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* Institute of Medical Microbiology and
Department of Clinical Immunology, Medical School Hannover, Hannover, Germany;
Division of Molecular Immunology, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229;
Department of Immunology, Georg August University Goettingen, Goettingen, Germany;
¶ Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands;
|| Austin Research Institute, Austin and Repatriation Medical Center, Heidelberg, Victoria, Australia;
# Ina Sue Perlmutter Laboratory, Children’s Hospital, Harvard Medical School, Boston, MA 02115; and
** Department of Cell Biology and Immunology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R. In this study, we demonstrate that activation of the complement system, specifically generation of C5a, initiates the neutrophilic inflammation in IC peritonitis. We show that ablation of C5a receptor signaling abrogates neutrophil recruitment in wild-type mice and prevents the enhancement of neutrophil migration seen in FcRIIB–/– mice, suggesting that C5aR signaling is the crucial initial event upstream of FcR signaling. We also provide evidence that C5a initiates the inflammatory cascade both directly, through C5aR-mediated effector functions on infiltrating and resident peritoneal cells, and indirectly, through shifting the balance between activating and inhibitory FcRs on resident cells toward an inflammatory phenotype. We conclude that complement activation and C5a generation are prerequisites for IC-induced inflammation through activating FcR, which amplifies complement-induced inflammation in autoimmunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R and a variety of complement receptors. Both classes of receptors have been implicated in immune adherence of opsonized particles, phagocytosis, IC clearance, and signal transduction. When clearance mechanisms are overwhelmed, IC can become an important cause of tissue damage (2). In such settings, IC can lead to the generation of destructive proinflammatory processes marked by the chemotaxis and activation of myeloid cells at sites of IC deposition. In the prototypic experimental model of soluble IC disease, the Arthus reaction, IC activation of local resident cells results in edema, hemorrhage, and neutrophil infiltration. Analysis of the experimental Arthus reaction in various tissues has provided considerable insight into the mechanisms underlying IC-mediated inflammation (3, 4).
Data obtained from the Arthus model indicate that IC-mediated activation of FcR is essential to the neutrophilic inflammatory response (5, 6, 7). Mice have three different FcR: 1) a high affinity activating receptor, FcRI (FcR type I for IgG (CD64)), expressed by monocytes, macrophages (M
), and dendritic cells; 2) a low affinity inhibitory receptor, FcRIIB, with a broad distribution pattern; and 3) a low affinity activating receptor, FcRIII (FcR type III for IgG (CD16)), expressed by M, dendritic cells, neutrophils, mast cells (MC), and NK cells. The aggregation of activating FcRs (FcRI and FcRIII) induces proinflammatory effects through an activating ITAM motif, whereas aggregation of activating and inhibitory FcR (FcRIIB) inhibits such effects through an ITIM motif. In the Arthus model, a strict requirement for activating FcRs has been found along with clear evidence of regulatory control by inhibitory FcRIIB (5, 6, 7, 8). In fact, FcRIIB–/– mice show a strikingly enhancement of cutaneous and pulmonary Arthus reactions (8).
Activation of the complement system also contributes significantly to IC-induced inflammation. Complement activation by IC generates IC-bound ligands for complement receptors such as CR1 and CR3, along with C3a and C5a, which are potent chemoattractants for myeloid cells and also up-regulate the production of a variety of proinflammatory mediators (9). C3a and C5a act through G protein-coupled receptors, C3aR (10, 11) and C5aR (CD88), respectively (12, 13), expressed by a variety of cells, including M, neutrophils, and MCs, all important effectors in the Arthus model. Recently, the orphan receptor C5L2 has been described as a second receptor for C5a (14, 15, 16). In contrast to CD88, C5L2 is uncoupled from G proteins. C5L2 ligation does not result in degranulation, increased intracellular Ca2+, or receptor internalization, suggesting that C5L2 signaling does not follow the classical pathways of chemoattractant receptors. Using complement-deficient mice (C3–/– or C5aR–/–) or C5aR antagonists, we have found that complement activation contributes significantly to neutrophil recruitment, edema formation, and hemorrhage in different Arthus models (7, 17, 18, 19, 20, 21, 22, 23, 24).
Although it is clear that IC initiate the inflammatory response in the Arthus setting, the network of downstream events is poorly understood. In particular, the receptor pathways activated in response to complement activation and the relative roles of complement and FcR remain unclear. The ablation of IC-mediated inflammation in mice that lack the ability to signal through activating FcRs and the lack of an effect of genetic deficiencies in complement factors on such inflammation suggested that FcR initiate the inflammatory cascade (reviewed in Ref. 25). In this study, we show that blocking the C5aR suppresses neutrophilic inflammation in wild-type mice and ablates the up-regulation of inflammation seen in FcRIIB–/– mice, strongly suggesting that 1) C5aR signaling is the dominant complement pathway mediating the inflammatory response; and 2) FcR activation is downstream of complement activation. Further, we show that C5aR signaling modulates the balance between activating and inhibitory FcR, providing a plausible mechanism for the crucial role of C5a in initiating the inflammatory response. Finally, we demonstrate that CXC chemokine release from peritoneal cells is a critical downstream mechanism of C5aR signaling, which is amplified by the interaction of IC with FcR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free female BALB/c, FcRIIB–/–, FcR -chain–/– (all on a BALB/c background), WBB6F-1 KitW/W-v, and WBB6-F1-Kit+/+ mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions. FcRIII–/– mice (on the C57BL/6 background) were purchased from The Jackson Laboratory and backcrossed to the BALB/c background (six generations). FcRI–/– (26) and C5aR–/– (27) mice were generated as described and were backcrossed to the BALB/c background (six generations). All mice were used at 8–10 wk of age. Animal care was provided in accordance with National Institutes of Health guidelines. Animal studies were approved by either the Bezirksregierung Hannover (Hannover, Germany) or the Cincinnati Children’s Hospital Medical Center (Cincinnati, OH) institutional animal care and use committee.
Peritoneal reverse passive Arthus reaction
OVA (20 mg/kg body weight; Sigma-Aldrich, Munich, Germany) was injected i.v., followed by i.p. injection of IgG rich in Ab to OVA (800 µg/mouse; ICN, Costa Mesa, CA) exactly as previously described (20). Mice were killed at the indicated time points after injury, and the peritoneal cavity was lavaged with 2 ml of ice-cold PBS and 0.1% BSA. Cytospin slides (200 µl) were prepared from lavage fluid and stained using Diff-Quick (Baxter Merz & Dade, Dudingen, Switzerland). PMN numbers per microscopic field were counted (
20 different fields). To block the C5aR and C5L2 in BALB/c mice, animals were treated with the C5aRA A8
71–73 (28) (10–5 M, 200 µl i.v.); to exclusively block the C5aR (CD88), Fab of the neutralizing anti-C5aR mAb 20/70 (29) (2 x 10–6M, 200 µl i.v.) were administered 10 min (A871–73) or 30 min (Fab 20/70) before the initiation of IC peritonitis and 2 h after IC challenge. To block the C3aR, the C3a receptor antagonist (C3aRA) N2-[(2,2-diphenylethoxy)-acetyl]-L-arginine (SB290157) (30) was given once (500 µg dissolved 200 µl of PBS and 0.5% DMSO i.p.) 1 h before initiation of the Arthus reaction. Neutrophil numbers in controls receiving 200 µl of PBS and 0.5% DMSO were as high as those in Ab controls. To block the functional activity of cytokine-induced neutrophil chemoattractant (KC) and/or MIP, 100 µg each of anti-KC and/or anti-MIP-2 polyclonal Abs or of control polyclonal Abs (R&D Systems, Wiesbaden, Germany) were injected i.p. 90 min before initiation of the Arthus reaction.
Reduction of peritoneal M using clodronate
To selectively reduce the number of M, liposomes containing dichloromethylene-bisphosphonate (clodronate; gift from Roche, Mannheim, Germany) were used according to published procedures (31). In brief, mice were treated twice i.p. with 100 µl of clodronate preparation or with 100 µl of PBS (72-h interval). The number of M in peritoneal lavage fluid was determined 24, 48, 72, and 96 h after the last injection of clodronate or PBS by staining cytospins (55 x g, 10 min) of peritoneal lavage fluid with Turk’s solution. In addition, differential cell counts were determined by staining cytospins with Diff-Quick (Baxter Merz & Dade). We found >85% reduction of peritoneal M 24–96 h after the last injection of clodronate, but no reduction of peritoneal M in PBS-treated controls. The reduction of peritoneal M was confirmed by flow cytometry using PE-conjugated F4/80 mAb or an isotype-matched control. Samples were analyzed on a FACSCalibur flow cytometer and were evaluated using the CellQuest software (BD Biosciences, San Diego, CA). Profound neutrophil accumulation was found in clodronate-treated mice, which declined to zero during the next 3 days. Thus, IC challenge was performed 4 days after the last clodronate treatment.
Chemokine and cytokine ELISAs
Immunoreactive KC and MIP-2 in peritoneal lavage fluid were quantified using commercially available ELISA kits (R&D Systems) according to the manufacturer’s protocol. The detection limit of the assays was 15.6 pg/ml.
CXC chemokine release from elicited peritoneal M
BALB/c mice were injected i.p. with thioglycolate (3%, 2 ml). After 72 h, peritoneal cells were harvested from lavage fluid as previously described (20). Peritoneal cells were transferred into six-well plates and cultured overnight in RPMI 1640 medium to allow M to adhere to the plastic surface. After washing twice with PBS and 0.1% BSA, cells were incubated with three different concentrations of recombinant human C5a (rhC5a; 10–7, 5 x 10–7, and 10–6 M; Sigma-Aldrich, St. Louis, MO) or IC (10, 50, and 100 µg/ml OVA/anti-OVA polyclonal rabbit Ab). Soluble IC were formed by incubation of OVA with rabbit anti-OVA in a molar ratio of 1:4 (32). The precipitate was centrifuged and then resuspended in medium at the indicated concentration. No detectable LPS was found in the C5a and IC preparations as determined by a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD; <3 pg/ml).
Real-time RT-PCR for KC and MIP-2 gene transcription
RNA from thioglycolate-elicited peritoneal M (1 x 106) was prepared using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to manufacturer’s recommendations. cDNA was generated from 2 µg of DNase-treated total RNA, using Superscript II reverse transcriptase (Invitrogen Life Technologies) following the manufacturer’s protocol. Gene expression levels were determined using iQ-SYBR Green Supermix (Bio-Rad, Hercules, CA) containing 5 µl of cDNA (diluted 1/30) and 500 nM primer in a total volume of 50 µl. Samples were amplified by 40 cycles at 58°C on an iCycler Real-Time PCR System (Bio-Rad). For data analysis, expression levels were normalized to 
-actin expression. The following oligonucleotides were used: mouse KC (72-bp fragment): forward primer, 5'-TTCTCTGTGCAGCGCTGCTG-3'; reverse primer, 5' CGCAGCTCATTGGCGATAGG-3'; and mouse MIP-2 (87-bp fragment): forward primer, 5'-TCAGTGCTGCACAGTTCACTG-3'; reverse primer, 5'-CATTGACAGCGCAGTTCACTG-3'.
Expression of C5aR and FcR on peritoneal resident cells
To determine the impact of C5aR signaling on FcR expression, naive BALB/c mice were injected with rhC5a (5 x 10–8 M i.p.). Binding studies with peritoneal M and hC5aR transfected RBL-2H3 cells revealed indistinguishable binding affinities of rhC5a to murine CD88 and human CD88 (2.7 ± 0.2 x 10–9 M (28) vs 2.9. ± 0.1 x 10–9 M; data not shown). Two hours later, mice were killed, and the peritoneal cavity was lavaged with 2 ml of ice-cold PBS and 0.1% BSA. Receptor expression levels on peritoneal resident cells were determined by flow cytometry. Cells obtained from peritoneal lavage (1 x 106) were fixed with paraformaldehyde and stained with the following Abs or isotype controls (Immunotech, Westbrook, ME): anti-F4/80 (PE-conjugated; Serotec, Raleigh, NC), anti-FcRIIB (Ly17.2, clone K9.361 (33) Alexa 488-conjugated; provided by F. D. Finkelman, Cincinnati, OH); anti-FcRIIB/III (clone 2.4G2. Alexa 488-conjugated; BD Pharmingen, San Diego, CA); and biotinylated anti-FcRI (clone X54-5/7.1) (34). Binding of anti-FcRI mAb was detected using streptavidin-FITC (BD Pharmingen).
To determine C5aR expression on resident peritoneal M or resident peritoneal MCs, the following Abs or isotype controls (Immunotech) were used: anti-CD117 (c-Kit, clone 2B8, PE-conjugated; Pharmingen); anti-F4/80 (PE-conjugated; Serotec), and anti-C5aR (clone 20/70 (29), Alexa 488-conjugated). Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences), and data were evaluated using CellQuest Pro software.
Statistical analysis
Statistical analysis was performed using the SigmaStat version 2.0 statistical package (Jandel, Erkrath, Germany). All data are given as the mean ± SEM. First we tested for a normal distributed population using the Kolmogoroff-Smirnov test. To analyze differences between two normally distributed groups, an unpaired t test was used. Comparison of the means of more than two groups was performed using one-way ANOVA. When the mean values of the groups showed a significant difference, pairwise comparison was performed using Tukey’s test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used the well-established reverse passive Arthus reaction peritonitis model to assess the importance of C5aR, C5L2, and C3aR signaling to initiation of the inflammatory response in IC disease. As a readout, we determined the recruitment of neutrophils into the peritoneal cavity, the hallmark of the Arthus reaction. The naive peritoneum consists mainly of M, lymphocytes, and a small population of MCs. In the course of IC peritonitis, neutrophils accumulate in the peritoneal cavity and comprise 40–50% of the cells in the peritoneum 6 h after IC challenge (data not shown). To assess the impact of C5aR ligation on neutrophil attraction, we blocked the C5aR using Fab of the neutralizing anti-C5aR mAb 20/70 (29) and used C5aR–/– mice (27). As shown in Fig. 1a, neutrophil recruitment was abrogated. We have recently shown that C5aRA A871–73 blocks the interaction of C5a with CD88 and with the second C5aR, C5L2 (28). Administration of C5aRA A871–73 blocked neutrophil migration completely. To assess whether C3a contributes to neutrophil recruitment in IC peritonitis, e.g., through the activation of peritoneal resident cells, we blocked C3aR by a specific C3aRA (30). No reduction of PMN numbers occurred, arguing against an important role for this anaphylatoxin in IC peritonitis.
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R to neutrophil recruitment, we used mice with engineered deficiencies of 1) FcRI, 2) FcRIII, or 3) the FcR -chain, which leads to a combined functional deficiency of FcRI and FcRIII. These mice were backcrossed onto the BALB/c background. In FcRI–/– mice, neutrophil accumulation in the peritoneum was slightly (although significantly) reduced by 25% compared with BALB/c controls, whereas the reduction of neutrophils in FcRIII–/– mice was only 15% (and did not reach statistical significance; Fig. 1b). However, neutrophil trafficking was abolished in FcR -chain–/– mice, confirming our previous results (obtained with FcR -chain–/– mice on the C57BL/6 background) (20). The fact that neither the absence of FcRIII nor the absence of FcRI (Fig. 1b) reduced neutrophil recruitment substantially (in contrast to FcR -chain–/– mice) strongly suggests a codominant role for activating FcRI and FcRIII in neutrophil recruitment.
To assess a possible interrelationship between C5aR and FcR signaling, we investigated whether C5a affects FcR-induced neutrophil recruitment. Deficiency of FcRIIB resulted in a strikingly enhanced accumulation of neutrophils during IC peritonitis (Fig. 1c). Neutrophil numbers were twice as high as in BALB/c controls. Notably, blocking of C5aR signaling prevented the increase in neutrophil migration into the peritoneum of FcRIIB–/– mice. These data strongly suggest that C5a acts upstream of FcR signaling and that C5aR signaling is a prerequisite for FcR-mediated inflammatory responses.
C5aR signaling on peritoneal M regulates the expression of FcR
In a search for mechanisms that account for this crucial role of C5a, we hypothesized that C5a up-regulates the expression of activating FcR on peritoneal resident cells. To determine whether this is the case, we determined the impact of C5a challenge on the expression of activating FcRs (FcRI and FcRIII) on peritoneal M. Furthermore, we tested whether C5a down-regulates inhibitory FcRIIB. In fact, the expression of activating FcRs was up-regulated, whereas that of FcRIIB was down-regulated (Fig. 2) 2 h after i.p injection of C5a into BALB/c mice.
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and MCs contribute to PMN recruitment
Our finding that FcRI contributes to PMN recruitment led us to hypothesize that C5aR and FcR signaling in peritoneal resident cells is important for triggering neutrophil recruitment in IC peritonitis, because murine neutrophils do not express FcRI (34). We assessed the contribution of peritoneal M by depleting these cells using the well-established method of clodronate-induced apoptosis (31). To assess the importance of MCs, we used the MC-deficient mouse strain KitW/W-v along with MC-competent controls (Kit+/+). In Kit+/+ mice, neutrophil numbers increased substantially between 2 and 6 h after IC challenge. At 6 h, neutrophils comprised 40–50% of the cells in the peritoneum. Thereafter, neutrophil numbers declined over the next 24 h (Fig. 3a). Either genetic deficiency of MCs (KitW/W-v), or pharmacological depletion of M significantly inhibited neutrophil recruitment 4 and 6 h after IC challenge (Fig. 3, a and b). However, the kinetics of inhibition were different. In MC-deficient KitW/W-v mice, neutrophil influx was virtually abrogated during the first 4 h, but increased with a slope similar to that in Kit+/+ mice between 4 and 6 h. After depletion of M, neutrophils migrated almost normally during the first 4 h; however, their trafficking was essentially blocked between 4 and 6 h. These data suggest that MCs contribute mainly to early neutrophil recruitment, whereas M are needed to mediate neutrophil trafficking between 4 and 6 h after IC challenge. To ensure that the crucial effect of C5aR blocking is not restricted to the BALB/c background, we blocked C5aR signaling in Kit+/+ and KitW/W-v mice. In both cases, we found neutrophil migration to be abolished (Fig. 3b).
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and peritoneal MCs. Although some reports have suggested the presence of C5aR expression on murine MCs (35), the expression of C5aR on resident peritoneal MCs has not yet been determined. As shown in Fig. 3C, peritoneal-derived M and MCs clearly express the C5aR. Of the M population (F4/80+ cells), 98–99% of cells are C5aR+, whereas 80–85% of the MC population (c-Kit+; 2% of total cells) express C5aR. Thus, resident peritoneal cells express activating FcR (36, 37) as well as C5aR. CXC chemokines KC and MIP-2 are important secondary mediators of neutrophil recruitment
To begin to investigate the mechanism by which resident cells contribute to peritoneal recruitment of neutrophils, we determined the release of chemoattractant chemokines into the peritoneum during IC peritonitis. We focused on the CXC chemokines KC and MIP-2. Neither of the chemokines was detected during the first 10 min after IC challenge (Fig. 4), suggesting that these mediators are not released from preformed stores. Measurable amounts of KC and MIP-2 appeared after 1 h. Between 1 and 2.5 h, KC and MIP-2 concentrations continued to rise, thereafter declining to reach baseline levels after 6 h. In the absence of MCs, KC and MIP-2 levels started to increase later (at 2 h), reached only 30% of the maximum in MC-competent animals, and had returned to baseline levels after 4 h (Fig. 4, a and b). These data suggest that MCs contribute significantly to the early release of KC and MIP-2. M depletion did not affect KC release (Fig. 4a). However, it diminished the release of MIP-2 similar to that seen in the absence of MCs (Fig. 4b), indicating that M contribute significantly to MIP-2, but not to KC, release.
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and MCs contribute significantly to neutrophil recruitment during IC peritonitis by mechanisms that involve CXC chemokines.
C5aR signaling and FcR signaling in resident cells is critical to induce CXC chemokines
Our data suggest that MCs and M are an important source of KC and MIP-2 in IC peritonitis (Fig. 4). As we found C5aR expression on both cell types, we wondered whether C5aR signaling in these cells is an important trigger for CXC chemokine production and release. C5aR blockade decreased KC and MIP-2 concentrations by 66% 2.5 h after IC challenge (Fig. 5a). Of note, KC and MIP-2 levels did not increase at later time points, ruling out the possibility that C5aR blockade results in a delay of CXC chemokine release. Although these data provide no direct evidence, they strongly suggest that C5aR ligation on resident cells contributes to CXC chemokine release. Further, the data indicate that signaling through C5aR on MCs triggers the release of KC, as depletion of MCs, but not of M, impairs the release of KC (Fig. 5a, left panel). Finally, the release of MIP-2 is most likely to depend on C5aR ligation on MCs and M, because both cell populations contribute to MIP-2 secretion (Fig. 5a, right panel).
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R to KC and MIP-2 release. KC and MIP-2 levels in FcR -chain–/– mice were reduced by 60% compared with those in BALB/c mice (Fig. 5b). However, KC and MIP-2 levels in FcRI–/– or FcRIII–/– mice were unchanged. These data suggest a significant role for FcRI and FcRIIII in CXC chemokine release. To ascertain that the chemokine release in Kit+/+ mice (Fig. 4) and BALB/c mice is comparable, we assessed the kinetics and the concentrations of KC and MIP-2 in BALB/c mice. We found that they were almost identical with those determined in Kit+/+ mice (data not shown).
Cross-talk between C5aR and FcR signaling amplifies effector functions in peritoneal M
To assess whether the induction of CXC chemokine release through C5a in vivo results from direct C5aR signaling on peritoneal resident cells, thioglycolate-elicited peritoneal M were stimulated with different concentrations of C5a. We found dose- and time-dependent production of MIP-2 (Fig. 6, a and b) and KC (similar to MIP-2; data not shown). To assess whether C5a regulates the production of the chemokines at the transcriptional level, we performed real-time RT-PCR. We found some mRNA induction as early as 15 min after C5a stimulation, which strongly increased over time. Two hours (MIP-2) or 1 h (KC) after C5a challenge, mRNA transcripts were 35.4 ± 4.2-fold (MIP-2) or 30.3 ± 3.8-fold (KC) higher compared with medium controls (Fig. 6c). These data provide evidence that C5a induces de novo synthesis of KC and MIP-2 from peritoneal M and regulates the production of these chemokines at the mRNA level.
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. Similar to C5a, we found a dose- and time-dependent induction of both chemokines (Fig. 6, a and b). Finally, we stimulated peritoneal M with a combination of C5a and IC. As shown in Fig. 6a, MIP-2 and KC release increased substantially. In fact, it exceeded by far the sum of the amounts induced by C5aR or FcR ligation alone. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs, and neutrophils. It is obvious that ICs initiate the reaction. However, our knowledge about the importance of the complement system and of FcRs is still sketchy, as is our understanding of the mechanisms downstream of complement and FcR activation. Studies in different models of IC disease, such as anti-glucose-6-phosphate isomerase arthritis (1), autoimmune vitiligo (38), autoimmune hemolytic anemia (39), as well as cutaneous and pulmonary Arthus reactions (7, 19, 20, 21, 22, 23, 24), suggest codominant and redundant roles for the complement system and FcRs in IC inflammation.
Critical importance of complement and both activating FcRs in neutrophil recruitment
All available data suggest that the main contribution of complement to IC inflammation is through C5aR (CD88) signaling (4). However, recent findings that a second C5aR, C5L2, provides high affinity binding sites for C5a and its degradation product, C5adesArg (15), raises the possibility that C5L2 signaling accounts for some of the C5a effects. In particular, blocking or deletion of C5aR (CD88) does not rule out that the reduced inflammatory response results from inhibitory signaling through C5L2 (similar to the inhibitory effect of FcRIIB on activating FcRs). Our findings that ablation of C5aR signaling as well as ablation of C5aR- and C5L2-signaling abrogate neutrophil recruitment argue against such an inhibitory role of C5L2.
C3a is not a chemoattractant for neutrophils. However, it is a powerful chemoattractant and activator of MCs and M, suggesting a pathogenetic role for C3aR signaling in IC disease. In fact, C3a was shown to attract neutrophils through such indirect pathways (40). However, blocking of the C3aR pathway had no impact on neutrophil migration, suggesting that C3aR signaling plays no or at best a redundant role in the pathogenesis of IC peritonitis.
In most models of IC disease, signaling through FcRIII is critical for the inflammatory phenotype (1, 7, 23). Studies with FcRI–/– mice revealed a critical role for FcRI in inflammation and hypersensitivity (26, 41). A role for FcRI in IC peritonitis was suggested by comparison of two mouse strains: 1) mice with a combined functional deficiency of FcRI and FcRIII (lacking the common -chain), and 2) FcRIII–/– mice (20). Data from this study suggested a dominant role for FcRI and only a minor role for FcRIII in IC peritonitis. However, because the -chain is not exclusively used to transmit signals downstream of activating FcRs, but for many other receptors as well (e.g., activating isoforms of the paired Ig receptor (42), and certain subsets of Ig-like transcripts (43)), these data do not necessarily reflect the role of FcRI. Further, codominant effects of both activating FcRs may not be adequately addressed by this approach. Comparing the impacts of deletion of FcRI, FcRIII, and FcR -chain on neutrophil recruitment, we found that deletion of either activating FcR had only a minor effect on neutrophil attraction; however, deletion of the -chain abrogated neutrophil migration. In contrast to other IC disease models (1, 7), these data suggest a codominant role for both activating FcRs in the inflammatory response, although we cannot rule out at this point that the activation of other receptors, the signaling of which is -chain dependent, may account for this effect as well.
Complement activation is a crucial event upstream of FcR activation
Much of the data available from experimental IC disease support a model in which C5aR signaling and FcR signaling form a network that orchestrates the inflammatory phenotype (1, 7, 19, 20, 21, 22, 23, 24, 38, 39). In contrast, some data suggest an exclusive role for FcR (5, 6, 8, 44), which has led to the imputation of distinct roles for complement and FcR in autoimmunity and infection (25). Our recent data (24) along with that from the current study may help resolve this apparent discrepancy. In fact, we found a direct regulatory effect of C5aR signaling on the balance of activating (FcRIII) and inhibitory (FcRIIB) FcR expression in IC alveolitis (24). C5a up-regulates FcRIII and down-regulates FcRIIB on alveolar M, thereby reducing the threshold for FcR activation. In this study, we show that the same regulatory pathway is present in peritoneal M, and that C5aR signaling up-regulates the expression of FcRI as well. Thus, C5a modulates the entire FcR network, suggesting a general mechanism in IC disease. Although other factors have been described that regulate the balance between activating FcRs (such as TNF-
and IFN-) and inhibitory FcRs (IL-4), they play no role in the initial events leading to IC-induced inflammation. As we have shown previously (20), TNF- blockade has no impact on PMN recruitment. Further, we found no IFN- or IL-4 in peritoneal lavage samples taken during the first 6 h after IC challenge (our unpublished observations). In further support of a critical role for C5a in regulating FcR expression, we found that C5aR blockade in FcRII–/– mice (which have a markedly increased inflammatory phenotype) reduced neutrophil recruitment to the level in wild-type mice. Together these data suggest a model in which C5a acts upstream of FcR activation by setting the threshold for FcR activation in IC disease (Fig. 7). This model integrates the findings that the inflammatory phenotype in anti-glucose-6-phosphate isomerase arthritis (1), autoimmune vitiligo (38), autoimmune hemolytic anemia (39), and the various models of the Arthus reaction (4) depend on both C5aR and FcR signaling. In a broader sense, data that link the development of many autoimmune disease to impaired FcR regulation and function (for review, see Ref. 45), such as Goodpasture’s syndrome, autoimmune arthritis, and systemic lupus erythematosus, may be integrated by including complement activation into the network of FcR regulation.
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R signaling: resident peritoneal cells are important
The pathways downstream of C5aR and FcR signaling are poorly understood, as are the qualitative and quantitative contributions of resident tissue cells, such as MCs and M. Depletion of MCs results in significant reduction of the inflammatory response in Arthus reaction models (46, 47, 48, 49). In the absence of MCs, neutrophil recruitment is delayed, and the maximum response is only 50% of that in controls (Ref. 50 and this study). The pathways that activate MCs are incompletely understood. Experiments in which MCs from FcR -chain-deficient mice were adoptively transferred to wild-type mice indicated a crucial role for FcRIII in a cutaneous Arthus model (37). Depletion of complement by cobra venom factor or genetic ablation of C5 prevents degranulation of peritoneal MCs, suggesting a mechanistic role for C5 (35). However, C5aR expression has only been described on distinct subtypes of MCs, including skin MCs (51), a subfraction of cardiac MCs (52), synovial MCs in rheumatoid arthritis (53), and neoplastic MCs in patients with MC neoplasms (and the human MC line HMC-1) (52). By contrast, no expression of C5aR was found on lung MCs or MCs in other visceral organs. In this study, we demonstrate C5aR expression on resident peritoneal MCs. These data along with our finding that specific blockade of the C5aR reduces KC release in Kit+/+ mice to the same extent as MC depletion (in KitW/W-v) and our data showing that KC release is critically dependent on the presence of MCs suggest an important role for C5aR signaling in MC activation in vivo (see below).
The remaining neutrophil migration seen in the absence of MCs points toward another cell type contributing to neutrophil migration. The codominant roles of FcRI and FcRIII in IC peritonitis imply a significant contribution of tissue M in neutrophil attraction. In support of this view, we found a significant reduction of neutrophil recruitment after M depletion. Although previous studies (19, 20, 21) suggested a significant role for tissue M, we now provide evidence that this is indeed the case. Of note, M depletion had a negative impact on neutrophil elicitation that became evident only >4 h after IC challenge, thus highlighting the important role of M in late (>4 h) neutrophil recruitment.
Downstream of C5aR and FcR signaling: CXC chemokines are critical
Ligation of C5aR and activating FcRs results in pleiotropic proinflammatory effector functions, including the release of cytokines and chemokines (3, 9). Of particular interest are the CXC chemokines that harbor the conserved glutamic acid-leucine-arginine tripeptide motif, such as KC and MIP-2. These chemokines bind to CXCR2, which belongs to the group of chemoattractant, G protein-coupled receptors present on neutrophils (54), MCs (55), and M (56). Substantial release of these chemokines has been demonstrated in mouse (23, 24) and rat (32) models of IC alveolitis, something that was markedly reduced in the absence of C5aR signaling (24, 32). In line with these data, we found huge local accumulation of KC and MIP-2 in IC peritonitis that was reduced by C5aR blockade. These in vivo data were mirrored by in vitro data showing that C5a is a strong, FcR-independent inductor of KC and MIP-2 release from peritoneal M (in contrast to alveolar M (23)). Furthermore, we found that both activating FcRs (FcRI and FcRIII) promote the synthesis of MIP-2 and KC in vivo and in vitro, matching our data for neutrophil recruitment. Again, these data are in contrast to IC alveolitis, in which FcRIII was found to be crucial for MIP-2 release. Only when both chemokines were blocked in vivo did neutrophil migration decrease substantially, suggesting that a certain amount of CXCR2 triggering is sufficient to mediate efficient neutrophil migration. However, because neutrophil recruitment was affected by, at best, 50%, other CXCR2 ligands (e.g., LPS-inducible CXX chemokine) may contribute to neutrophil migration as well. Consequently, the contribution of CXCR2 signaling to neutrophil trafficking may be underestimated. Importantly, combined activation of C5aR and FcR synergized in promoting MIP-2 and KC release from peritoneal M in vitro. This synergism may result from C5a-mediated reduction of the threshold for FcR activation and/or a positive feedback between C5aR signaling and signaling pathways of activating FcRs. Together our data demonstrate that CXCR2 signaling is a critical effector mechanism downstream of C5aR/FcR signaling. Furthermore, our data provide a mechanistic link between C5aR/FcR signaling, M activation, CXC chemokine release, and neutrophil recruitment.
In summary, we propose a model in which C5a acts upstream of FcRs, initiating the inflammatory response by acting as a chemoattractant for neutrophils and by activating local MCs and M to release CXC chemokines. In this model, C5a functions as the dominant regulator of FcR expression decreasing the threshold for FcR activation (Fig. 7). This regulation is the prerequisite for adequate inflammatory function of the FcR system, because it shifts FcR signaling from the inhibitory, ITIM-dominated phenotype to the activating, ITAM-dominated phenotype. Understanding the mechanisms underlying the complex network of C5aR and FcR regulation is likely to lead to novel, urgently needed therapeutics for autoimmune diseases.
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Acknowledgments |
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Footnotes |
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1 This work was supported by Cincinnati Children’s Hospital Research Foundation funding, Deutsche Forschungsgemeinschaft Grant KO1245/1-1, and National Institute of Health Grant R21AI59306-01 (to J.K.). 
2 J.G. and T.H. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jörg Köhl, Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, MLC 7021, Cincinnati, OH 45229. E-mail address: joerg.koehl{at}chmcc.org
4 Abbreviations used in this paper: IC, immune complex; KC, cytokine-induced neutrophil chemoattractant; KitW/W-v, WBB6F1 KitW/W-v; MC, mast cell; M, macrophage; rh, recombinant human.
Received for publication April 23, 2004. Accepted for publication June 25, 2004.
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