The anaphylatoxin C3a has been reported to have immunomodulatory effects on a number of different cell types. In this study we investigated the effects of C3a and C3adesArg on gene expression and protein secretion of IL-6 in human PBMCs, either alone or in combination with LPS or IL-1β. C3a or C3adesArg alone exhibited no effect on the expression or secretion of IL-6. However, when PBMC were stimulated with LPS or IL-1β, both C3a and C3adesArg were found to enhance IL-6 release by PBMC in a dose-dependent manner. Since C3a has been shown to induce PGE2 production by monocytes, and PGE2 has been shown to influence cytokine production, we investigated the potential role of PGE2 in C3a-mediated enhancement of LPS- and IL-1β-induced IL-6 production. Indomethacin blocked PGE2 release, but had no influence on the observed effects of C3a, suggesting that the effects of C3a on IL-6 production are independent of PGE2 formation by monocytes. Northern blot analysis showed that C3a as well as C3adesArg enhanced LPS-induced mRNA levels for IL-6. Pretreatment of PBMCs with pertussis toxin blocked the functions of C3a and C3adesArg, indicating that the actions of these two molecules are mediated by a G protein-coupled pathway. Furthermore, we investigated the effects of C3a and C3adesArg on induction of NF-κB and activating protein-1 binding. Both molecules enhanced LPS-induced NF-κB and activating protein-1 binding activity. These results demonstrate the capacity of intact C3a and its circulating des-Arg form to exert immunmodulatory effects in vitro.

Activation and/or dysregulation of the complement system has been shown to be involved in several clinical situations, such as sepsis (1, 2), burn injury (3), adult respiratory distress syndrome (4), and autoimmune diseases (5).

Activation of the blood complement system via either the classical or alternative pathways results in the production of one or more of the anaphylatoxins, i.e., C3a, C4a, and/or C5a (6, 7, 8). C5a, the best characterized of the anaphylatoxins, is a potent proinflammatory mediator that induces chemotactic migration, increases oxidative metabolism and the release of lysosomal enzymes in leukocytes, stimulates the release of numerous inflammatory mediators including histamine and cytokines, and enhances cellular adhesion (8, 9, 10, 11, 12). C3a also possesses proinflammatory properties, which overlap with C5a in a number of activities. C3a is spasmogenic, induces the release of PGE2 from human macrophages, induces degranulation and chemotaxis of eosinophils, and is one of the most effective chemotactic mediators yet identified for human mast cells (13, 14, 15, 16). Very recently, we demonstrated a direct immunmodulatory effect of C3a (as well as C3adesArg) on human tonsil-derived resting B lymphocytes (17). Furthermore, it has been shown that C3a and C3adesArg can regulate TNF-α and IL-1β synthesis in LPS-stimulated human PBMC (15).

The immune system is essential for protection of the host organism from microbial invasion, tumorigenesis, and environmental insult. This task requires concerted actions of cellular responses that are regulated by a complicated network of cytokines, other humoral factors, and cell-cell interactions. Inflammatory cytokines, including TNF-α, IL-1β, IL-8, and IL-6 have been implicated in the pathogenesis of several diseases that are also associated with complement activation. Like TNF-α and IL-1, IL-6 is a pleiotropic mediator produced by numerous cell types in response to a variety of stimuli, including LPS, platelet-derived growth factor, and the cytokines TNF-α, IL-1, and IL-2 (18, 19, 20, 21). IL-6 is known to promote differentiation and maturation of B lymphocytes, to augment T lymphocyte responses, to stimulate hemopoiesis, and to induce the production of acute phase proteins (22, 23). Increased serum levels of IL-6 have been reported in patients with a variety of diseases, including acute bacterial infections, meningcoccal septic shock, systemic lupus erythematosus, Castleman’s disease, rheumatoid arthritis, and AIDS (24, 25).

The effects of C5a on cytokine production are well characterized. We and others have demonstrated that C5a is a potent inducer of gene expression and protein synthesis for TNF-α, IL-1, IL-6, and IL-8 (10, 11, 12, 26). On the other hand, the characterization of C3a as an inducer of cytokine production has just begun. It is the purpose of the present study to further explore the mechanism(s) by which C3a and C3adesArg influence immune and inflammatory responses. C3a and C3adesArg were assessed for their ability to modulate LPS- and IL-1β-induced IL-6 production in human PBMC cultures. In this report we show that C3a as well as C3adesArg are potent modulators of IL-6-specific mRNA and protein synthesis in human PBMCs. Our results suggest that C3a and C3adesArg contribute to both inflammation and the regulation of immune function by modulating IL-6, which possesses potent pleiotropic functions.

All chemicals were obtained from Sigma (St. Louis, MO) unless indicated otherwise. Ficoll was purchased from Pharmacia (Piscataway, NJ). RPMI medium was obtained from BioWhittaker (Walkersville, MD). IL-1β was purchased from R&D Systems (Minneapolis, MN). Capture and detection Abs for ELISA were obtained from PharMingen (San Diego, CA). The synthetic C3a analogue peptide 71/53 (13, 30) (WWGKKYRASKLGLAR) was synthesized, and both purity and sequence were confirmed in the protein/nucleotide core facilities of The Scripps Research Institute. The human IL-6 cDNA probe used in these experiments was provided by Dr. Edward Morgan (The La Jolla Cancer Institute, La Jolla, CA).

Human C3a was purified according to the method described previously (27). The concentration of C3a was determined by amino acid analysis. Based on tracer experiments with [125I]C5a, contamination of the C3a preparation with C5a was <0.0017%. By using the Limulus amebocyte lysate assay (BioWhittaker), LPS contamination of C3a, C3adesArg, or the C3a analogue 71/53 was not detectable. C3adesArg was obtained by treating the highly purified C3a with 1% (w/w) carboxypeptidase B for 30 min at 37°C in amonium bicarbonate buffer at pH 8.0. The conversion of C3a to C3adesArg was confirmed by mass spectometry, demonstrating homogeneous peaks of 9093 and 8933 mass units, respectively, for C3a and C3adesArg (mass spectometry core facilities of The Scripps Research Institute).

PBMCs were isolated from peripheral blood of healthy human donors as described previously (26). Briefly, blood was drawn into EDTA-containing syringes to achieve a final EDTA concentration of 10 mM. Thirty-five milliliters of whole blood was layered over 15 ml of Ficoll-Hypaque (Pharmacia) and centrifuged at 600 × g for 20 min. The mononuclear cell layer was recovered and washed twice in Earle’s balanced salt solution containing 10 mM MOPS at pH 7.3. The final pellet was resuspended in RPMI 1640 supplemented with 2 mM l-glutamine, 100 U penicillin, and 100 μg/ml streptomycin (Bio-Whittaker, Walkersville, MD).

For induction of IL-6 protein, triplicate cultures were established in 96-well microculture plates (Corning, NY); each well contained 5 × 105 PBMCs. LPS from Escherichia coli (strain ECO55B5; Sigma) was added to a final concentration of 500 ng/ml in the presence or the absence of C3a or C3adesArg. The plates were then incubated at 37°C in a humidified atmosphere containing 5% CO2. Culture supernatants were collected after 18 h and stored at −20°C until assayed for IL-6. For isolation of total RNA and extracts of nuclear proteins, 5 × 106 cells were plated in 1 ml of culture medium in six-well plates and incubated for various times under the conditions described above.

Total RNA was extracted with TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. Eight micrograms of total RNA was subjected to electrophoresis in agarose gels containing 0.22 M formaldehyde and transferred to the Hybond-N Plus membranes (Amersham, Arlington Heights, IL) by diffusion. The probe for human β-actin was generated by RT-PCR, using human β-actin specific primers (Stratagene, La Jolla, CA). To confirm that the amplified product reflects the human β-actin mRNA, it was eluted from the gel and cloned in the TA cloning vector pCRTM2.1. (Invitrogen, La Jolla, CA) following the manufacturer’s instructions, and the nucleotide sequence was determined. Sequencing was performed in the protein/nucleotide core facilities of The Scripps Research Institute. The sequence obtained was identical with those published. The human IL-6 cDNA probe used in these experiments was provided by Dr. Edward Morgan. For hybridization, the fragments were labeled with [α-32P]dCTP to a sp. act. of >2 × 108 cpm/μg DNA using a random primer DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). Membranes were prehybridized in QuikHyb solution (Stratagene, La Jolla, CA) at 68°C for 20 min and further hybridized with the labeled probes (1 × 106 cpm/ml) for 2 h at the same temperature. Filters were washed twice with 2× SSC and 0.1% SDS at room temperature for 15 min followed by another wash with 0.1× SSC and 0.1% SDS at 60°C for 30 min. The membranes were exposed to a PhosphorImager screen and analyzed by a PhosphorImaging system (Molecular Dynamics, Sunnyvale, CA).

Nuclear extracts were prepared by a modified method of Dignam et al. (28). Aliquots of PBMC (5 × 106/sample) were stimulated with different agent as indicated in the text, after which the cells were washed three times with ice-cold PBS, harvested, and resuspended in 0.4 ml of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM DTT, 0.1 mM each of EDTA and EGTA, and 1 mM PMSF). After 10 min, 23 μl of 10% Nonidet P-40 was added and mixed for 2 s. Nuclei were separated from cytosol by centrifugation at 13,000 × g for 20 s and were resuspended in 50 μl of buffer B (10 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM PMSF). After 30 min at 4°C, lysates were separated by centrifugation at 13,000 × g for 40 s, and the supernatants containing nuclear proteins were transferred into new vials. Protein concentrations were measured using a protein dye reagent (Bio-Rad, Richmond, CA) with BSA as standard, and samples were diluted to equal concentrations in buffer B. Samples were stored at −70°C.

EMSAs were performed essentially as previously described (29). Briefly, 5 μg of nuclear extracts were added to 12 μl of binding buffer (5 mM HEPES (pH 7.9), 5 mM MgCl2, 50 mM KCl, 0.5 mM DTT, 0.4 mg/ml poly(dI-dC), 0.1 mg/ml sonicated salmon sperm DNA, and 10% glycerol) and incubated for 15 min at room temperature. Approximately 40 fmol of 32P-labeled oligonucleotide probe (Stratagene) was then added, and the reaction was continued at room temperature for 15 min. For reactions involving competitor oligonucleotides, the unlabeled and labeled probes were added simultaneously to the reaction mixture. The samples were analyzed on 5% acrylamide gel, made with buffers containing 50 mM Tris borate and 1 mM EDTA. After pre-electrophoresis for 1 h at 8 V/cm, samples were applied, and electrophoresis was conducted at the same voltage for 1.5–2 h. The gels were transfered to Whatman paper (Clifton, NJ), dried, exposed to a PhosphorImager screen, and analyzed by a PhosphorImaging system (Molecular Dynamics).

IL-6 production in supernatants from human PBMC cultures was measured by sandwich ELISA using paired mAb (PharMingen), following the instructions of the manufacturer.

Recently, we and others have demonstrated immunmodulatory effects of C3a and C3adesArg on cytokine synthesis in human PBMCs and human tonsil-derived B lymphocytes (15, 17). These studies prompted us to investigate whether C3a and C3adesArg can modulate the production of a related cytokine, i.e., IL-6, under various experimental conditions.

Unstimulated human PBMCs or PBMCs stimulated with 1 μM C3a or C3adesArg alone did not release detectable levels of IL-6 (Fig. 1). However, at concentrations from 1 μM to 10 nM, both C3a and C3adesArg enhanced LPS-induced IL-6 release from human PBMCs in a dose-dependent manner. At a concentration of 1 μM, C3a and C3adesArg significantly increased IL-6 levels in culture supernatants of PBMCs stimulated with LPS (100 ng/ml) by 400 ± 30 and 328 ± 19%, respectively (Fig. 1, A and B). At a concentration at or below 10 nM, neither C3a nor C3adesArg induced a significant increase in IL-6. We next asked whether the observed increase in IL-6 production by C3a and C3adesArg is specific for LPS stimulation or whether activation of cells by other stimuli (i.e., IL-1β (25 ng/ml)) in combination with C3a or C3adesArg would also result in enhanced IL-6 release. As shown in Fig. 1 (C and D), both C3a and C3adesArg enhanced IL-1β-induced IL-6 release in PBMCs. At the highest concentrations of C3a and C3adesArg (1 μM) the increases in IL-1β-induced IL-6 release were 310 ± 40 and 293 ± 38%, respectively. Surprisingly, but in agreement with former studies, C3adesArg was almost as effective as C3a itself (15, 17). However, at a concentration of 500 nM an apparent difference was observed between C3a and C3adesArg that was not observed at other concentrations.

FIGURE 1.

Effects of C3a and C3adesArg on IL-6 production by human PBMCs. PBMCs were cultured at 5 × 105 cells/ml and stimulated with LPS (100 ng/ml; A and B) or IL-1β (25 ng/ml; C and D) in the absence or the presence of different concentrations of C3a or C3adesArg. Supernatants were harvested after 18 h of culture. Levels of IL-6 were determined by cytokine-specific ELISA. Differences in IL-6 levels were analyzed by Student’s t test (the asterisk indicates p < 0.02). n.d., not detectable. The data are expressed as the mean ± SD of six independent experiments using different donors.

FIGURE 1.

Effects of C3a and C3adesArg on IL-6 production by human PBMCs. PBMCs were cultured at 5 × 105 cells/ml and stimulated with LPS (100 ng/ml; A and B) or IL-1β (25 ng/ml; C and D) in the absence or the presence of different concentrations of C3a or C3adesArg. Supernatants were harvested after 18 h of culture. Levels of IL-6 were determined by cytokine-specific ELISA. Differences in IL-6 levels were analyzed by Student’s t test (the asterisk indicates p < 0.02). n.d., not detectable. The data are expressed as the mean ± SD of six independent experiments using different donors.

Close modal

To prove that the observed increases in IL-6 production were specific effects of C3a and C3adesArg and not due to contaminants in the C3a preparations used in our experiments, we tested the synthetic C3a analogue peptide 71/53 (peptide sequence WWGKKYRASKLGLAR). Previous studies have shown that this peptide exhibits the same biological activities as native C3a (13, 30). PBMCs were stimulated with LPS (100 ng/ml) and different concentrations of C3a analogue 71/53. As shown in Fig. 2, the peptide analogue was also found to increase LPS-induced IL-6 production in a dose-dependent manner. The concentration of C3a analogue peptide required to produce approximately the same enhancement was 5–10 times higher than that of native human C3a. This is in agreement with former studies of this peptide (30). Taken together, these results indicate that the observed enhancing effects of C3a on IL-6 production in PBMCs are specific effects of the molecule.

FIGURE 2.

Effect of the synthetic analogue C3a peptide 71/53 (peptide sequence: WWGKKYRASKLGLAR) on IL-6 production by human PBMCs. PBMCs were cultured at 5 × 105 cells/ml and stimulated with LPS (100 ng/ml) in the absence or the presence of different concentrations of 71/53 peptide. Supernatants were harvested after 18 h of culture. Levels of IL-6 were determined by cytokine-specific ELISA. Differences in IL-6 levels were analyzed by Student’s t test (the asterisk indicates p < 0.02). n.d., not detectable. The data are expressed as the mean ± SD of three independent experiments using different donors.

FIGURE 2.

Effect of the synthetic analogue C3a peptide 71/53 (peptide sequence: WWGKKYRASKLGLAR) on IL-6 production by human PBMCs. PBMCs were cultured at 5 × 105 cells/ml and stimulated with LPS (100 ng/ml) in the absence or the presence of different concentrations of 71/53 peptide. Supernatants were harvested after 18 h of culture. Levels of IL-6 were determined by cytokine-specific ELISA. Differences in IL-6 levels were analyzed by Student’s t test (the asterisk indicates p < 0.02). n.d., not detectable. The data are expressed as the mean ± SD of three independent experiments using different donors.

Close modal

To investigate whether the effects of C3a and C3adesArg on IL-6 are accompanied by increased IL-6 mRNA levels, PBMCs were stimulated with LPS (100 ng/ml) plus 1 μM C3a or C3adesArg, and RNA was prepared for Northern blotting. A time course from 0–24 h demonstrated that neither C3a nor C3adesArg alone can induce IL-6 mRNA in PBMCs (data not shown). However, when cells were stimulated with both LPS plus C3a or C3adesArg, we found an increase in IL-6 mRNA at 3 h compared with that in cells stimulated with LPS alone (Fig. 3). Standardization of IL-6 mRNA levels to β-actin mRNA levels revealed that IL-6 mRNA increased by 60 and 63%, respectively, for C3a and C3adesArg costimulated with LPS.

FIGURE 3.

Effects of C3a and C3adesArg on IL-6 gene expression in human PBMCs. Northern blot with total RNA (8 μg/lane) isolated from PBMCs (5 × 106/ml) treated as indicated below. 1a, Unstimulated cells, cells stimulated with C3a (1 μM) or C3adesArg (1 μM); 1b, PBMCs stimulated with LPS (100 ng/ml) alone or in combination with C3a (1 μM), PBMCs stimulated with LPS (100 ng/ml) alone or in the presence of C3adesArg (1 μM); 1d–1f, β-actin controls. These results are representative of three independent experiments using different donors.

FIGURE 3.

Effects of C3a and C3adesArg on IL-6 gene expression in human PBMCs. Northern blot with total RNA (8 μg/lane) isolated from PBMCs (5 × 106/ml) treated as indicated below. 1a, Unstimulated cells, cells stimulated with C3a (1 μM) or C3adesArg (1 μM); 1b, PBMCs stimulated with LPS (100 ng/ml) alone or in combination with C3a (1 μM), PBMCs stimulated with LPS (100 ng/ml) alone or in the presence of C3adesArg (1 μM); 1d–1f, β-actin controls. These results are representative of three independent experiments using different donors.

Close modal

A previous study demonstrated that C3a stimulates PGE2 production in human macrophages (14). Furthermore, it has been shown that PGE2 leads to increased intracellular cAMP levels (31). Since both PGE2 and cAMP are involved in the regulation of proinflammatory cytokines, we addressed the question of whether endogenous PGE2 contributes to and/or influences these effects of C3a and C3adesArg on IL-6 regulation in LPS- or IL-1β-stimulated PBMCs. Indomethacin at a concentration of 1 μg/ml totally inhibited the production of PGE2 in PBMC cultures stimulated with LPS (100 ng/ml), C3a (1 μM), or LPS in combination with C3a (data not shown). Furthermore, indomethacin had no effect on IL-6 release in human PBMCs stimulated with LPS or IL-1β alone or in combination with C3a or C3adesArg (Fig. 4, A and B). Indomethacin itself did not induce IL-6 release (data not shown). These data demonstrate that the observed effects of C3a and C3adesArg are not mediated via the production of endogenous PGE2.

FIGURE 4.

Influence of indomethacin on the effects of C3a and C3adesArg on IL-6 production by human PBMCs. Cells were cultured at 5 × 105 cells/ml, prestimulated with indomethacin (1 μg/ml) for 0.5 h (open bars), and stimulated with LPS (100 ng/ml; A) or IL-1β (B) in the absence or the presence of C3a or C3adesArg. Supernatants were harvested after 18 h of culture. Levels of IL-6 were determined by cytokine-specific ELISA. The data are expressed as the mean ± SD of four independent experiments using different donors.

FIGURE 4.

Influence of indomethacin on the effects of C3a and C3adesArg on IL-6 production by human PBMCs. Cells were cultured at 5 × 105 cells/ml, prestimulated with indomethacin (1 μg/ml) for 0.5 h (open bars), and stimulated with LPS (100 ng/ml; A) or IL-1β (B) in the absence or the presence of C3a or C3adesArg. Supernatants were harvested after 18 h of culture. Levels of IL-6 were determined by cytokine-specific ELISA. The data are expressed as the mean ± SD of four independent experiments using different donors.

Close modal

C3a is known to bind to a receptor that is functionally coupled to G proteins (32, 33). In addition, cloning of the human C3a receptor confirmed that it possesses a structure typical of a G protein-coupled receptor. Therefore, we addressed the question of whether the observed effects of C3a and C3adesArg are subject to inhibition by PTX. Pretreatment of cells with PTX is known to interfere with signals mediated through Gi-type guanine nucleotide binding proteins. Our results shown in Fig. 5 indicate that pretreatment of PBMCs with PTX for 3 h almost completely abolished C3a- or C3adesArg-mediated enhancement of IL-6 release in LPS- or IL-1β-stimulated PBMCs. We next examined whether the observed inhibitory effect of PTX could also be demonstrated at the mRNA level. As shown in Fig. 6 pretreatment of cells with PTX blocked C3a- as well as C3adesArg-mediated increases in IL-6 mRNA. Taken together, these data indicate that C3a and C3adesArg mediate their effects via a specific G protein-coupled pathway.

FIGURE 5.

Influence of PTX on the effects of C3a and C3adesArg on IL-6 release by human PBMCs. Cells were cultured at 5 × 105 cells/ml, pretreated with PTX (500 ng/ml) for 3 h (open bars), and stimulated with LPS (100 ng/ml; A) or IL-1β (B) in the absence or the presence of C3a or C3adesArg. Supernatants were harvested after 18 h of culture. Levels of IL-6 were determined by cytokine-specific ELISA. Differences in IL-6 levels were analyzed by Student’s t test (the asterisk indicates p < 0.01). The data are expressed as the mean ± SD of four independent experiments using different donors.

FIGURE 5.

Influence of PTX on the effects of C3a and C3adesArg on IL-6 release by human PBMCs. Cells were cultured at 5 × 105 cells/ml, pretreated with PTX (500 ng/ml) for 3 h (open bars), and stimulated with LPS (100 ng/ml; A) or IL-1β (B) in the absence or the presence of C3a or C3adesArg. Supernatants were harvested after 18 h of culture. Levels of IL-6 were determined by cytokine-specific ELISA. Differences in IL-6 levels were analyzed by Student’s t test (the asterisk indicates p < 0.01). The data are expressed as the mean ± SD of four independent experiments using different donors.

Close modal
FIGURE 6.

Influence of PTX on C3a and C3adesArg up-regulation of IL-6 mRNA expression by human PBMCs. A Northern blot with total RNA (8 μg/lane) isolated after treatment of PBMCs (5 × 106/ml), as indicated below, is shown. Cells were prestimulated with PTX (500 ng/ml) for 3 h and thereafter stimulated with LPS (100 ng/ml) in the absence or the presence of C3a (1 μM) or C3adesArg (1 μM) for 3 h. β-Actin was used as the control. These results are representative of three independent experiments using different donors.

FIGURE 6.

Influence of PTX on C3a and C3adesArg up-regulation of IL-6 mRNA expression by human PBMCs. A Northern blot with total RNA (8 μg/lane) isolated after treatment of PBMCs (5 × 106/ml), as indicated below, is shown. Cells were prestimulated with PTX (500 ng/ml) for 3 h and thereafter stimulated with LPS (100 ng/ml) in the absence or the presence of C3a (1 μM) or C3adesArg (1 μM) for 3 h. β-Actin was used as the control. These results are representative of three independent experiments using different donors.

Close modal

Previous reports indicated that the NF-κB binding site located between positions −72 and −63 on the IL-6 gene is important for the induction of IL-6 (34, 35). Another important transcription factor involved in the regulation of IL-6 transcription is AP-1, with a consensus binding sequence found at position −283 to −277 in the IL-6 promoter (36). To examine whether the observed enhanced responses seen after costimulation of PBMCs with LPS and C3a were related to a different induction of the transcription factors NF-κB and AP-1, we measured the DNA binding activity of these factors using EMSAs. As expected, treatment of PBMCs with LPS (100 ng/ml) alone caused induction of NF-κB and AP-1 binding activities. However, costimulation of PBMCs with LPS (100 ng/ml) and C3a (1 μM) resulted in an additional increase in the binding activities of NF-κB and AP-1 (Fig. 7). Surprisingly, nuclear extracts from PBMCs stimulated with C3a (1 μM) alone showed increased DNA binding activity compared with the activity in untreated cells (Fig. 8). To verify that these DNA-protein interactions were specific, we performed competition experiments with unlabeled oligonucleotides. NF-κB and AP-1 binding activities in nuclear extracts from PBMCs stimulated with LPS (100 ng/ml) plus C3a (1 μM) were completely abrogated in the presence of a 100-fold molar excess of unlabeled oligonucleotides, but remained intact in the presence of a 100-fold molar excess of Oct-1 oligonucleotide (Fig. 9). These results indicate that the enhancing effects of C3a are associated with an increased binding activity of the transcription factors NF-κB and AP-1.

FIGURE 7.

Effects of C3a on NF-κB and AP-1 DNA binding activity in human PBMCs. Shown are autoradiographs of EMSA results. Nuclear extracts of cells stimulated with LPS (100 ng/ml) in the absence or the presence of C3a (1 μM) for 1 h were analyzed. 32P-labeled 22 mer, containing the consensus sequences for NF-κB (GGGACTTTCC; A) or AP-1 (TGACTCA; B), was used for EMSA. These results are representative of three independent experiments using different donors.

FIGURE 7.

Effects of C3a on NF-κB and AP-1 DNA binding activity in human PBMCs. Shown are autoradiographs of EMSA results. Nuclear extracts of cells stimulated with LPS (100 ng/ml) in the absence or the presence of C3a (1 μM) for 1 h were analyzed. 32P-labeled 22 mer, containing the consensus sequences for NF-κB (GGGACTTTCC; A) or AP-1 (TGACTCA; B), was used for EMSA. These results are representative of three independent experiments using different donors.

Close modal
FIGURE 8.

Effects of C3a on NF-κB and AP-1 DNA binding activity in human PBMCs. Shown are autoradiographs of EMSA results. Nuclear extracts of cells stimulated with LPS (100 ng/ml) or C3a (1 μM) for 1 h were analyzed. 32P-labeled 22-mer, containing the consensus sequences for NF-κB (GGGACTTTCC; A) or AP-1 (TGACTCA; B), was used for EMSA. These results are representative of three independent experiments using different donors.

FIGURE 8.

Effects of C3a on NF-κB and AP-1 DNA binding activity in human PBMCs. Shown are autoradiographs of EMSA results. Nuclear extracts of cells stimulated with LPS (100 ng/ml) or C3a (1 μM) for 1 h were analyzed. 32P-labeled 22-mer, containing the consensus sequences for NF-κB (GGGACTTTCC; A) or AP-1 (TGACTCA; B), was used for EMSA. These results are representative of three independent experiments using different donors.

Close modal
FIGURE 9.

Specificity of NF-κB and AP-1 binding activity. Nuclear extracts prepared from PBMCs stimulated with LPS (100 ng/ml) plus C3a (1 μM) were used for analysis of the NF-κB (A) or AP-1 (B) binding activity by EMSA in the absence (left lanes) or the presence of competitive unlabeled oligonucleotides (100-fold excess). Middle lanes, Competition with an oligonucleotide containing the NF-κB consensus sequence (A) or the AP-1 consensus sequence (B). Right lanes (A and B), Competition with an oligonucleotide containing the Oct-1 binding sequence. These results are representative of two independent experiments using different donors.

FIGURE 9.

Specificity of NF-κB and AP-1 binding activity. Nuclear extracts prepared from PBMCs stimulated with LPS (100 ng/ml) plus C3a (1 μM) were used for analysis of the NF-κB (A) or AP-1 (B) binding activity by EMSA in the absence (left lanes) or the presence of competitive unlabeled oligonucleotides (100-fold excess). Middle lanes, Competition with an oligonucleotide containing the NF-κB consensus sequence (A) or the AP-1 consensus sequence (B). Right lanes (A and B), Competition with an oligonucleotide containing the Oct-1 binding sequence. These results are representative of two independent experiments using different donors.

Close modal

Involvement of anaphylatoxins in the processes of inflammation and immunoregulation is supported by well-established phenomena (8, 9). The purpose of the present study was to investigate the molecular basis for the biological effects of C3a and C3adesArg on human PBMCs. The effects of C3a on cytokine synthesis are poorly defined. A previous report demonstrated that C3a stimulated human PBMCs to produce IL-1 (37). However, another study failed to show IL-1 synthesis in PBMCs in response to C3a (38). Regarding C3a as an inducer of cytokine synthesis, this latter finding is in agreement with our results as well as other published data (15). Neither C3a nor C3adesArg induced IL-6 specific mRNA or release of IL-6 in human PBMCs. Although rapid expression of cytokine mRNA in monocytes can be induced by adherence without subsequent translation, C3a or C3adesArg alone did not induce or modify IL-6 mRNA synthesis in our in vitro model. However, here we demonstrate that C3a as well as C3adesArg increase IL-6-specific mRNA and protein synthesis by human PBMCs after stimulation with LPS or IL-1β. The C3a analogue peptide 71/53 also enhanced IL-6 release, suggesting that the observed effects of C3a are specific. Recently, Takabayshi et al. reported that C3a and C3adesArg modulate LPS-induced mRNA and protein synthesis of TNF-α and IL-1β in human PBMCs (15). In their study they showed that synthesis of these cytokines was up-regulated in adherent PBMCs, whereas they were down-regulated in nonadherent PBMCs. Very recently, these studies were extended to demonstrate the same effects on IL-6 production (39). In our study we did not observe a difference between adherent and nonadherent PBMCs, independent of the concentration of LPS employed (data not shown). It is currently unclear what differences in our experimental systems are likely to account to the discrepancies in these studies.

In a recent study we demonstrated C3a-induced down-regulation of IL-6 and TNF-α release in human tonsil-derived B lymphocytes (40). Taken together, these findings suggests that C3a can modulate cytokine synthesis in a cell-specific manner. Similar effects have been reported for other mediators, such as IL-4, which is known to inhibit IL-6 production in a tissue-specific manner (41).

C3a is known to induce PGE2 production in human monocytes, leading to increased cAMP levels by stimulation of adenylate cyclase activity. These signals are known to regulate cytokine production. In our experiments indomethacin, an inhibitor of cyclo-oxgenase, totally suppressed PGE2 production, but did not affect C3a- and C3adesArg-mediated increases in IL-6 expression or release in human PBMCs. This suggests that the effects of C3a and C3adesArg on IL-6 production in LPS- or IL-1β-stimulated PBMCs are independent of endogenous PGE2 synthesis. These data are in agreement with those published by Takabayashi et al. (15, 39).

Recently, the receptor for human C3a was cloned and was found to belong to the family of seven-transmembrane receptors (32, 33, 42). Moreover, several biological responses of C3a can be blocked by PTX, suggesting that the C3a receptor acts as a Gi-coupled receptor (16). In the present study we show that the effects of both C3a and C3adesArg on IL-6 synthesis were totally blocked by PTX, indicating that these molecules act through a G protein-dependent pathway, possibly involving the C3a receptor. However, the biological activity of C3adesArg as well as the concentrations of C3a and C3adesArg required to elicit enhancement of IL-6 production are inconsistent with the currently known properties of the receptor on other cell types (33, 50). Definitive assignment of a receptor-mediated mechanism currently awaits the generation of specific receptor antagonists or blocking Abs to the C3a receptor.

A number of previous studies have revealed that NF-κB and AP-1 are important transcription factors involved in the control and regulation of IL-6 transcription. The major form of NF-κB is a heterodimer composed of p50 and p65 subunits that exist in the cytoplasm in an inactive form due to association with the specific inhibitor protein IκB (43, 44). Phosphorylation of IκB by candidate kinases leads to the release and subsequent nuclear translocation of NF-κB, which then binds to a decameric DNA sequence originally identified in the κ light chain enhancer of Ig (45). The transcription factor AP-1 is a heterodimer formed by a product of the fos and jun families (46). In the present study we show that the enhancing activities of C3a on IL-6 expression in LPS-stimulated PBMCs are associated with increased binding of NF-κB and AP-1 to their consensus sequences. Unexpectedly, nuclear extracts from PBMCs stimulated with C3a alone also showed elevated DNA binding activities of NF-κB and AP-1 compared with those from untreated cells. Despite this observation, C3a was not able to induce IL-6 expression. Furthermore, in the studies by Takabayashi et al., C3a alone did not induce IL-1β, TNF-α, or IL-6 (15, 39). A possible explanation could be that other transcription factors shown to be important for IL-6 expression (e.g., NF-IL-6 and SP-1) are not inducible by C3a (47). A study by Matsusaka et al. (48) showed that in the absence of NF-IL-6, NF-κB (p50/p65), or any other combination of p50 and p65, IL-6 expression was not inducible in a cotransfection system. These results indicate that a cooperative effect exists among these transcription factors (48). Recently, LeClair et al. (49) found that p50 and NF-IL-6 directly associate with each other via the b-Zip domain and the Rel homology domain. These data raise the possibility that C3a alone might be competent to induce the transcription of yet uninvestigated genes by inducing binding activity of NF-κB and AP-1. To our knowledge, this is the first report showing that the anaphylatoxin C3a is capable of inducing activation of transcription factors, namely NF-κB and AP-1, which have been shown to be important regulators of many immune response and acute phase response genes, including IL-8, TNF-α, IL-1β, granulocyte CSF, Ig κ light chain, and serum amyloid A-1. Therefore, it is possible that C3a is involved in the regulation of a number of immunological functions in vivo.

Surprisingly, C3adesArg shared the ability to enhance IL-6 production with C3a. C3adesArg, which lacks the C-terminal arginine, has generally been regarded to be biologically inactive (13, 14, 50, 51). C3adesArg is not chemotactic for eosinophils (13), does not bind to specific receptors on guinea pig platelets (50) or on a human mast cell line (52), and does not induce calcium mobilization in either human mast cells (52) or human monocytes (53), all properties that have been demonstrated by the intact molecule. Because the C-terminal arginine is rapidly cleaved by serum carboxypeptidase N to convert C3a to C3adesArg, this enzymatic process has been considered a major mechanism for controlling C3a function in vivo (51). However, recent studies have reported biological activities for C3adesArg, including inhibition of cytotoxicity by human NK cells (54), induction of histamine release from rat peritoneal mast cells (55), regulation of TNF-α and IL-1 in human PBMC (15), and regulation of B cell function (17). Both C3a and C3adesArg are highly cationic molecules that can bind to anionic components on the cell membrane, leading to nonspecific cell activation (55). This nonspecific effect, which depends on the net charge of the molecules, could explain why C3adesArg and C3a share some biological activities. There is also evidence that C3a (and possibly C3adesArg) binds to the β-chain of the Fcε receptor on mast cells (56). Similar Fc interactions on monocytes could influence the signaling induced by LPS. However, the question remains as to why C3adesArg activity is observed in some, but not all, cell types responding to C3a. A possible explanation would be differences in the cell surface of these cell types. An even more speculative possibility would be that there is a second receptor for C3a and/or C3adesArg. Two groups who have cloned the C3aR found a second band in their Northern blots supporting this possibility (33, 42). Our findings that a synthetic peptide analogue of C3a mimics the effects of C3a and C3adesArg and that both the effects of C3a and C3adesArg are inhibited by PTX support a receptor-mediated signaling pathway. Nevertheless, more studies are necessary to identify receptor vs nonreceptor mechanisms of cellular activation by C3a. Regardless of the mechanism, the enhancing effect of C3a/C3adesArg on IL-6 production in LPS- and IL-1β-stimulated PBMCs is readily demonstrated.

In conclusion, by influencing IL-6 production in B cells (17) and PBMCs, C3a and C3adesArg may contribute to the regulation of both immune responses and inflammation. To our knowledge, this is the first report showing a biological function of C3a or C3adesArg that is associated with increased binding activities of the transcription factors NF-κB and AP-1.

We thank Philippe Pfeifer for purification of the C3a, and Alicia Palestini for her help in preparing this manuscript.

1

This work was supported in part by National Institutes of Health Grants DE10992 and AI41670, and by Deutsche Forschungsgemeinschaft (to W.H.F.). Blood drawing was performed by the General Clinical Research Center facility supported by National Institute of Health Grant M01RR00833. This is Publication 11653-IMM from the Department of Immunology, The Scripps Research Institute.

5

Abbreviations used in this paper: EMSA, electrophoretic mobility shift assay; PTX, pertussis toxin; AP-1, activating protein-1.

1
Weinberg, P. F., M. A. Matthay, O. Webster, K. V. Roskos, I. M. Goldstein, J. F. Murray.
1984
. Biologically active products of complement and acute lung injury in patients with sepsis syndrome.
Am. Rev. Respir. Dis.
130
:
791
2
Bengston, A., M. Heideman.
1988
. Anaphylatoxin formation in sepsis.
Arch. Surg.
123
:
645
3
Heideman, M., T. E. Hugli.
1984
. Anaphylatoxin generation in multisystem organ failure.
J. Trauma
24
:
1038
4
Zilow, G., T. Joka, U. Obertacke, U. Rother, M. Kirschfink.
1992
. Generation of anaphylatoxin C3a in plasma and bronchoalveolar lavage fluid in trauma patients at risk for adult respiratory distress syndrome.
Crit. Care Med.
20
:
468
5
Belmont, H. M., P. Hopkins, H. S. Edelson, H. B. Kaplan, R. Ludewig, G. Weissmann, S. Abramson.
1986
. Complement activation during systemic lupus erythematosus: C3a and C5a anaphylatoxins circulate during exacerbations of disease.
Arthritis Rheum.
29
:
1085
6
Hugli, T. E..
1981
. The structural basis for anaphylatoxin and chemotactic functions of C3a, C4a, and C5a.
Crit. Rev. Immunol.
1
:
321
7
Hugli, T. E..
1989
. Structure and function of C3a anaphylatoxin.
Curr. Top. Microbiol. Immunol.
153
:
181
8
Köhl, J., D. Bitter-Suermann.
1993
. Anaphylatoxins. K. Whaley, and M. Loos, and J. M. Weiler, eds.
Complement in Health and Disease
299
-324. Kluwer Academic Publishers, Lancaster.
9
Goldstein, I. M. 1992. Complement: biologically active products. In Inflammation: Basic Principles and Clinical Correlates. J. I. Gallin, I. M. Goldstein, and R. Snyderman, eds. Raven Press, Ltd. New York.
10
Goodman, M. G., D. E. Chenoweth, W. O. Weigle.
1982
. Induction of interleukin-1 secretion and enhancement of humoral immunity by binding of C5a to macrophage surface C5a receptors.
J. Exp. Med.
156
:
912
11
Scholz, W., M. R. McClurg, G. J. Cardenas, M. Smith, D. J. Noonan, T. E. Hugli, E. L. Morgan.
1990
. C5a-mediated release of interleukin-6 by human monocytes.
Clin. Immunol. Immunopathol.
57
:
297
12
Schindler, R., J. A. Gelfand, C. A. Dinarello.
1990
. Recombinant C5a stimulates transcription rather than translation of IL-1 and TNF: translational signal provided by LPS or IL-1 itself.
Blood
76
:
1631
13
Daffern, P. J., P. H. Pfeifer, J. A. Ember, T. E. Hugli.
1995
. C3a is a chemotaxin for human eosinophils but not for neutrophils. I. C3a stimulation of neutrophils is secondary to eosinophil activation.
J. Exp. Med.
181
:
2119
14
Morgan, E. L..
1987
. The role of prostaglandins in C3a-mediated suppression of human in vitro polyclonal antibody responses.
Clin. Immunol. Immunopathol.
44
:
1
15
Takabayashi, T., E. Vannier, B. D. Clark, N. H. Margolis, C. A. Dinarello, J. F. Burke, J. A. Gelfand.
1996
. A new biologic role for C3a and C3a desArg: regulation of TNF-alpha and IL-1beta synthesis.
J. Immunol.
156
:
3455
16
Nilsson, G., M. Johnell, C. H. Hammer, H. L. Tiffany, K. Nilsson, D. D. Metcalfe, A. Siegbahn, P. M. Murphy.
1996
. C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway.
J. Immunol.
157
:
1693
17
Fischer, W. H., T. E. Hugli.
1997
. Regulation of B cell function by C3a and C3adesArg: suppression of TNF-α, IL-6, and the polyclonal immune response.
J. Immunol.
159
:
4279
18
Van Damme, J., S. Cayphas, G. Opdenakker, A. Billiau, J. Van Snick.
1987
. Interleukin-1 and poly(rI):poly(rC) induce production of a hybridoma growth factor by human fibroblasts.
Eur. J. Immunol.
17
:
1
19
Sironi, M., F. Breviario, P. Proserpio, A. Biondi, A. Vecchi, J. Van Damme, E. Dejana, A. Mantovani.
1989
. IL-1 stimulates IL-6 production in endothelial cells.
J. Immunol.
142
:
549
20
Jablons, D. M., J. J. Mule, J. K. Macintosh, P. B. Sehgal, L. T. May, C. M. Huang, S. A. Rosenberg, M. T. Lotze.
1989
. IL-6/IFN-β2 as a circulating hormone: induction by cytokine administration in humans.
J. Immunol.
142
:
1542
21
Kohase, M., D. Henrikson-DeStefano, L. T. May, J. Vileck, P. B. Sehgal.
1986
. Induction of β2-interferon by tumor necrosis factor: a homeostatic mechanism in the control of cell proliferation.
Cell
45
:
659
22
Le, J., J. Vilcek.
1989
. Interleukin-6: a multifunctional cytokine regulating immune reaction and the acute phase protein response.
Lab. Invest.
61
:
588
23
Van Snick, J..
1990
. Interleukin-6: An overview.
Annu. Rev. Immunol.
8
:
253
24
Scholz, W..
1996
. Interleukin-6 in diseases: cause or cure?.
Immunopharmacology
31
:
131
25
Paquet, P., G. E. Pierard.
1996
. Interleukin-6 and the skin.
Int. Arch. Allergy Immunol.
109
:
308
26
Ember, J. A., S. D. Sanderson, T. E. Hugli, E. L. Morgan.
1994
. Induction of Interleukin-8 synthesis from monocytes by human C5a anaphylatoxin.
Am. J. Pathol.
144
:
393
27
Hugli, T. E., C. Gerard, M. Kawahara, M. E. Scheetz, R. Barton, S. Briggs, G. Koppel, S. Russell.
1981
. Isolation of three separate anaphylatoxins from complement-activated human serum.
Mol. Cell. Biochem.
41
:
59
28
Dignam, J. P., R. M. Lebovitz, R. G. Roeder.
1983
. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11
:
1475
29
Kravchenko, V. V., Z. Pan, J. Han, J.-M. Herbert, R. J. Ulevitch, R. D. Ye.
1995
. Platelet-activating factor induces NF-κB activation through a G protein-coupled pathway.
J. Biol. Chem.
270
:
14928
30
Ember, J. A., N. L. Johansen, T. E. Hugli.
1991
. Designing synthetic superagonist of C3a anaphylatoxin.
Biochemistry
30
:
3603
31
Knudsen, P. J., C. A. Dinarello, T. B. Strom.
1986
. Prostaglandins posttranscriptionally inhibit monocyte expression of interleukin 1 activity by increasing intracellular cyclic adenosine monophosphate.
J. Immunol.
137
:
3189
32
Ames, R. S., H. M. Sarau, P. Nuthulaganti, J. J. Foley, C. Ellis, Z. Zeng, K. Su, A. J. Jurewicz, R. P. Hertzberg, D. J. Bergsma, et al
1996
. Molecular cloning and characterization of the human anaphylatoxin C3a receptor.
J. Biol. Chem.
271
:
20231
33
Crass, T., U. Raffetseder, U. Martin, M. Grove, A. Klos, J. Köhl, W. Bautsch.
1996
. Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells.
Eur. J. Immunol.
26
:
1944
34
Shimizu, H., T. Kishimoto.
1990
. Involvement of a NF-κB-like transcription factor in the activation of the interleukin-6 gene by inflammatory lymphokines.
Mol. Cell. Biol.
10
:
561
35
Libermann, T. A., D. Baltimore.
1990
. Activation of interleukin-6 gene expression through the NF-κB transcription factor.
Mol. Cell. Biol.
10
:
2327
36
Haegeman, G., J. Content, G. Volkaert, R. Derynck, J. Tavernier, W. Fiers.
1986
. Structural analysis of the sequence encoding for an inducible 26-kDa protein in human fibroblasts.
Eur. J. Biochem.
159
:
625
37
Haeffner-Cavaillon, N., J. M. Cavaillon, M. Laude, M. D. Kazatchkine.
1987
. C3a (C3ades-Arg) induces production and release of interleukin 1 by cultured human monocytes.
J. Immunol.
139
:
794
38
Arend, W. R., R. J. Massoni, M. A. Niemann, P. C. Giclas.
1989
. Absence of induction of IL-1 production in human monocytes by complement fragments.
J. Immunol.
142
:
173
39
Takabayashi, T., E. Vannier, J. F. Burke, R. G. Tompkins, J. A. Gelfand, B. D. Clark.
1998
. Both C3a and C3adesArg regulate Interleukin-6 synthesis in human peripheral blood mononuclear cells.
J. Infect. Dis.
177
:
1622
40
Fischer, M. B., M. Ma, S. Goerg, X. Zhou, J. Xia, O. Finco, S. Han, G. Kelsoe, R. G. Howard, T. L. Rothstein, et al
1996
. Regulation of the B cell response to T-dependent antigens by classical pathway complement.
J. Immunol.
157
:
549
41
Donnelly, R. P., L. J. Crofford, S. L. Freeman, J. Buras, E. Remmers, R. L. Wilder, M. J. Fenton.
1993
. Tissue-specific regulation of IL-6 production by IL-4: differential effects of IL-4 on nuclear factor-κB activity in monocytes and fibroblasts.
J. Immunol.
151
:
5603
42
Roglic, A., E. R. Prossnitz, S. L. Cavanagh, Z. Pan, A. Zou, R. D. Ye.
1996
. cDNA cloning of a novel G protein-coupled receptor with a large extracellular loop structure.
Biochim. Biophys. Acta
1305
:
39
43
Baeuerle, P. A., T. Henkel.
1994
. Function and activation of NF-κB in the immune system.
Annu. Rev. Immunol.
12
:
141
44
Baeuerle, P. A., D. Baltimore.
1996
. NF-κB: ten years after.
Cell
87
:
13
45
Sen, R., D. Baltimore.
1986
. Multiple nuclear factors interact with the immunglobulin enhancer sequences.
Cell
46
:
705
46
Curran, T., B. R. Franza.
1988
. Fos and Jun: the AP-1 connection.
Cell
55
:
395
47
Kang, S.-H., D. A. Brown, I. Kitajima, O. X. X., S. Heidenreich, S. Gryaznov, M. Nerenberg.
1996
. Binding and functional effects of transcriptional factor Sp1 on the murine interleukin-6 promotor.
J. Biol. Chem.
271
:
7330
48
Matsusaka, T., K. Fujikawa, Y. Nishio, N. Mukaida, K. Matsushima, T. Kishimoto, S. Akira.
1993
. Transcription factors NF-IL6 and NF-κB synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8.
Proc. Natl. Acad. Sci. USA
90
:
10193
49
LeClair, K. P., M. A. Blanar, P. A. Sharp.
1992
. The p50 subunit of NF-κB associates with the NF-IL6 transcription factor.
Proc. Natl. Acad. Sci. USA
89
:
8145
50
Fukuoka, Y., T. E. Hugli.
1988
. Demonstration of a specific C3a receptor on guinea pig platelets.
J. Immunol.
140
:
3496
51
Bokisch, V. A., H. J. Müller-Eberhard.
1970
. Anaphylatoxin inactivator of human plasma: its isolation and characterization as a carboxypeptidase.
J. Clin. Invest.
49
:
2427
52
Zwirner, J., O. Götze, A. Sieber, A. Kapp, G. Begemann, T. Zuberbier, T. Werfel.
1998
. The human mast cell line HMC-1 binds and responds to C3a but not C3a(desArg).
Scand. J. Immunol.
47
:
19
53
Zwirner, J., O. Götze, A. Mosier, A. Sieber, A. Kapp, G. Begemann, J. Elsner, T. Werfel.
1997
. Blood- and skin-derived monocytes/macrophages respond to C3a but not to C3a(desArg) with a transient release of calcium via a pertussis toxin-sensitive signal transduction pathway.
Eur. J. Immunol.
27
:
2317
54
Charriaut, C., A. Senik, J.-P. Kolb, M. Barel, R. Frade.
1982
. Inhibition of in vitro natural killer activity by the third component of complement: role for the C3a fragment.
Proc. Natl. Acad. Sci. USA
79
:
6003
55
Mousli, M., T. E. Hugli, Y. Landry, C. Bronner.
1992
. A mechanism of action for anaphylatoxin C3a stimulation of mast cells.
J. Immunol.
148
:
2456
56
Erdei, A., I. Pecht.
1996
. Complement peptides and mast cell triggering.
Immunol. Lett.
54
:
109