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-Primed and Lipopolysaccharide-Challenged Human Monocytes1
*
Department of Dermatology and Allergology, Hannover Medical University, Hannover, Germany; and
Department of Immunology, University of Göttingen, Göttingen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or
GM-CSF is required for expression of IL-12p70 by cells in which IL-12
is inducible by bacterial products such as LPS. We here show for the
first time that the production of bioactive IL-12 by human monocytes
can be significantly suppressed by C5a if applied to IFN--primed
monocytes before LPS stimulation. There was a dose-dependent
suppression by IL-12 (p70) on the levels of intracellular cytokine
production and cytokine secretion. mRNA studies consistently showed a
reduction of IL-12p40 and IL-12p35 expression by stimulation in the
presence of C5a. The results of several different experimental
approaches suggest that IL-12 down-regulation was not due to endogenous
IL-10, IL-4, or PGE2 production induced by C5a. Moreover,
stimulation of IFN--primed monocytes with C5a did not lead to a
down-regulation of the CD14 Ag, which is an LPS receptor. These
findings show that the anaphylatoxin C5a has the capacity to directly
interact with the complex regulation of IL-12. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 is a 75-kDa heterodimeric cytokine that plays a crucial role in both the innate and the acquired immune response. The major cells producing IL-12 were found to be macrophages/monocytes, but other cell types have also been reported to produce IL-12. These include dendritic cells, neutrophils, keratinocytes, and murine mast cells (9).
The two disulfide-linked N-glycosylated polypeptide chains of approximately 40 kDa (p40) and 35 kDa (p35) are encoded by two separate genes (10) located on different chromosomes and are independently regulated (9, 11). Both subunits of IL-12 have to be produced within the same cell to obtain the biologically active dimer (10).
Although initially described as a cytokine activating cytotoxic
lymphocytes, the major biological significance of IL-12 secretion is
probably based on its effects on Th cells. IL-12 is known to drive Th1
reactions in physiological and pathological immune responses, mainly
mediated by its capacity to stimulate growth and IFN- production in
T cells and NK cells (12, 13, 14). Some authors even consider this cytokine
to be an obligatory factor for Th1 generation and proliferation (15, 16).
In addition to the promotion of Th1 cell development, IL-12 has a broad range of biologic activities, including the regulation and proliferation of T and NK cells, the differentiation of CD8+ T cells, and the induction of hemopoiesis (17). Its involvement in autoimmunity has been shown in different diseases. When applied in vivo, IL-12 was shown to enhance the resistance to bacterial and parasitic infections, to promote antitumor immunity, and to influence antiviral responses, including HIV, in vivo or in vitro (18, 19, 20). Modulation of IL-12-dependent signaling may provide a therapeutic option for altering the Th1-Th2 balance in allergic and autoimmune diseases as well as in other conditions.
The most potent inhibitor of IL-12 synthesis by macrophages/monocytes appears to be IL-10, which acts at the protein as well as the mRNA level. IL-10 negatively regulates IL-12p40 expression (21, 22). Similar to IL-10, addition of PGE2 to cultures of LPS-stimulated human PBMC down-regulates IL-12 production (23). IL-4, IL-13, and TGF-ß can also suppress the production of IL-12 when added simultaneously with bacterial inducers. In contrast, pretreatment with IL-4 or IL-13 followed by activation with LPS enhances IL-12 production (24).
In this study we show that the induction of IL-12 production by LPS in
IFN--primed monocytes can be efficiently inhibited by the
anaphylatoxin C5a.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cytokines were used as purified recombinant human
preparations. Human IFN- was provided by Dr. Karl Thomae GmbH
(Biberach an der Riss, Germany), LPS was derived from Escherichia
coli serotype 055:B5 (Sigma, Deisenhofen, Germany), and GM-CSF was
obtained from Genzyme (Rüsselsheim, Germany). The anti-IL-4 Ab
used for neutralization of human IL-4 bioactivity (R&D Systems,
Wiesbaden, Germany) and the neutralizing rat anti-human IL-10 mAb
(PharMingen, Hamburg, Germany) both contain a low endotoxin level.
Indomethacin was obtained form Serva (Braunschweig, Germany), and
D,L-2-mercaptomethyl-3-guanidino-ethylthiopropanoic
acid (MERGETPA)3 was purchased
from Calbiochem (Frankfurt, Germany).
mAb C17/5 (mouse IgG1, 
) was generated as previously described (25).
The Ab is specific for a C5 activation-dependent neo-epitope expressed
only on C5a and C5a(desArg) and has been shown to be an effective
inhibitor of C5a (4).
Recombinant human C5a (rhC5a) was produced as previously described (26) and was further purified by affinity chromatography using the C17/5 Ab or by FPLC (the endotoxin content of this C5a preparation was 7 pg/ml as determined by the Limulus amebocyte lysate assay). As a control rhC5a obtained from Sigma was used in some experiments in parallel to C5a produced in our laboratory.
Isolation and culture of human monocytes
PBMC from healthy donors were separated by Ficoll-Hypaque
density gradient centrifugation and were resuspended in Iscove’s
medium supplemented with 4% human heat-inactivated AB serum (IAB
medium). Monocytes were isolated by adherence on petri dishes (Heraeus,
Hannover, Germany). After 3 h (37°C, 5% CO2) the
nonadherent cells were removed by several washes with PBS, and the
adherent cell population was detached. Detached cells were washed and
resuspended in IAB medium and then incubated in round-bottom 96-well
microtiter plates overnight. Monocytes were subsequently primed with
IFN- (100 U/ml) for 2 h before the addition of C5a and LPS (20
ng/ml; 10 min after C5a) as indicated in Results. When
indicated, human peripheral blood monocytes purified by centrifugal
counterflow elutriation as previously described (27) were used. The
resulting cell preparations contained up to 90% monocytes as assessed
by CD14 staining and FACS analysis.
Priming of monocytes with LPS and IFN-
In unstimulated cells IL-12p70 was undetectable with the ELISA
used. Stimulation of monocytes was performed with 100 U/ml IFN-
2 h before addition of 20 ng/ml LPS, which has previously been
shown to increase IL-12 production at the mRNA as well as at the
protein level (28). At the intracellular level, a marked IL-12
(p40/p70) production was detectable upon IFN-/LPS stimulation (i.e.,
>10% cells stained positive for IL-12) in all experiments evaluated
in this study.
mRNA isolation and RT
mRNA was isolated from 100,000 monocytes/well using an mRNA isolation kit (Boehringer Mannheim, Mannheim, Germany) according to the supplier’s instructions. Resulting poly(A)+ RNA was stored at -80°C. RNA was then subjected to first-strand cDNA synthesis using oligo(dT)15 for full-length cDNA synthesis. The RT reaction mixture contained a final concentration of 50 U of Expand-RT (Boehringer Mannheim), 20 U of RNase inhibitor (RNase out, Life Technologies, Eggenheim, Germany), 10 mM DTT, 1x first-strand RT buffer for Expand-RT, 0,5 mM of each dNTP (Boehringer Mannheim), and 80 pmol of oligo(dT)15 (Boehringer Mannheim). To control for genomic DNA contamination, cDNA synthesis was performed in the absence of reverse transcriptase. First-strand cDNA was stored at -20°C.
PCR
For PCR amplification the resulting cDNA was amplified. The PCR mixture contained a final concentration of 1 mM MgCl, 0.1 µM of the specific primers, and 0.25 U of Taq polymerase (Life Technologies). Primers specific for IL-12p40 and p35 were purchased from BioSource (Ratingen, Germany) and Stratagene (Heidelberg, Germany), respectively. For ß-actin amplification the following primers were used (forward, 5'-GAGCGGGAAATCGTGCGTGACATT; reverse, 5'-GAAGGTAGTTTCGTGGATGCC). PCRs were conducted for 28 cycles, with annealing temperatures of 52°C for p40, 62°C for p35, and 60°C for ß-actin. To control for saturation effects of the PCR, preliminary experiments were assayed at different cycle numbers by removing part of the reaction at appropriate times. An aliquot of each PCR was subjected to electrophoresis on a 2% agarose gel (Qualex Gold, AGS, Heidelberg, Germany) stained with ethidium bromide, visualized, and photographed under UV illumination. The expected sizes of amplified fragments were 225 bp for ß-actin, 290 bp for p40, and 414 bp for p35.
Flow cytometric analysis of intracellular cytokines
Intracellular staining and quantification of cytokines was
conducted with modifications as previously described (29). During the
stimulation procedure brefeldin (Sigma) was added at 3 µg/ml. Cells
were harvested, washed twice in PBS, then fixed with 4% ice-cold
phosphate-buffered paraformaldehyde for 15 min at 4°C, and washed in
PBS. To facilitate diffusion of Ab through the cell membranes, cells
were permeabilized in PBS with 0.1% saponin (Riedel de Haen, Seelze,
Germany) for 15 min. Thereafter, pretitrated cytokine-specific mAb
diluted in the permeabilization buffer (PBS-saponin) were added and
incubated for 45 min at 4°C. The PE-conjugated cytokine-specific mAb
and IgG1 isotype control mAb were used at final concentrations of 2
µg/ml (monoclonal mouse anti-human-IL-12 (p40/p70), this Ab
reacts with human IL-12p40 monomer and with the p70 heterodimer, but
not with the p35 monomer; monoclonal rat-anti-human-IL-6,
PharMingen). After subsequent washings in permeabilization buffer,
cells were resuspended and measured in PBS by flow cytometric analysis.
Expression of surface Ags on monocytes was assessed using FITC-labeled
C5aR Ab (P12/1 (IgG1/) as described previously (30)) and labeled
CD14 (Coulter-Immunotech, Hamburg, Germany). Samples were analyzed on a
FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany). Results
were analyzed using CellQuest software (Becton Dickinson).
Cytokine determinations
IL-12p70 ELISA was performed using an IL-12p70 detection kit (R&D Systems), which recognizes only the IL-12p70 heterodimer. The same samples were analyzed in another ELISA specific for p40 (R&D Systems) to determine the amount of non-p70-bound p40.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stimulation of IFN- (100 U/ml)-primed monocytes with LPS (20
ng/ml) for 24 h resulted in about 30% (mean value) of monocytes
stained positive for intracellular IL-12 (p40/p70) expression (vs
<0.5% in unstimulated cells) and a mean IL-12p70 content of about 70
pg/ml in the supernatants as described previously (28).
Exposure of IFN--primed monocytes to C5a before the addition of LPS
significantly decreased the production of IL-12. As shown in Fig. 1
, incubation with C5a suppressed
intracellular IL-12 (p40/p70) production to <30% of the positive
control value (i.e., stimulation with IFN-/LPS) in elutriated
monocytes. Stimulation caused 39.2 ± 5.4% (±SEM) of the cells
to stain positively for IL-12 (p40/p70), whereas upon addition of C5a
to IFN--primed monocytes the number of positive cells was reduced to
10.5 ± 2.1%. Suppressive effects were observed with rhC5a
preparations purified by affinity chromatography or by FPLC (Fig. 1).
Moreover, C5a suppressed IL-12 (p40/p70) in monocytes isolated by
elutriation from PBMC (Fig. 1) or by an adherence step. Markedly lower
IL-12 (p40/p70) production upon stimulation was detectable in monocytes
isolated by an adherent step. Only 18.6 ± 1.3% (±SEM) of these
cells stained positively for IL-12 (p40/p70) upon challenge with
IFN-/LPS; C5a reduced the number of cells positive for IL-12
(p40/p70) to 10.2 ± 1.0% in these cells.
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), with a marked suppression of
intracellular IL-12 (p40/p70) production using C5a doses as low as 1.5
ng/ml.
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). The
inhibition of serum carboxypeptidase N with MERGETPA resulted in an
increase in C5a-induced IL-12 suppression of >30%. This indicates
that C5a(desArg) may be the active component in our experimental
system.
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).
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-primed monocytes after addition of C5a compared with 60.52%
(SD = 9.74, n = 3) CD14+
IFN--primed monocytes that had not been exposed to C5a. IFN- (100
U/ml) did not reduce the expression of C5aR on monocytes significantly;
in two different staining experiments the decrease in C5aR expression
upon stimulation with IFN- (100 U/ml) was <20%.
A C5a-induced reduction of IL-12p70 and p40 secretion was observed in
supernatants of IFN-/LPS-stimulated monocytes (Fig. 5).
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for monocyte priming, addition of C5a caused a 75%
reduction in intracellular IL-12 production (data not shown). These
results indicate that C5a may specifically interfere with intracellular
events induced during the monocyte priming process. Both IL-12 subunits are suppressed by C5a
Fig. 5
shows that both p40 and p70 were suppressed by C5a. P40
production after stimulation (IFN- plus LPS) was 25- to 40-fold
higher than p70 production, as described by others (9, 22).
As shown in Fig. 6 a down-regulation of
IL-12p40 and p35 mRNA accumulation was observable upon stimulation of
IFN--primed monocytes with C5a and LPS for 5 h.
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IL-10, IL-4, and PGE2 have been shown to down-regulate
IL-12 production under defined experimental conditions. We therefore
tested whether endogenous production of these mediators might be
responsible for the C5a-induced IL-12 down-regulation using
indomethacin or neutralizing Abs for IL-4 or IL-10. The down-regulatory
effect of C5a remained stable in the presence of neutralizing
anti-IL-10 and anti-IL-4 Abs during preincubation, with IL-12
secretion diminished by about 70%. Fig. 7 summarizes the results obtained for
intracellular cytokine staining. The NO synthase inhibitor
N-methyl-L-arginine (data not shown) as well as
the PG synthesis inhibitor indomethacin had no effect on the reduction
of IL-12 upon stimulation in the presence of C5a (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- or GM-CSF-primed human monocytes
with C5a before addition of LPS. The ability of C5a to inhibit the
induction of IL-12 is a new finding. This may be of particular
importance because of the crucial role of IL-12 in situations where C5a
is generated, such as in acute bacterial infections, in autoimmune
disorders, or in the development of specific immunity against a number
of intracellular pathogens. The effect of C5a was specific, since it could be inhibited by an anti-C5a Ab that has been shown to block C5a-induced degranulation of granulocytes (4). The inhibition of the C5a effect on monocytes was not complete, which might be explained by an intrinsic activity of the Ab (complete Ab was used) or a low affinity of the C5a-specific Ab that led to partial C5a binding to monocytes.
Our results demonstrate that C5a treatment does not result in a down-modulation of all proinflammatory cytokines, as IL-6 was not affected by C5a-mediated down-regulation.
In vivo, C5a is rapidly degraded to C5a(desArg) by the ubiquitous
carboxypeptidase N (31). This plasma enzyme significantly reduces C5a
activity by removing the C-terminal arginyl residue. In our experiments
relatively high amounts of C5a were used. Since we used culture medium
supplemented with human serum (containing the carboxypeptidase N) the
observed effects were probably due to C5a(desArg). This speculation is
supported by experiments performed in the presence of MERGETPA, a
potent competitive inhibitor of the serum carboxypeptidase N. In the
presence of MERGETPA a 
30% increase in the IL-12 down-regulatory
effect was observable. C5a(desArg) is the natural ligand of C5aR (CD88)
and is generally less active than C5a (32).
C5a(desArg) serum levels of healthy individuals have been shown to range from 6.4–16.8 ng/ml (33). Müller et al. (34, 35) have shown that in different pathological conditions, such as renal allograft rejection and CMV diseases, plasma and/or urinary C5a levels may show a mean increase of 251 or 379%, respectively. Since even higher concentrations can be expected at the local site of complement activation, the concentrations of C5a/C5a(desArg) tested in our system are probably in a range that may occur in vivo.
Moreover, C5a used in a low (pathophysiological) nanomolar range (1.5
ng/ml = 0.18 nM) had a marked suppressive effect (
50% suppression)
on IL-12 production (Fig. 2).
To exclude an indirect effect of C5a or C5a(desArg), respectively, on the production of IL-12 we examined whether the endogenous production of IL-12 antagonists could play a role in the suppression of IL-12 by C5a. The most potent inhibitor of IL-12 synthesis by monocytes/macrophages appears to be IL-10, which acts at the protein as well as at the mRNA level (21, 22). We did not observe an enhancement of IL-12 production by neutralization of endogenously produced IL-10 in our experimental setting.
IL-4 is another antagonist of proinflammatory monokines, which has been
shown to down-regulate IL-12 (22, 24) and the expression of CD14 in
normal human monocytes (36). Similar to IL-10, the addition of
neutralizing anti-IL-4 Ab to monocyte cultures failed to overcome
the C5a-mediated suppression of IFN-/LPS-induced IL-12 release.
Moreover, C5a-induced IL-12 down-regulation was observable in highly
purified monocytes containing only small amounts of potentially
IL-4-producing lymphocytes, which suggests that T cell-derived IL-4 is
not the cause of the observed effects.
Recently, it was reported that PGE2 is a potent down-regulator of IL-12p40 subunit production by LPS-stimulated monocytes in whole blood cultures (23). Both IL-12 and PGE2 are secreted by monocytes/macrophages and other APCs in response to a variety of compounds, including bacterial products (37). Indomethacin did not reverse the suppressive effect of C5a, which excludes a role of endogenously produced PGE2 in our experiments. Using the NO synthase inhibitor N-methyl-L-arginine, we were further able to exclude that endogenously produced NO during the preincubation time interfered with the observed C5a-induced IL-12 down-regulation.
Enhanced expression of IL-12p40 leads to the formation of the p40
homodimer, which has been shown in a murine cell system to antagonize
IL-12 activities (38). Experiments with recombinant (p40)2
indicated that an approximately 10- to 100-fold excess of
IL-12(p40)2 over IL-12 is required to achieve a 50–90%
inhibition of the effects of IL-12 on Th1 cells (9). In accordance with
the findings of others (11, 22) we found that both subunits of IL-12
are inducible by stimulation with IFN-/LPS. In this study we provide
evidence that both subunits are down-regulated by C5a; at the protein
level we found that p40 parallels the down-regulation of p70, and at
the mRNA level a reduction of p35 and p40 accumulation was observed.
We used amplification by RT-PCR to facilitate detection of the mRNA species, arguing that although RT-PCR is a nonquantitative method, large differences in the intensity of the bands obtained with the same primer set should nonetheless reflect significant differences in the expression levels of the amplified mRNA. This semiquantitative method does not allow an interpretation concerning absolute mRNA amounts expressed, but detects differences in expression levels within the same experiment.
C5a obviously prevented IFN--primed monocytes to respond to an LPS
signal. Interestingly, C5a did not induce any down-regulation of IL-12
if given before the IFN--priming procedure. We excluded that
C5a-induced IL-12 suppression was due to the down-modulation of the LPS
receptor CD14. All C5a preparations used in this study were virtually
LPS free as detected by the Limulus assay. This is
important, since LPS can induce a state of hyporesponsiveness to its
own effects, a phenomenon mediated by monocytes/macrophages (39).
LPS-induced tolerance has recently been described by us (28) with
respect to IL-12 production and has previously been described for TNF,
IL-6, IL-8, IL-10, NO, and other cytokines (40, 41, 42, 43, 44, 45) and is
characterized by a diminished synthesis of these mediators by
monocytes.
LPS and C5a have been shown to act synergistically in the induction of a number of proinflammatory cytokines. Montz et al. (4) demonstrated that IL-6 production by LPS-stimulated human PBMC was substantially potentiated by C5a in a dose-dependent manner. Cavaillon et al. (46) were able to show that rC5a enhances IL-1 and TNF release by LPS-stimulated monocytes and macrophages. These experiments had been performed without a priming step before incubation of monocytes with LPS.
Monocytes/macrophages require such a priming signal before LPS
stimulation for optimal production of IL-12 (11, 47). Interestingly,
C5a did not exert a suppressive effect if applied before the priming
procedure. This finding suggests that C5a renders primed monocytes
insensitive to subsequent stimulation with the second signal LPS,
probably by interfering with signal transduction pathways important for
an optimal response to LPS. In this context, Marth and Kelsall (48)
have shown in a recent study that iC3b (and CR3 Ab) binding to CR3
(CD11b/CD18) on human macrophages prestimulated with
Staphylococcus aureus cells (Cowan I strain) caused a
decrease in IL-12 production. They speculated that CR3 Abs may suppress
IL-12 production by the inhibition of IFN--induced tyrosine
phosphorylation.
We cannot exclude interference of C5a with IFN- signal transduction
pathways. Since IL-12 suppression could also be observed after
replacing IFN- by GM-CSF, C5a does not exclusively interfere with
the IFN- signal transduction pathway. Preincubation of C5a before
GM-CSF or IFN- priming did not result in IL-12 down-regulation.
Therefore, our hypothesis is that C5a may render primed monocytes
insensitive to a subsequent second signal provided by LPS.
Sutterwala et al. (49) have demonstrated that a number of ligands for
phagocytic receptors, such as the scavenger and the Fc receptor, can
down-modulate IL-12 mRNA synthesis and protein secretion. They discuss
that this effect is dependent on receptor ligations that cause a
calcium influx. In this context C5a is another agent that increases
intracellular calcium levels upon binding to its receptor (50).
The C5aR has previously been shown to be expressed in situ on resident macrophages or dendritic cells (30). The C5a-induced down-modulation of IL-12 shown here may be important as an endogenous counter-regulation of potentially harmful inflammatory reactions in situ, but the generation of C5a may also favor a type 2 cytokine milieu in acute inflammatory or allergic reactions.
The complete elucidation of the cellular events induced by C5a may aid in understanding inflammatory processes, especially the status of primed monocytes/macrophages and may potentially lead to novel therapeutical strategies in sepsis, shock, and allergic and autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Miriam Wittmann, Department of Dermatology and Allergology, Hannover Medical University, Ricklinger Str. 5, D-30449 Hannover, Germany. E-mail address:
3 Abbreviations used in this paper: MERGETPA, D,L-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid; rh, recombinant human; FPLC, fast protein liquid chromatography; IAB medium, Iscove’s medium supplemented with 4% human heat-inactivated AB serum; NO, nitric oxide.
Received for publication December 3, 1998. Accepted for publication March 10, 1999.
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T. Werfel, K. Kirchhoff, M. Wittmann, G. Begemann, A. Kapp, F. Heidenreich, O. Gotze, and J. Zwirner Activated Human T Lymphocytes Express a Functional C3a Receptor J. Immunol., December 1, 2000; 165(11): 6599 - 6605. [Abstract] [Full Text] [PDF]
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 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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M. C. Braun, E. Lahey, and B. L. Kelsall Selective Suppression of IL-12 Production by Chemoattractants J. Immunol., March 15, 2000; 164(6): 3009 - 3017. [Abstract] [Full Text] [PDF]
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M. Wittmann, V.-A. Larsson, P. Schmidt, G. Begemann, A. Kapp, and T. Werfel Suppression of Interleukin-12 Production by Human Monocytes After Preincubation With Lipopolysaccharide Blood, September 1, 1999; 94(5): 1717 - 1726. [Abstract] [Full Text] [PDF]
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