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Immune Cell Interaction Unit, Mucosal Immunity Section, Laboratory for Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, RANTES, or eotaxin, inhibited IL-12 p70
production in response to stimulation with Staphylococcus
aureus, Cowan strain 1 (SAC), and IFN-
. The production of
TNF- and IL-10, however, was minimally affected by any of the
chemoattractants. The degree of inhibition of IL-12 p70 production by
MCP-1 to -4 was donor dependent and was affected by the autocrine
inhibitory effects of IL-10. In contrast, C5a profoundly suppressed
IL-12 production in an IL-10-independent fashion. Neither TGF-ß1 nor
PGE2 was important for the suppression of IL-12 by any of
the chemoattractants tested. The accumulation of mRNA for both IL-12
p35 and p40 genes was inhibited by chemokine pretreatment.
Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by
monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and
IFN- or by SAC and IFN-, suggesting that these factors may act at
the site of inflammation to suppress IL-12 and IFN- production
rather than in the lymph node to affect T cell priming. Despite the
inability of C5a to inhibit IL-12 production by DCs, the receptor for
C5a (CD88) was expressed by these cells, and recombinant C5a induced a
Ca2+ flux. Taken together, these results define a range of
chemoattractant molecules with the ability to suppress IL-12 production
by human monocytes and have broad implications for the regulation of
immune responses in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK cells and T cells
and is vital for the induction of Th1 T cell responses (reviewed in
Refs. 1 and 2). The genes for the p35 and p40
chains of IL-12 are located on separate chromosomes (3)
and are regulated independently. The IL-12 p35, but not the p40, chain
is constitutively produced at a low level by a variety of cell types,
and transcription of both genes as well as production of IL-12 p70
heterodimer are inducible (4, 5, 6). IL-12 is normally
produced by monocyte/macrophages and dendritic cells in response to
infection by bacteria or parasites, or following exposure to their
products. In this regard, LPS and fixed Staphylococcus
aureus, Cowan strain 1
(SAC),2 have been used
extensively for the study of IL-12 production, as they have been shown
to provide an important stimulatory signal for the transcription of
both the p35 and p40 genes and the production of the active IL-12 p70
heterodimer. In addition to microbial products, engagement of CD40 by
CD40 ligand (CD40L) expressed on activated T cells has been shown to be
important in the induction of IL-12 production during cognate APC-T
cell interactions (7, 8, 9, 10, 11). Finally, it has recently been
shown that low m.w. fragments of hyaluronan that are produced in areas
of acute inflammation can stimulate IL-12 production, thus providing a
mechanism by which macrophages may be induced to produce IL-12 in the
absence of either bacteria or T cells (12). IFN-
provides an important costimulus for the production of IL-12 (13, 14). Thus, while IFN- alone has little direct effect on IL-12
production, in combination with LPS, SAC, or CD40L it can dramatically
enhance p35 and p40 gene transcription as well as IL-12 p70
production. In studies of monocyte/macrophages stimulated in vitro with
LPS, this augmentation is enhanced by preincubation with IFN- up to
24 h before the LPS stimulus (6).
In contrast to these positive regulators of IL-12 production, it has
become clear that signals that inhibit IL-12 production from APCs may
be important for the regulation of cell-mediated immune responses.
Initially described were cytokine inhibitors of IL-12, such as IL-10,
TGF-ß1, IL-4, and IL-13, the latter two having more complex effects
than the former, in that preincubation with these cytokines can also
enhance IL-12 production (15, 16). Recently, the
engagement of noncytokine receptors by microbes (17, 18, 19),
IgG in the form of Ig-opsonized bacteria (20) or immune
complexes (21), complement components (19, 20, 22), adrenergic agonists (23), and
1,25-dihydroxyvitamin D3 (24) has
been shown to inhibit IL-12 p70 production by human monocytes; the
latter two factors were also shown to inhibit CD40-induced IL-12
production by human monocyte-derived dendritic cells. Interestingly,
all these inhibitors, despite acting through discrete surface
receptors, appear to inhibit IL-12 in a more or less selective fashion,
in that the production of other proinflammatory cytokines, such as
TNF-, remain largely unaffected.
We previously demonstrated that Abs to complement receptor 3 (CR3, MAC-1, CD11b/CD18) as well as CR3 ligands, such as Histoplama capsulatum and iC3b-coated RBCs, suppress the ability of human monocytes to produce IL-12 in response to a variety of stimuli (22). Because the binding of ligands to CR3 depends on its surface conformation, we initially sought to determine whether chemoattractants known to stimulate CR3-mediated adhesion would affect the ability of CR3 ligands to inhibit IL-12 production by human monocytes. This possibility was suggested by a recent report describing the ability of MCP-1 to enhance oral tolerance, possibly via its ability to inhibit IL-12 production (25). Somewhat surprisingly, by screening a panel of chemokines and natural chemoattractants for their ability to suppress IL-12 production, we found that a selective set of C-C chemokines, the macrophage chemoattractant proteins (MCP-1 to -4), as well as the natural chemoattractant C5a themselves, i.e., in the absence of exogenous CR3 ligands, were potent inhibitors of IL-12 production by human monocytes. This study thus defines a new range of chemokines with suppressive effects on IL-12 production. In addition, it identifies the natural chemoattractant C5a as a particularly potent molecule that suppresses IL-12 production at the level of gene transcription. The findings with C5a contribute to the growing body of information suggesting that an inverse relationship exists between complement activation and the generation of Th1 T cell responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following recombinant human chemokines were obtained from
PreproTech (Rocky Hill, NJ): MCP-1, MCP-2, MCP-3, MCP-4, RANTES,
eotaxin, monocyte inflammatory protein-1 (MIP-1), MIP-1ß, and
stromal-derived factor-1 (SDF-1) (lot-specific endotoxin concentrations
were 0.03, 0.02, 0.03, 0.01, 0.001, 0.01, 0.06, and 0.001 EU/µg,
respectively). Recombinant human C5a (lot-specific endotoxin
concentration, 0.005 EU/µg) and indomethacin were obtained from Sigma
(St. Louis, MO). SAC was supplied by Calbiochem (San Diego, CA).
Pertussis toxin was obtained from List Biological Laboratories
(Campbell, CA). Recombinant human IL-4 and GM-CSF were purchased from
Genzyme (Cambridge, MA). Neutralizing Ab to human TGF-ß1 (polyclonal
chicken Ig) and control Ab (normal chicken Ig), and neutralizing Ab to
human IL-10 (clone 23738.11) and isotype control (clone 20116.11) were
obtained from R&D Systems (Minneapolis, MN). Recombinant trimerized
human CD40L (CD154) was provided by Immunex (Seattle, WA).
Isolation and stimulation of human monocytes
Human monocytes were obtained from normal healthy donors (total
n = 30) by standard leukopheresis and purified by
counterflow centrifugation (elutriation), which yielded cells of
uniform forward/side scatter that were 95–99%
CD14+ by flow cytometry. Cells were cultured at a
density of 2 x 106 cells/ml in 1 ml of RPMI
1640 (Biofluids, Rockville, MD) supplemented with 10% FCS (Biofluids),
100 µg/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml
gentamicin, 5% NCTC-109 medium (Biofluids), 15 mM HEPES, and 200 mM
glutamine (cRPMI) at 37°C and 6% CO2. For
measurement of cytokine production, human monocytes were preincubated
with medium alone or varying concentrations of chemokines or C5a for
1 h at 37°C before stimulation with SAC (0.01%, w/v) and
IFN- (100 ng/ml); 24 h later culture supernatants were
collected and stored at -20°C until assayed for cytokines.
To determine the role of autocrine inhibitors of IL-12 in the monocyte
cultures, elutriated monocytes were preincubated for 1 h with
chemokines (100 nM), C5a (100 nM), or medium alone and one of the
following: neutralizing Ab to TGF-ß1 (10 µg/ml) or control Ab (10
µg/ml), neutralizing Ab to IL-10 (2.5 µg/ml) or isotype-matched
control Ab (2.5 µg/ml), or indomethacin (1 x
10-5 M). SAC (0.01%) and IFN- (100 ng/ml)
were then added; following 24 h of culture, supernatants were
harvested and stored at -20°C until assayed for IL-12 p70
production. For studies with pertussis toxin, elutriated monocytes were
cultured for 2 h with 250 ng/ml of purified pertussis toxin before
incubation with chemokines and stimulation with SAC/IFN- as
described above.
Generation and stimulation of human dendritic cells (DC)
DC were derived from elutriated monocytes as previously
described (26). Briefly, monocytes were cultured for 7
days in cRPMI supplemented every other day with IL-4 (100 ng/ml) and
GM-CSF (100 ng/ml). Nonadherent cells were harvested by gentle washing,
and the majority (70–90%) were demonstrated by flow cytometry to
express high levels of CD1a (clone HI149, PharMingen, San Diego, CA)
and low levels of CD83 (clone HB15e, PharMingen), consistent with prior
reports (26). Greater than 95% of the cells excluded
trypan blue and demonstrated characteristic dendrite formation on
examination with phase-contrast light microscopy. The DC were
resuspended at a density of 1 x 106
cells/ml in cRPMI and treated with various chemokines (100 nM), C5a
(100 nM), or medium alone for 1 h before stimulation with
either SAC (0.01%) and IFN- (100 ng/ml), or CD40L (3 µg/ml) and
IFN- (100 ng/ml). Supernatants were collected after 24 h of
culture and stored at -20°C until assayed for cytokines.
Flow cytometry for C5a and IL-10 receptor expression
C5a receptor expression on monocyte-derived DC (after 7 days in
culture with GM-CSF and IL-4) was performed as follows. Cells (1
x 106) in 100 µl of staining buffer (PBS
containing 0.2% BSA and 0.1% sodium azide) were sequentially
incubated with human IgG as a blocking agent for 15 min, followed by
either FITC- or PE-conjugated isotype control Abs (PharMingen) or mouse
anti-human CD88 FITC-conjugated mAb (clone W17/1, RDI, Flanders,
NJ) or mouse anti-human CD1a PE-conjugated mAb (PharMingen) for 45
min. IL-10R expression was performed on 1 x
106 fresh elutriated human monocytes cultured for
1, 2, 4, 6, or 12 h with 100 nM MCP-1- to 4 or C5a with and
without SAC/IFN- stimulation. At the given time, the cells were
harvested, washed twice with ice-cold staining buffer, and stained with
either PE-conjugated isotype matched control Ab (clone R35-95,
PharMingen) or PE-conjugated rat anti-human IL-10R mAb (clone 3F9,
PharMingen) as described above. All incubations were performed at 4°C
in staining buffer, and cells were washed twice with staining buffer
before and after incubations. The stained cells were analyzed on a
FACScan flow cytometer using CellQuest software (Becton Dickinson, San
Jose, CA).
Cytokine ELISAs
Cell culture supernatants were assayed for cytokines by ELISA
using matched Ab pairs according to the manufacturer’s suggestions.
ELISA reagents for human IL-12 p70 were purchased from R&D Systems and
for IL-10 and TNF- from BioSource International (Camarillo, CA); the
respective capture and detection Abs were as follows: IL-12 p70, clone
24945.11; and polyclonal goat IgG, IL-10 clones AHC8102 and AHC7109,
and TNF- clones AHC3712 and AHC3419. Briefly, the capture Ab was
bound to 96-well ELISA plates (Immulon 4, Dynatech, Chantilly, VA) in
the appropriate buffer overnight at 4°C; capture Ab for IL-12 p70 was
used at a concentration of 4 µg/ml diluted in PBS; IL-10 and TNF-
capture Abs were used in a concentration of 2 µg/ml diluted in
bicarbonate buffer. The plates were then washed (three times) with PBS
with 0.05% Tween-20, and blocked for 2 h at room temperature; 1%
BSA, 5% sucrose, and 0.05% sodium azide in PBS (pH 7.3) was used to
block the IL-12 p70 plates, while 3% BSA in PBS was used for the
remaining cytokines. Cytokine standards and supernatants were diluted
as necessary; 20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, and 0.1%
BSA was used as the diluent for the IL-12 p70 assay, and the IL-10 and
TNF-, ELISAs used 3% BSA in PBS as a diluent. The ELISA plates were
then incubated overnight at 4°C. The plates were washed, and bound
cytokine was revealed with a biotin-labeled detecting Ab (2 h at room
temperature). IL-12 p70 detection Ab was used at 300 ng/ml, while IL-10
and TNF- detection Abs were used at 500 ng/ml. This was followed by
HRP-conjugated streptavidin (Zymed, South San Francisco, CA; 1/1000 for
30 min at room temperature) and the substrate
o-phenylenediamine dihydrochloride (Sigma) at 0.5 mg/ml in
phosphate-citrate buffer (pH 5.0) and 0.03%
H2O2. The OD of the
individual wells was determined at 450 nm using an automated ELISA
reader (Dynatech). The lower limit of sensitivity of the assays was 32
pg/ml for IL-12 p70 and IL-10, and 64 pg/ml for TNF-. Assays for
TGF-ß1 were performed using ELISA kits from Genzyme according to the
manufacturer’s instructions. TGF-ß1 levels were measured following
acidification, and therefore reflect both the active and latent forms
of TGF-ß1.
RT-PCR
Elutriated monocytes (2.0 x 107) were stimulated for 16 h as described above in the presence or the absence of 100 nM MCP-1, MCP-2, MCP-3, MCP-4, or C5a. Total RNA was obtained using STAT-60 (Tel-Test, Friendswood, TX) according to the manufacturer’s instructions. RNA concentrations were determined by measuring the OD at 260 nm. mRNA for each experimental condition was reverse transcribed with oligo(dT) priming to first-strand cDNA using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD). Briefly, 1 µg of total RNA suspended in RNase-free water was added to 16 µl of an RT reaction mixture consisting of 20 mM Tris-HCl, 2.5 mM MgCl2, 50 mM KCl, 10 mM DTT, and 1 mM dNTPs. After an initial incubation of 5 min at 42°C, 200 U of Superscript II RT was added, the reaction was continued for 50 min at 42°C and was then terminated at 70°C for 10 min. The samples were chilled on ice for 15 min. RNase H (2 U) was added, and the reaction was incubated for 20 min at 37°C. The first-strand cDNA was then stored at -80°C before PCR amplification.
PCR amplification was performed using 2 µl of cDNA template and 50 µl of a reaction mixture consisting of 20 mM Tris-HCl, 50 mM KCl, 0.2 mM dNTPs, 1.25 mM MgCl2, and 2.5 U Taq DNA polymerase (Life Technologies). The following primer pairs were used at a concentration of 1 µM: IL-12 p40: sense, 5'-AGAGGCTCTTCTGACCCCCAG-3'; antisense, 5'-CTCTTGCTCTTGCCCTGGACCTG-3'; IL-12 p35: sense, 5'-TCAGCAACATGCTCCAGAAGGC-3'; antisense, 5'-TGCATTCATGGTCTTGAACTCCACC-3'; and GAPDH: sense, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'; antisense, 5'-CATGTGGGCCATGAGGTCCACCAC-3'. PCR amplification was performed under the following conditions: initial denaturation at 94°C for 2 min, followed by cycles of 94°C for 45 s, 60°C for 60 s, and 70°C for 90 s and a final elongation step of 70°C for 5 min. The number of amplification cycles was determined by prior experiments to ensure linear phase amplification of cDNA template (data not shown). GAPDH was amplified for 25 cycles, IL-12 p40 for 30 cycles, and IL-12 p35 for 32 cycles. Amplified PCR products were resolved by gel electrophoresis on a 1.5% agarose gel and stained with ethidium bromide.
Statistics
Statistical analysis was performed by paired t testing using SigmaStat software (Jandel, San Raphael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially determined whether the exposure of elutriated human
monocytes to chemoattractants in vitro could inhibit the production of
IL-12 p70 production in response to a known IL-12 stimulus. Thus, we
preincubated monocytes for 60 min with a panel of C-C (MIP-1,
RANTES, MCP-1 to -4, and eotaxin) and C-X-C (SDF-1) chemokines and
natural chemoattractants (C5a). Following stimulation with SAC (0.01%,
w/v) and IFN- (100 ng/ml) for 24 h, IL-12 p70 as well as other
cytokines were measured in the culture supernatants by ELISA. As shown
Table I
, we found that MCP-1, MCP-3,
MCP-4, and C5a significantly suppressed the production of IL-12 p70.
MCP-2 also suppressed IL-12 p70 production, although this effect was
more variable. Thus, despite significant suppression of IL-12 p70
production from cells from individual donors (Fig. 1), when multiple donors were included in
the analysis (Table I) this suppression failed to reach statistical
significance. Interestingly, the degree of IL-12 suppression by a given
MCP and by the MCPs as a group was donor dependent, which may reflect
interdonor variations in receptor-ligand affinity or postreceptor
signaling pathways (Fig. 1). The MCP proteins were less suppressive
overall compared with the consistent and virtually complete suppression
of IL-12 by C5a (Fig. 1). The other chemokines tested had minimal to no
effect on IL-12 p70 production. In addition, as might be expected, the
higher the baseline IL-12 production (e.g., donor 2, Fig. 1), the less
effect overall of the MCP proteins; however, even in this case C5a
suppressed production by >80%.
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, the production of
IL-10, TGF-ß1, and TNF- was not significantly affected by any of
the inhibitors. In addition, the chemokines and chemoattractants
themselves did not directly stimulate the production of IL-10 or any
other of the cytokines measured (data not shown). The fact that IL-10,
TGF-ß1, and TNF- production was not affected suggested that the
suppression of IL-12 seen was not due to any generalized toxic effects
of the chemoattractants. Indeed, we found no significant differences
between any of the culture conditions with respect to the total number
of monocytes/macrophages present after 24 h in culture or the
viability of the cultured cells, as determined both by trypan blue
exclusion of cells directly in the culture dish and by flow cytometric
analysis of propidium iodide-stained cells removed from the culture
plates.
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, the maximal suppressive effects of C5a
occurred at a dose of less than 10 nM, and those of MCP-1 at
concentrations <20 nM. These levels, as well as those of the other MCP
proteins used, are consistent with prior reports of the doses required
to induce chemotaxis and other biological functions (27, 28).
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To determine whether these mediators suppress IL-12 production at
the level of gene transcription, we performed semiquantitative RT-PCR
for IL-12 p35 and p40 mRNA isolated from monocytes stimulated with SAC
and IFN- with and without prior treatment with MCP-1 to -4 or C5a.
As shown in Fig. 4, pretreatment with
MCP-1 or C5a resulted in marked suppression of mRNA accumulation for
both the p35 and p40 genes. MCP-2 to -4 had a more modest effect on
mRNA accumulation, as is reflected by the protein data. This suggests
that signaling by these chemokines and C5a acts at the level of gene
transcription. Interestingly, C5a suppressed p35 transcription more
than p40 transcription, similar to the effects of other IL-12
inhibitors, such as 1,25-dihydroxyvitamin D3
(24).
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As the production of IL-10 and TGF-ß1 in response to SAC and
IFN- stimulation was largely unaffected by pretreatment with
chemoattractants, we sought to determine whether chemoattractant
pretreatment might be affecting monocyte sensitivity to autocrine
inhibition of IL-12 by those factors. Accordingly, we added
neutralizing Abs to IL-10 or TGF-ß1, or indomethacin, to prevent
PGE2 induction, before exposure to
chemoattractants and stimulation with SAC and IFN-. As shown in Fig. 5A, anti-IL-10 partially
reversed the suppression of IL-12 seen with MCP-1 to -4. Anti-IL-10 had
minimal, but reproducible, effects on reversing the suppression seen
with C5a. The reversal of suppression by anti-IL-10 with the MCP
proteins was not statistically significant when multiple donors were
combined for analysis (Fig. 5A) as there was significant
interdonor variation in this effect. However, for cells from many
individual donors this effect was remarkable. In contrast to
anti-IL-10, neither anti-TGF-ß1 nor indomethacin
significantly reversed the inhibition induced by any of the
chemoattractant proteins. We then pretreated monocytes with a
combination of anti-IL-10, anti-TGF-ß1, and indomethacin to
determine whether there might be additive inhibitory effects of IL-10,
TGF-ß1, and PGE2 induced by the
chemoattractants. We found little enhancement of the reversal of
inhibition over that seen with anti-IL-10 alone, demonstrating that
IL-10 was predominantly responsible for this effect (data not shown).
In addition, we performed initial studies to address the possibility
that the sensitivity of donor cells to IL-10 might be affected by
pretreatment with chemoattractants, as the levels of IL-10 produced in
response to stimulation were similar with C5a and MCP-1 to -4
pretreatment, yet the degree of reversal with anti-IL-10 was
remarkable only with select MCP proteins. We measured expression of the
IL-10R on chemoattractant or medium-treated monocytes by flow
cytometry. Pretreatment with MCP-1 to -4 or C5a did not affect cell
surface receptor expression by monocytes directly or following
stimulation with SAC and IFN- (data not shown), suggesting that
differences in sensitivity to IL-10 may be due to alterations in
postreceptor signaling.
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The chemoattractant proteins are known to transmit signals via
seven-transmembrane domain receptors that couple to G proteins of the
Gi and possibly Gq classes
(29). To determine the extent to which the inhibitory
signals for IL-12 production are dependent on signaling via
Gi, we pretreated monocytes with the
Gi inhibitor pertussis toxin before exposure to
the chemoattractants and simulation with SAC and IFN-. As shown in
Fig. 6, we found that pertussis toxin
partially prevented the inhibition of IL-12 production
chemoattractants. Despite using high doses of pertussis toxin (up to
500 ng/ml) and long preincubation times (up to 4 h), complete
reversal of the inhibitory effects of any of the chemoattractants was
never observed (data not shown).
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We next sought to determine whether the inhibition of IL-12 p70
production with MCP-1 to -4 and C5a seen with monocytes also applied to
DC. For these experiments we cultured elutriated monocytes in GM-CSF
and IL-4 for 7 days to obtain DC (26). In addition, we
cultured these cells for an additional 3 days with either a
monocyte-derived supernatant (26) or soluble CD40L trimer
(30), which resulted in DC with a more mature or
differentiated phenotype. We exposed both these populations of DC to a
range of chemokines, including MCP-1 to -4 and C5a before stimulation
with either CD40L and IFN-, or SAC and IFN-. As shown in Fig. 7, we found that none of the
chemoattractants significantly suppressed the production of IL-12 p70
by the less mature cells in response to either stimulus. As shown in
Fig. 8, this lack of suppression did not
appear to be due to a lack of functional expression of chemokine
receptors by these cells. C5a receptor (CD88) expression was clearly
demonstrable by flow cytometry. In addition, MCP-1 to -4 as well as C5a
readily induced an intracellular Ca+2 flux in the
less mature DCs (data not shown), consistent with prior reports
(31, 32, 33). Also consistent with prior studies, we found
that maturation of DCs by exposure to either CD40L or
monocyte-conditioned medium severely reduced their capacity to produce
IL-12 p70 (34). Similar to the findings with the less
mature DCs, however, incubation of the differentiated DCs with MCP-1 to
-4 or C5a before stimulation had no effect on the minimal production of
IL-12 p70 by these cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
TGF-ß1, or IL-10, in response to a variety of stimuli
(22). These findings suggest that the binding of
iC3b-opsonized micro-organisms to CR3 on human monocytes or DC may
provide a mechanism by which certain pathogens, such as
Leishmania species (17, 18, 35), inhibit a host
immune response that is important for their elimination. Because the
binding of ligands to CR3 depends on its surface conformation, we
initially sought to determine whether chemoattractants known to induce
CR3-mediated adhesion would have effects on the inhibitory signals
provided through CR3. Surprisingly, we found that a select group of
chemoattractants themselves, i.e., in the absence of additional CR3
ligands, suppressed IL-12 production by human monocytes.
We initially screened a panel of chemokines from the C-C (MCP-1 to -4,
MIP-1, RANTES, and eotaxin) and C-X-C (SDF-1) chemokine families as
well as the chemoattractant C5a for their abilities to affect the
production of IL-12 as well as other cytokines by human monocytes in
response to a well-known monocyte stimulus, SAC and IFN-. We found
that many chemokines had insignificant effects on IL-12 production from
monocytes isolated from multiple donors. This group included chemokines
known to bind to the chemokine receptors CCR1 (MIP-1), CCR3
(eotaxin, RANTES), CCR4 (MIP-1, RANTES), and CCR5 (MIP-1, RANTES)
as well as to CXCR4 (SDF-1). Of these receptors, all are known to be
expressed on human monocytes with the exception of CCR3, which cannot
be detected in this cell type at the protein or mRNA level (36, 37).
In contrast, the MCP family of C-C chemokines (MCP-1 to -4), all of
which bind to the CCR2-receptors CCR2a and CCR2b, inhibited IL-12 p70
production. This inhibition was specific, in that production of IL-10
and TNF- was largely unaffected, and the degree of inhibition was
donor dependent. In addition, as shown in Fig. 1
, the degree of
inhibition was MCP dependent, in that different members of the MCP
family could have quite different effects on cells from a given donor.
Finally, we demonstrated that the suppression of IL-12 by the MCP
proteins is due in part to the effects of paracrine/autocrine
production of IL-10 (and not TGF-ß1 or PGE2) by
the stimulated mononuclear cells (Fig. 5). Because the levels of IL-10
in the culture supernatants were only minimally affected by the MCPs
(Fig. 2), and the chemokines failed to either induce the production of
IL-10 in the absence of other stimuli or directly alter IL-10R
expression, it is possible that the reversal of suppression by
anti-IL-10 reflects differences in individual donor sensitivity to
IL-10.
The suppression of IL-12 by MCP family members is consistent with prior
studies performed in mice. Thus, it has been shown that treatment of
mice with MCP-1 results in the enhancement of systemic tolerance
induced by oral protein feeding and that this correlated to a reduction
in the production of IL-12 within mucosal tissues. In addition, it was
shown that treatment with anti-MCP-1 prevented oral tolerance
induction, which correlated with the enhanced production of IL-12
(25). This ability of MCP-1 to regulate IL-12 at mucosal
surfaces is of particular interest for several reasons. Firstly, the
presence of low levels of IL-12 appears to correlate with
noninflammatory and tolerizing conditions within the intestine, while
the presence of high levels of IL-12 has the opposite effect (25, 38, 39). Secondly, intestinal epithelial cells produce a number
of chemokines, including MCP-1 (40, 41), implying that
these cells are a potential direct source of MCPs in the intestine.
Finally, it has recently been shown that mice with targeted deletion of
the G protein Gi2 develop a spontaneous
Th1-mediated inflammatory bowel disease (42). In this
model, inflamed colonic tissue expresses high levels of IL-12 p40 mRNA,
and cultured intestinal tissue and purified intestinal T cells
spontaneously produce high levels of IFN- and IL-1ß. Since MCP-1
signals via a G-linked receptor and results in the suppression of IL-12
production, it is logical to hypothesize that
Gi2 knockout mice develop Th1-mediated
colitis, because they have poorly regulated production of IL-12. Taken
together, these prior studies suggest that the production of MCP
proteins within the intestine, possibly by epithelial cells,
contributes to a generally suppressive environment at this site. That
this suppressive environment is mediated by IL-10 is supported by the
findings of the current study and is consistent with the results of
prior studies demonstrating the presence of high levels of IL-10 within
the intestinal tract (43, 44, 45). In addition, it is
consistent with the fact that IL-10 mice develop Th1-mediated
enterocolitis (46, 47).
Studies in mice also imply a role for MCP-1 in the regulation of IL-12 at nonmucosal sites. Thus, in a mouse model of endotoxic shock, it was shown that the administration of exogenous MCP-1 significantly protected mice from endotoxin-induced lethality (similar to the administration of anti-IL-12 in other studies (48, 49)), while the administration of anti-MCP-1 enhanced lethality (50). Importantly, enhanced lethality in this system correlated with high serum levels of IL-12 and low levels of IL-10, while its inhibition by MCP-1 correlated with high IL-10 and low IL-12 levels. Finally, it has been suggested that MCP-1 enhances the development of Th2 granuloma in the lungs of mice injected i.v. with eggs from Shistosoma mansoni, and that such enhancement may be partially related to its ability to suppress the production of IL-12 (51).
Our findings have significantly extended these prior murine studies. We now demonstrate the effects of MCP proteins on IL-12 production by human cells, show that the suppressive effect extends to four members of this family, MCP-1 to -4, and demonstrate that the degree of suppression is both donor and MCP dependent. In addition, we demonstrate that the mechanism of IL-12 suppression by such proteins is largely dependent on IL-10 and may be mediated not by absolute levels of IL-10, but by alterations in the sensitivity of cells to this cytokine.
In contrast to the variable effects of the MCPs, striking and
consistent suppression of IL-12 p70 production was found with C5a, a
classical chemoattractant that binds to the C5a receptor
(29). Greater than 10-fold reduction in the production of
IL-12 was consistently seen in all donors with C5a. The suppression of
IL-12 by C5a was specific for IL-12 (Fig. 1), occurred maximally at
concentrations as low as 10 nM (Fig. 3) and acted at the level of gene
transcription for both p35 and p40 chains (Fig. 4), and as with the
MCPs, autocrine production of TGF-ß1 and PGE2
appeared to have a minimal role (Fig. 5, B and
C). In contrast to the MCPs, however, the suppression of
IL-12 was only modestly reversed by anti-IL-10 (Fig. 5A). Thus, C5a appears to suppress IL-12 production directly
or via the induction of a novel, as yet unidentified autocrine
inhibitory factor(s). These findings with C5a are consistent with a
recent paper by Wittman et al. (52), who showed that C5a
suppressed IL-12 p40 and p70 production by IFN--primed human
monocytes stimulated with LPS. In contrast to this prior report,
however, we found that suppression of IL-12 production following
stimulation with SAC and IFN- is partially, although minimally,
dependent on IL-10. In addition, we explored the ability of MCP-1 to -4
and C5a to inhibit the production of IL-12 p70 by DCs and determined
that these chemoattractants had no effect on IL-12 p70 production by
these cells. The lack of effect was not due to the inability of these
agents to induce an intracellular calcium flux in DCs.
The inhibition of IL-12 by C5a initially appeared to contradict the facts that these agents are known to potently induce chemotaxis, phagocytosis, and respiratory burst capacities of human monocytes and neutrophils (29), and that blocking C5a activity with a neutralizing antiserum reduced mortality in a rat model of septic shock (53). These effects of C5a, however, are not necessarily inconsistent with the findings reported here. In fact, chemoattractants such as C5a as well as the MCP proteins may act to attract monocyte/macrophages into sites of acute inflammation and at the same time act to prevent potentially harmful effects of high levels of IL-12, such as acute toxicity, and the triggering of potentially deleterious autoreactive Th1 immune responses (1). In this fashion, these chemoattractants could act as ideal signals for macrophage scavenging functions.
The ability of MCP-1 to -4 and C5a to suppress IL-12 p70 production
from monocytes, but not DC, is also consistent with a role for these
factors in local environments, rather than in the priming of T cells in
lymph nodes, a function normally ascribed to DC. In this sense, these
factors may affect the production of IL-12 p70 from
monocyte/macrophages and resulting IFN- production from NK cells as
well as affect the survival of effector Th1 cells in inflamed tissues.
Interestingly, the inability of MCP-1 to -4 and C5a to suppress IL-12
production from DC is unique, in that other IL-12 inhibitory signals,
such as vitamin D3,
ß2-adrenergic agonists, and cholera toxin,
affect both cell types equivalently (23, 24, 54). To begin
to address the reasons why C5a does not effect DC production of IL-12,
we analyzed the expression of the C5a receptor (CD88) on DC by flow
cytometry. The lack of suppression clearly cannot be attributed to lack
of receptor expression, as DC express the C5a receptor (Fig. 8).
Interestingly, as determined by flow cytometry, the expression of CD88
by the less mature population of CD1a+ DCs was
bimodal, suggesting that there is heterogeneity of receptor expression
by these cells.
Finally, the signaling pathway by which chemoattractants suppress IL-12
was explored. Signaling by chemoattractants is known to occur via
seven-transmembrane domain receptors via the release of coupled G
proteins from their heterotrimeric complexes. The fact that pertussis
toxin reversed the inhibition of the chemoattractants (Fig. 6)
demonstrates that the Gi is involved in the
suppressive signals. Because activation of Gi is
known to inhibit adenylate cyclase activity in many cell systems
(reviewed in Ref. 55), this finding suggests that
inhibition of IL-12 by chemoattractants is not dependent on the
induction of high levels of intracellular cAMP. This finding is in
contradistinction to the suppression of IL-12 by other factors that
also signal through seven-transmembrane receptors such as
PGE2 (56) or
ß2-adrenergic agonists (23). The
inhibitory effects of these factors appear to be directly related to
their ability to induce high levels of cAMP. Our findings are
consistent, however, with what is known regarding the families of G
proteins that couple to chemoattractant as opposed to other
seven-transmembrane domain receptors, since Gs
family members, which activate adenylate cyclase, do not appear to
couple to chemoattractant receptors. What this implies is that the many
factors that selectively suppress IL-12 production (15, 16, 19, 20, 21, 22, 23, 24, 56, 57, 58, 59), which act via disparate surface receptors and
can induce discrete proximal intracellular signals, have a common
downstream signaling pathway, the end result of which is the inhibition
of IL-12 gene transcription.
In summary, our findings indicate that a select group of chemoattractants may be active participants in the development of T cell-mediated immune responses by acting to alter the balance of cytokines in the environment of differentiating T cells. They suggest that these chemoattractants may act as the ideal signals for scavenging functions of macrophages by functioning to attract and active these cells at the same time as preventing potentially harmful Th1 responses and toxic effects of IL-12. In conjunction with the previous finding of signaling via receptors for complement opsonins C3b or iC3b (19, 20, 22), the findings presented here with C5a contribute to the hypothesis that a reciprocal relationship exists between complement activation and the development of Th1 T cell responses. Finally, these studies have implications for understanding immunity to organisms that bind to chemokine receptors for their entry (60), produce chemokine analogues in the process of immune evasion (61), or generate active complement components, either directly or following opsonization with specific Ig.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: SAC, Staphylococcus aureus Cowan strain 1; CD40L, CD40 ligand; MCP, monocyte chemoattractant protein; MIP, monocyte inflammatory protein; SDF, stromal-derived factor; DC, dendritic cell.
Received for publication July 30, 1999. Accepted for publication January 11, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
of lipopolysaccharide-inducible p35 and p40 genes. Blood 86:646. in monocytic cells. J. Exp. Med. 183:147., and IL-1ß production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 83:1847.-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041. production. J. Exp. Med. 181:537.
B downregulation in transcriptional repression of the p40 gene. J. Clin. Invest. 101:252.[Medline]
and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells. J. Immunol. 162:3231.)i2-deficient mice with colitis exhibit a local increase in memory CD4+ T cells and proinflammatory Th1-type cytokines. J. Immunol. 158:1068.[Abstract]
production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
interferon. Infect. Immun. 62:4244.-primed and lipopolysaccharide-challenged human monocytes. J. Immunol. 162:6763. receptor type I. J. Exp. Med. 188:217.This article has been cited by other articles:
|
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 |
|
  M. Del Corno, A. Michienzi, A. Masotti, L. Da Sacco, G. F. Bottazzo, F. Belardelli, and S. Gessani CC chemokine ligand 2 down-modulation by selected Toll-like receptor agonist combinations contributes to T helper 1 polarization in human dendritic cells Blood, July 23, 2009; 114(4): 796 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. N. Lalli, M. G. Strainic, F. Lin, M. E. Medof, and P. S. Heeger Decay Accelerating Factor Can Control T Cell Differentiation into IFN-{gamma}-Producing Effector Cells via Regulating Local C5a-Induced IL-12 Production J. Immunol., November 1, 2007; 179(9): 5793 - 5802. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Moulton, M. A. Mashruwala, A. K. Smith, D. R. Lindsey, R. A. Wetsel, D. L. Haviland, R. L. Hunter, and C. Jagannath Complement C5a anaphylatoxin is an innate determinant of dendritic cell-induced Th1 immunity to Mycobacterium bovis BCG infection in mice J. Leukoc. Biol., October 1, 2007; 82(4): 956 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Byrnes, D.-Y. Li, K. Park, D. Thompson, C. Mocilnikar, P. Mohan, J. P. Molleston, M. Narkewicz, H. Zhou, S. F. Wolf, et al. Modulation of the IL-12/IFN-{gamma} axis by IFN-{alpha} therapy for hepatitis C J. Leukoc. Biol., March 1, 2007; 81(3): 825 - 834. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Melendi, S. J. Hoffman, R. A. Karron, P. M. Irusta, F. R. Laham, A. Humbles, B. Schofield, C.-H. Pan, R. Rabold, B. Thumar, et al. C5 Modulates Airway Hyperreactivity and Pulmonary Eosinophilia during Enhanced Respiratory Syncytial Virus Disease by Decreasing C3a Receptor Expression J. Virol., January 15, 2007; 81(2): 991 - 999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. O. Flores-Villanueva, J. A. Ruiz-Morales, C.-H. Song, L. M. Flores, E.-K. Jo, M. Montano, P. F. Barnes, M. Selman, and J. Granados A functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis J. Exp. Med., December 19, 2005; 202(12): 1649 - 1658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Wenderfer, B. Ke, T. J. Hollmann, R. A. Wetsel, H. Y. Lan, and M. C. Braun C5a Receptor Deficiency Attenuates T Cell Function and Renal Disease in MRLlpr Mice J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3572 - 3582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Appel, V. Mirakaj, A. Bringmann, M. M. Weck, F. Grunebach, and P. Brossart PPAR-{gamma} agonists inhibit toll-like receptor-mediated activation of dendritic cells via the MAP kinase and NF-{kappa}B pathways Blood, December 1, 2005; 106(12): 3888 - 3894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. W. DePaolo, R. Lathan, B. J. Rollins, and W. J. Karpus The Chemokine CCL2 Is Required for Control of Murine Gastric Salmonella enterica Infection Infect. Immun., October 1, 2005; 73(10): 6514 - 6522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. N. Waggoner, M. W. Cruise, R. Kassel, and Y. S. Hahn gC1q Receptor Ligation Selectively Down-Regulates Human IL-12 Production through Activation of the Phosphoinositide 3-Kinase Pathway J. Immunol., October 1, 2005; 175(7): 4706 - 4714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. la Sala, M. Gadina, and B. L. Kelsall Gi-Protein-Dependent Inhibition of IL-12 Production Is Mediated by Activation of the Phosphatidylinositol 3-Kinase-Protein 3 Kinase B/Akt Pathway and JNK J. Immunol., September 1, 2005; 175(5): 2994 - 2999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Elhofy, J. Wang, M. Tani, B. T. Fife, K. J. Kennedy, J. Bennett, D. Huang, R. M. Ransohoff, and W. J. Karpus Transgenic expression of CCL2 in the central nervous system prevents experimental autoimmune encephalomyelitis J. Leukoc. Biol., February 1, 2005; 77(2): 229 - 237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Antoniazi, H. P. Price, P. Kropf, M. A. Freudenberg, C. Galanos, D. F. Smith, and I. Muller Chemokine Gene Expression in Toll-Like Receptor-Competent and -Deficient Mice Infected with Leishmania major Infect. Immun., September 1, 2004; 72(9): 5168 - 5174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. H. J. Kim, I. D. Dimitriou, M. C. H. Holland, D. Mastellos, Y. M. Mueller, J. D. Altman, J. D. Lambris, and P. D. Katsikis Complement C5a Receptor Is Essential for the Optimal Generation of Antiviral CD8+ T Cell Responses J. Immunol., August 15, 2004; 173(4): 2524 - 2529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhou, H. Xiong, H. Li, S. E. Plevy, P. D. Walden, M. Sassaroli, G. D. Prestwich, and J. C. Unkeless Microtubule-Associated Serine/Threonine Kinase-205 kDa and Fc{gamma} Receptor Control IL-12 p40 Synthesis and NF-{kappa}B Activation J. Immunol., February 15, 2004; 172(4): 2559 - 2568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Klein, L. Izikson, T. Means, H. D. Gibson, E. Lin, R. A. Sobel, H. L. Weiner, and A. D. Luster IFN-Inducible Protein 10/CXC Chemokine Ligand 10-Independent Induction of Experimental Autoimmune Encephalomyelitis J. Immunol., January 1, 2004; 172(1): 550 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Taube, Y.-H. Rha, K. Takeda, J.-W. Park, A. Joetham, A. Balhorn, A. Dakhama, P. C. Giclas, V. M. Holers, and E. W. Gelfand Inhibition of Complement Activation Decreases Airway Inflammation and Hyperresponsiveness Am. J. Respir. Crit. Care Med., December 1, 2003; 168(11): 1333 - 1341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. deSchoolmeester, M. C. Little, B. J. Rollins, and K. J. Else Absence of CC Chemokine Ligand 2 Results in an Altered Th1/Th2 Cytokine Balance and Failure to Expel Trichuris muris Infection J. Immunol., May 1, 2003; 170(9): 4693 - 4700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Soruri, Z. Kiafard, C. Dettmer, J. Riggert, J. Kohl, and J. Zwirner IL-4 Down-Regulates Anaphylatoxin Receptors in Monocytes and Dendritic Cells and Impairs Anaphylatoxin-Induced Migration In Vivo J. Immunol., March 15, 2003; 170(6): 3306 - 3314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zaph and P. Scott Interleukin-12 Regulates Chemokine Gene Expression during the Early Immune Response to Leishmania major Infect. Immun., March 1, 2003; 71(3): 1587 - 1589. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Dalwadi, B. Wei, M. Schrage, T. T. Su, D. J. Rawlings, and J. Braun B Cell Developmental Requirement for the G{alpha}i2 Gene J. Immunol., February 15, 2003; 170(4): 1707 - 1715. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Drouin, D. B. Corry, T. J. Hollman, J. Kildsgaard, and R. A. Wetsel Absence of the Complement Anaphylatoxin C3a Receptor Suppresses Th2 Effector Functions in a Murine Model of Pulmonary Allergy J. Immunol., November 15, 2002; 169(10): 5926 - 5933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Omata, M. Yasutomi, A. Yamada, H. Iwasaki, M. Mayumi, and Y. Ohshima Monocyte Chemoattractant Protein-1 Selectively Inhibits the Acquisition of CD40 Ligand-Dependent IL-12-Producing Capacity of Monocyte-Derived Dendritic Cells and Modulates Th1 Immune Response J. Immunol., November 1, 2002; 169(9): 4861 - 4866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Locati, U. Deuschle, M. L. Massardi, F. O. Martinez, M. Sironi, S. Sozzani, T. Bartfai, and A. Mantovani Analysis of the Gene Expression Profile Activated by the CC Chemokine Ligand 5/RANTES and by Lipopolysaccharide in Human Monocytes J. Immunol., April 1, 2002; 168(7): 3557 - 3562. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Lipscomb and B. J. Masten Dendritic Cells: Immune Regulators in Health and Disease Physiol Rev, January 1, 2002; 82(1): 97 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. KRUG, T. TSCHERNIG, V. J. ERPENBECK, J. M. HOHLFELD, and J. KOHL Complement Factors C3a and C5a Are Increased in Bronchoalveolar Lavage Fluid after Segmental Allergen Provocation in Subjects with Asthma Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): 1841 - 1843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Huang, D. Liu, P. Majewski, L. C. Schulte, J. M. Korn, R. A. Young, E. S. Lander, and N. Hacohen The Plasticity of Dendritic Cell Responses to Pathogens and Their Components Science, October 26, 2001; 294(5543): 870 - 875. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Drouin, D. B. Corry, J. Kildsgaard, and R. A. Wetsel Cutting Edge: The Absence of C3 Demonstrates a Role for Complement in Th2 Effector Functions in a Murine Model of Pulmonary Allergy J. Immunol., October 15, 2001; 167(8): 4141 - 4145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Gerber and D. M. Mosser Reversing Lipopolysaccharide Toxicity by Ligating the Macrophage Fc{{gamma}} Receptors J. Immunol., June 1, 2001; 166(11): 6861 - 6868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Braun, J. M. Wang, E. Lahey, R. L. Rabin, and B. L. Kelsall Activation of the formyl peptide receptor by the HIV-derived peptide T-20 suppresses interleukin-12 p70 production by human monocytes Blood, June 1, 2001; 97(11): 3531 - 3536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.-F. Guo, A. B. Lentsch, R. L. Warner, M. Huber-Lang, J. V. Sarma, T. Hlaing, M. M. Shi, N. W. Lukacs, and P. A. Ward Regulatory Effects of Eotaxin on Acute Lung Inflammatory Injury J. Immunol., April 15, 2001; 166(8): 5208 - 5218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Corinti, C. Albanesi, A. la Sala, S. Pastore, and G. Girolomoni Regulatory Activity of Autocrine IL-10 on Dendritic Cell Functions J. Immunol., April 1, 2001; 166(7): 4312 - 4318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Matsunaga, T. W. Klein, C. Newton, H. Friedman, and Y. Yamamoto Legionella pneumophila Suppresses Interleukin-12 Production by Macrophages Infect. Immun., March 1, 2001; 69(3): 1929 - 1933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Nakachi, K. Matsunaga, T. W. Klein, H. Friedman, and Y. Yamamoto Differential Effects of Virulent versus Avirulent Legionella pneumophila on Chemokine Gene Expression in Murine Alveolar Macrophages Determined by cDNA Expression Array Technique Infect. Immun., October 1, 2000; 68(10): 6069 - 6072. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. He, S. Gurunathan, A. Iwasaki, B. Ash-Shaheed, and B. L. Kelsall Primary Role for GI Protein Signaling in the Regulation of Interleukin 12 Production and the Induction of T Helper Cell Type 1 Responses J. Exp. Med., May 1, 2000; 191(9): 1605 - 1610. [Abstract] [Full Text] [PDF]
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