
10-week-infected mice and evaluated for the ability of recombinant
murine IL-10 to inhibit LPS-induced synthesis of TNF- and IL-6. As
seen in Figure 2
, TNF- production by normal alveolar macrophages stimulated with
LPS for 24 h was inhibited nearly to background levels by
preincubation for 2 h with recombinant-IL (rIL)-10 at 2.5 and 5
ng/mL. In contrast, TNF- synthesis was only marginally reduced by
IL-10 treatment of infected macrophages. Similarly, IL-6 production was
inhibited 68 and 73% by preincubation of normal alveolar macrophages
with 2.5 and 5 ng/mL of IL-10, respectively, whereas infected
macrophages were inhibited by only 23 and 26% by the same respective
concentrations of IL-10. Thus, alveolar macrophages from
LP-BM5-infected mice displayed partial resistance to the
immunosuppressive effects of IL-10.
 View larger version (13K): [in a new window] |
Figure 2. Resistance of alveolar macrophages to IL-10. Alveolar macrophages from
normal and 10-week-infected mice were incubated for 2 h in the
indicated concentrations of murine rIL-10, after which LPS (10 µg/mL)
was added to the cultures. Twenty-four hours later, the culture
supernatants were analyzed for TNF- and IL-6 by ELISA. Data were
analyzed by one-way ANOVA, and multiple comparisons versus a control
were performed with Bonferroni’s test. *, P  0.05;
**, P 0.01; n.s., not significant. Data are
representative of three separate experiments.
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and IL-6 production (Fig. 3
). A similar pattern of IL-10 resistance was observed in peritoneal
macrophages from normal versus infected mice. Synthesis of both TNF-
and IL-6 was low in both normal and infected macrophages in the absence
of any in vitro stimulation. Culture for 24 h in the presence of
LPS induced the synthesis of TNF- and IL-6 in both normal and
infected cells with the production of cytokines generally being greater
in macrophages from infected mice. Prior incubation of normal
macrophages in concentrations of mouse rIL-10 from 1.25 to 5 ng/mL
reduced TNF- and IL-6 synthesis to levels near those observed in
unstimulated macrophages. In contrast, preincubation of infected
macrophages with IL-10 also reduced cytokine production; however,
substantial synthesis still occurred and often was above that seen in
normal macrophages stimulated with LPS in the absence of IL-10. Thus,
infection of mice with LP-BM5 retrovirus resulted in the systemic
appearance of macrophages that showed pronounced resistance to the
inhibitory effects of IL-10.
 View larger version (15K): [in a new window] |
Figure 3. Resistance of peritoneal macrophages to IL-10. Peritoneal macrophages
from normal and 10-week-infected mice were incubated for 2 h in
the indicated concentrations of murine rIL-10, after which LPS (10
µg/mL) was added to the cultures. Twenty-four hours later the culture
supernatants were analyzed for TNF- and IL-6 by ELISA. Data were
analyzed by one-way ANOVA, and multiple comparisons versus a control
were performed with Bonferroni’s test. *, P 0.05;
pa*, P 0.01; n.s., not significant. Data are
representative of three separate experiments.
|
 View larger version (14K): [in a new window] |
Figure 4. Responsiveness of bone marrow-derived macrophages to IL-10. Normal and
LP-BM5-infected bone marrow macrophages were incubated for 2 h in
the indicated concentrations of murine rIL-10, after which LPS (10
µg/mL) was added to the cultures. Twenty-four hours later, the
culture supernatants were analyzed for TNF- and IL-6 by ELISA. Data
from infected versus normal groups were analyzed for each concentration
of IL-10 using Student’s t-test. *, P
0.05; **, P 0.01; n.s., not significant. Data are
the means of three separate experiments.
|
and
TNF- are expressed persistently during infection with LP-BM5
[22
, 26
]. Preexposure of normal macrophages
to IFN- was therefore evaluated for the ability to induce resistance
to IL-10 (Fig. 5
). Stimulation of normal peritoneal macrophages with LPS alone
induced IL-6 synthesis, whereas 2-h preincubation with IL-10 inhibited
LPS-induced IL-6 synthesis by approximately 50%. Neither IL-10 alone
nor IFN- alone led to induction of IL-6 synthesis. Preexposure of
macrophages to IFN- for 24 h at doses of 2, 4, and 8 ng/mL had
no effect on LPS-induced IL-6 synthesis. More importantly, that
preexposure to IFN- for 24 h also had no effect on the ability
of IL-10 to inhibit IL-6 synthesis in LPS-stimulated macrophages. This
suggests that the expression of IFN- in infected mice, which
occurred prior to expression of IL-10 (Fig. 1)
, is not the likely
reason for development of IL-10 resistance. Similar studies were
performed to determine whether prior exposure to TNF- could
contribute to IL-10 resistance (Fig. 6
). IL-10 (10 ng/mL) was able to inhibit IL-6 synthesis in response
to LPS by 80.6%. Preexposure to TNF- at concentrations of 1 ng/mL
and 1 µg/mL for 24 h partially reduced the inhibitory effect of
IL-10 in that the same concentrations of IL-10 inhibited IL-6 synthesis
by only 68.5 and 49.0%, respectively. The effect of TNF preexposure
was not caused by any additive effect with LPS on IL-6 synthesis,
because TNF preexposure had no effect on the level of IL-6 synthesis
induced either by an optimal concentration (5 µg/mL) of LPS alone
(Fig. 6) or by a suboptimal concentration of LPS (1 µg/mL) (data not
shown). Studies were performed on the effect of preexposure to combined
IFN- and TNF-. The combined preexposure to IFN- and TNF-
neither enhanced nor diminished the effect of TNF- on IL-10 receptor
responsiveness (data not shown). The ability of TNF- to induce
resistance to IL-10 was usually less than that observed in macrophages
from infected mice. These data suggest that exposure of macrophages to
TNF- might contribute to the develop of IL-10 resistance but that
exposure to TNF- alone might not be sufficient to achieve the level
of resistance seen in infected macrophages.
 View larger version (17K): [in a new window] |
Figure 5. IFN- exposure does not induce IL-10 resistance in macrophages.
Normal peritoneal macrophages were cultured for 24 h in the
presence of the indicated concentrations of recombinant murine IFN-.
Cells were then exposed to murine rIL-10 (10 ng/mL) for 2 h, after
which LPS (10 µg/mL) was added to the cultures. Twenty-four hours
later, the culture supernatants were analyzed for TNF- and IL-6 by
ELISA. Data were analyzed by one-way ANOVA, and multiple comparisons
versus a control were performed with Bonferroni’s test. The control
for cells treated with LPS without IL-10 was bar 2; the control
for cells treated with LPS plus IL-10 was bar 3. *, P 0.05. Data are representative of three separate experiments.
|
 View larger version (18K): [in a new window] |
Figure 6. TNF- induces partial resistance to IL-10. Normal peritoneal
macrophages were cultured for 24 h in the presence of the
indicated concentrations of recombinant murine TNF-. Cells were then
exposed to murine rIL-10 (10 ng/mL) for 2 h after which LPS (10
µg/mL) was added to the cultures. Twenty-four hours later the culture
supernatants were analyzed for IL-6 by ELISA. Data were analyzed by
one-way ANOVA and multiple comparisons versus a control were performed
with Bonferroni’s test. *, P 0.05; **,
P 0.01; n.s., not significant. Data are
representative of three separate experiments.
|
chain and a
40-kDa ß chain [1
, 29
]. The receptor
density on macrophages is low, with only a few hundred receptors per
cell [1
]. RT-PCR analysis of the IL-10 and IL-10ß
chains showed that both chains of the receptor are expressed in normal
and infected peritoneal macrophages (Fig. 7
, insert). Expression of IL-10 and ß chain mRNA was also
observed in normal and infected alveolar macrophages, but at a lower
level than that observed in peritoneal macrophages (data not shown).
Surface expression of IL-10 receptors was evaluated by measuring the
ability of alveolar and peritoneal macrophages to bind FITC-conjugated
recombinant human IL-10 (Fluorokine; R&D Systems), compared with
binding of a negative control which consisted of IL-10 that was first
neutralized by preincubation with anti-human IL-10 (Fig. 7)
. Background
autofluorescence (Fig. 7
, shaded histogram) in normal
F4/80+ peritoneal macrophages stained with
antibody-neutralized IL-10 showed two populations, that were also seen
in unstained macrophages (data not shown). Normal peritoneal
macrophages (solid-line histogram) displayed about a 10-fold increase
in fluorescence intensity, demonstrating the presence of IL-10-binding
receptors on their plasma membranes. Peritoneal macrophages from
infected mice (dashed-line histogram) displayed an identical level of
IL-10 binding to that of normal macrophages, indicating that the
expression of IL-10 receptors was not changed in peritoneal macrophages
from infected mice. Similar results were observed with alveolar
macrophages from normal and infected mice (Fig. 7)
. The level of
background autofluorescence was significantly higher in alveolar
macrophages compared with peritoneal macrophages; however, this
difference was not caused by nonspecific binding of neutralized IL-10,
because the fluorescence pattern was identical to that of unstained
alveolar macrophages (data not shown). Alveolar macrophages displayed
binding of FITC-labeled IL-10 above background fluorescence, and
infection did not appear to change the level of IL-10 receptor
expression because both normal (solid-line histogram) and infected
(dashed-line histogram) alveolar macrophages had similar patterns of
fluorescence. Thus, resistance of infected macrophages to IL-10 is not
associated with a change in either the mRNA expression or surface
receptor expression of either the or ß chains of the IL-10
receptor.
 View larger version (29K): [in a new window] |
Figure 7. Expression of IL-10 receptor expression. Total RNA was isolated from
peritoneal macrophages from normal and 10-week-infected mice and was
analyzed by RT-PCR for expression of the and ß chains of the
IL-10 receptor. Expression of ß-actin was analyzed as a positive
control. Data are negative images of ethidium bromide-stained PCR
products. Binding of FITC-labeled human rIL-10 to normal (solid-line)
and infected (dashed-line) peritoneal and alveolar macrophages was
analyzed by flow cytometry and compared with binding of
anti-IL-10-neutralized IL-10 to normal macrophages as a negative
control (shaded histogram). The analyzed cells were first gated on
F4/80+ cells to identify macrophages in the alveolar- and
peritoneal-cell populations. Data are representative of two separate
experiments.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, suggesting that
endogenous production of IL-10 may be inefficient in vivo in
down-regulating proinflammatory cytokine expression
[14
]. A recent study by MacDonald et al.
[35
] showed that synovial dendritic cells from RA
patients were also partially resistant to the inhibitory effects of
IL-10, compared with blood dendritic cells. Individuals with SLE also
display elevated expression of IL-10, IFN-, and TNF- compared
with normal controls, suggesting an inability of IL-10 to effectively
inhibit cytokine synthesis in this autoimmune disease
[36
]. Moreover, studies by Mongan et al. showed that
recombinant IL-10 failed to inhibit production of IL-6 in vitro in
monocytes from SLE patients compared with those in normal controls
[15
]. Thus, in at least two different chronic autoimmune
diseases, endogenous production of IL-10 and exogenous treatment with
IL-10 might be insufficient to ameliorate inflammation and tissue
injury. Moreover, the inability of IL-10 to work appears to be related
to expression and/or function of the IL-10 receptor.
Septic shock is thought to be mediated by massive production of
proinflammatory cytokines by monocytes and macrophages, in particular
TNF-. A number of studies have shown that IL-10 is quite effective
in reducing proinflammatory secretion and mortality in endotoxin shock
models in rodents [5
, 6
]. However, only
marginal protection has been achieved with IL-10 in polymicrobial shock
models [38
, 39
]. In patients with septic
shock, coexpression of IL-10 with TNF- and IL-6 has been reported,
suggesting an inability of massive IL-10 synthesis in septic shock to
control production of proinflammatory mediators [40
,
41
]. The reason for the difference in effectiveness of
IL-10 for endotoxin versus polymicrobial shock is unknown. However, it
has been shown that the time of exposure to IL-10 relative to endotoxin
can affect the ability of IL-10 to down-regulate proinflammatory
cytokine synthesis in that delayed exposure to IL-10 after endotoxin
stimulation reduces the immunosuppressive ability of IL-10
[42
]. In other studies by Hart et al.
[43
], prior exposure of macrophages to GM-CSF inhibited
subsequent responsiveness of macrophages to IL-10. Thus, prior exposure
of macrophages to cytokines generated during polymicrobial sepsis can
contribute to the partial resistance of macrophages to IL-10. The
failure of IL-10 to reduce proinflammatory cytokine synthesis during
infection may not be unique for septic shock. Coexpression of high
amounts of IL-10 with IFN- or TNF- and the inability of IL-10 to
inhibit production of these cytokines have also been observed during
chronic infections with Plasmodium, Borrelia, Brucella,
Schistosoma, and Mycobacterium species
[18
, 19
, 44
45
46
47
].
Results presented here are the first to our knowledge
demonstrating that persistent coexpression of IL-10 with IFN- and
TNF- during a chronic viral infection is caused in part by
resistance of macrophages to the immunosuppressive effects of IL-10.
Other retrovirus infections have also been shown to display persistent
expression of IL-10, IFN-, and TNF-, including HIV, simian
immunodeficiency virus, and feline immunodeficiency virus
[20
, 21
, 48
49
50
51
52
]. Whether
cellular resistance to IL-10 is also a factor in cytokine expression
patterns in these retroviral infections is unknown, but it could be
affected by either direct virus infection of macrophages or other
cytokine-producing cells or, alternatively, by an effect of chronic
exposure of these cells to other cytokines or inflammatory mediators
during the course of infection. We were unable to demonstrate a role
for direct viral infection in our model. LPS-induced cytokine
production by bone marrow-derived macrophages infected with the LP-BM5
retrovirus was suppressed by IL-10 to the same extent as in uninfected
cells. Thus, we suggest that in this murine model previous exposure to
cytokines or inflammatory mediators rather than direct virus infection
may alter the responsiveness of macrophages to IL-10. Cytokines
expressed persistently after infection by LP-BM5 retrovirus are IFN-
and TNF-. Although prior in vitro exposure of peritoneal macrophages
to IFN- had no effect on the response to IL-10, TNF- preexposure
induced partial resistance to IL-10. However, TNF- was not usually
able to induce resistance to IL-10 to the same degree seen in
macrophages from infected mice. Therefore, although TNF- can
contribute to IL-10 resistance, other unidentified factors may
synergize with TNF- to further increase the resistance of
macrophages to IL-10. It is interesting that transgenic mice
overexpressing TNF- also express very high levels of IL-10
[53
]. In addition, a study by Bessis et al.
[54
] demonstrated that, in TNF--transgenic
mice engrafted with fibroblasts expressing IL-4, IL-10, or IL-13, only
IL-4 and IL-13 could effectively inhibit endogenous expression of
TNF- and IL-1, whereas IL-10 had a reduced capacity to inhibit these
cytokines. Thus, similar to our observations, persistent exposure to
TNF- in vivo also appears to partially reduce the ability of IL-10
to inhibit proinflammatory cytokine expression.
The molecular mechanism by which chronic exposure of macrophages to
cytokines or other inflammatory mediators during the course of LP-BM5
infection causes these cells to become resistant to the
immunosuppressive effects of IL-10 is unknown. Macrophages from
infected mice clearly express mRNA for both chains of the IL-10
receptor and display receptors on surface membranes, which are capable
of binding IL-10. Given that IL-10 receptor expression remains intact
after infection, it is probable that disruption of the IL-10 signal
transduction pathway may account for the defective IL-10
responsiveness. Engagement of the IL-10 receptor activates a Jak-Stat
signaling pathway involving Jak1 and Tyk2 kinases, which are associated
with the and ß chains of the receptor, respectively
[55
56
57
58
59
]. IL-10 receptor engagement also leads to the
binding of Stat3 to the receptor [59
]. It is important
that IL-10-mediated inhibition of TNF- production by macrophages is
also dependent on recruitment of Stat3 to the receptor complex
[60
]. A family of inhibitor proteins has been described,
suppressors of cytokine signaling (SOCS), which can inhibit the
responsiveness of several cytokine receptors to their ligands
[61
, 62
]. SOCS-3 has been implicated in
inhibition of IL-10 signaling [63
, 64
], and
recent data by Bode et al. [65
] demonstrate that
exposure of macrophages to TNF- induces the expression of SOCS-3,
thereby mediating inhibition of Stat3-dependent cytokine receptors.
Whether macrophages from LP-BM5-infected mice have elevated levels of
SOCS-3 or display other defects in signal transduction pathways for the
IL-10 receptor remains to be determined.
Received February 2, 2001; revised May 9, 2001; accepted May 11, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interleukin-10 are found in both the thymus and secondary lymphoid tissues of feline immunodeficiency virus-infected cats J. Infect. Dis. 181,564-575[Medline] transgenic mice Cytokine 10,124-131[Medline] induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages FEBS Lett 463,365-370[Medline]This article has been cited by other articles:
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 C. Herrero, X. Hu, W. P. Li, S. Samuels, M. N. Sharif, S. Kotenko, and L. B. Ivashkiv Reprogramming of IL-10 Activity and Signaling by IFN-{gamma} J. Immunol., November 15, 2003; 171(10): 5034 - 5041. [Abstract] [Full Text] [PDF]
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 J.-D. Ji, I. Tassiulas, K.-H. Park-Min, A. Aydin, I. Mecklenbrauker, A. Tarakhovsky, L. Pricop, J. E. Salmon, and L. B. Ivashkiv Inhibition of Interleukin 10 Signaling after Fc Receptor Ligation and during Rheumatoid Arthritis J. Exp. Med., June 2, 2003; 197(11): 1573 - 1583. [Abstract] [Full Text] [PDF]
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