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Rheumatoid arthritis culminates in joint destruction that in mouse models of disease, is supported by innate immune molecules including FcγRs and complement. However, the results may not predict outcomes in humans given the structural differences between murine and human activating FcγRs on neutrophils, a prominent component of joint exudates. In this study, we examined the role of the human neutrophil FcγRIIA in the development of arthritis and probed the underlying mechanism by which FcγRIIA initiated disease.
K/BxN serum transfer-induced arthritis was examined in mice that express FcγRIIA on neutrophils but lack their own activating FcγRs (γ-chain-deficient). The role of mast cells, complement (C3 and C5a) and CD18 integrins in FcγRIIA-initiated disease was examined using cell reconstitution approaches, inhibitors, and functional blocking antibodies respectively. Cross-talk between C5aR and FcγRIIA on neutrophils was evaluated in vitro.
Neutrophil FcγRIIA expression was sufficient to restore susceptibility to K/BxN serum induced neutrophil recruitment, synovitis and bone destruction in γ-chain-deficient mice. Joint inflammation was robust and proceeded even in the absence of mast cells and vascular permeability, shown to contribute to disease in wild-type mice. Neutrophil recruitment was dependent on CD18 integrin LFA-1 and C5aR. C5aR in addition significantly enhanced FcγRIIA mediated phagocytosis and oxidative burst in vitro.
Human and murine activating FcγRs on neutrophils are not functionally equivalent, and in humans may play a primary role in arthritis. Cross-talk between neutrophil C5aR and FcγRIIA is essential for disease thus highlighting a new aspect of complement during the effector phase of inflammatory arthritis.
Rheumatoid arthritis (RA) is an inflammatory disease of the joints with 1% prevalence in the industrialized world (1, 2). Neutrophils, lymphocytes, mast cells, macrophages, synovial tissue cells, and platelet microparticles present in the inflammed synovium have been implicated in the evolution of RA (3, 4). Circulating autoantibodies are present in a majority of patients and joint tissues are frequently covered with immune complexes (ICs) in RA (5, 6). Mechanisms of autoantibody driven disease have begun to be elucidated in mouse models such as the K/BxN arthritis model, which arises from the breakage of T cell tolerance followed by the generation of IgG autoantibodies to the glycolytic enzyme glucose-6-phosphate isomerase (GPI). K/BxN arthritis shares with RA certain clinical features including distal symmetric erosive polyarthritis and at the tissue level, pannus that is erosive into bone and cartilage, prominent vascular hyperplasia and neutrophil rich synovial fluid. The finding that disease can be passively transferred with autoantibodies from these animals to normal recipients together with results in knock-out mouse models support the view that although T and B cells are required for the initiation of disease, autoantibodies and innate immune mediators are the effector mechanisms that promote synovitis and joint destruction (7).
FcγRs for complexed IgG and the complement system play essential roles in disease progression. Polymorphisms in FcγRs are associated with RA (8, 9) and studies in mice suggest a significant role for these receptors in disease pathogenesis: mice deficient in the γ-chain, required for the expression and signaling of activating FcγRs, have reduced incidence and severity in models of arthritis, whereas a deficiency in the inhibitory receptor FcγRII exacerbates disease (10). Mast cells are among the FcγR-bearing cell types that contribute to articular inflammation by releasing mediators that induce vascular permeability, promote neutrophil recruitment, and activate resident synovial fibroblasts and macrophages (3). Generation of complement components is a key event in the effector phase of disease. Complement C5a levels correlate with neutrophil accumulation in patients (11) and in the K/BxN model, mice lacking C5 or C3 are resistant to disease (12). C5a exerts its effects primarily by binding C5a receptor (CD88) expressed on a wide variety of hematopoietic cells including neutrophils, mast cells and macrophages (13). Synergism between complement C3 receptors and FcγRs has been long recognized to enhance adherence and ingestion of complement fragment C3b and IgG opsonized targets (14, 15). However, only recently has cross-talk between C5aR and FcγRs been appreciated. A hierarchical relationship between the two receptors has been suggested. C5a attracts FcγR bearing leukocytes and C5aR transcriptionally regulates expression of inhibitory and activating FcγRs on macrophages to lower the threshold of IC activation (13). In other cases, complement and FcγRs clearly play redundant roles (16) or one pathway dominates over the other (17).
Despite the significant strides made in mice, the results may not predict outcomes in humans as the repertoire of activating FcγRs on neutrophils that bind ICs differ significantly between the two species. Human neutrophils express two activating FcγRs, which are uniquely human. Human FcγRIIA contains an immunotyrosine activating motif (ITAM) and ligand binding domain in a single polypeptide chain, and FcγRIIIB is glycophosphatidylinositol-linked. In contrast, murine neutrophils contain a transmembrane FcγRIII and a species-specific FcγRIV that complex with an ITAM-containing common γ-chain. Moreover, the contribution of FcγRs alone to disease pathogenesis remains unclear since deletion of theγ-chain in mice eliminates more than activating FcγRs (18). Previous work suggests that human FcγRIIA and FcγRIIIB expression on neutrophils of mice lacking their endogenous activating FcγRs (i.e. γ-chain−/−) restores susceptibility to nephrotoxic serum nephritis and the Reverse Passive Arthus reaction (induced by soluble ICs) (19).
Here we explored the role of human FcγRIIA on neutrophils in inflammatory arthritis and probed the underlying mechanisms by which this receptor mediates disease.
Human FcγRIIA (FcγRIIA+/γ−/−) mice (19) and miR-CD18 mice (20) were previously described. Mice expressing human CD18 on neutrophils but lacking endogenous murine CD18 (hCD18+/mCD18−/−) were generated as follows. Human CD18 cDNA was subcloned into the BglII site of pUC18-hMRP8 vector (21). A HindIII-EcoRI fragment was released and injected into zygotes from C57Bl/6J mice. Transgenic mice were generated in the transgenic facility of Brigham & Women’s Hospital (Boston, MA). A high-expressing founder of hCD18 (hCD18+) was crossed with CD18 deficient mice (mCD18−/−) (22)on a C57Bl/6J-F12 background and bred to be hemizygous for the hCD18 transgene, and mCD18 deficient, as assessed by PCR of genomic DNA and flow cytometry analysis of cells.
All mice were maintained in a virus- and antibody-free facility at the NRB animal housing facility at Harvard Medical School. Mice used for each experiment were 8–10 weeks of age and sex matched. The Harvard Medical School Animal Care and Use Committee approved all procedures in this study.
25–150 μl of K/BxN serum was given intraperitoneally (i.p.) on day 0 and 2. Every second day up to day 14, clinical arthritis was graded in all four paws (clinical score) and ankle joint thickness was measured on both side using a micrometer (23, 24). Ankle joints were isolated and tissue specimens were stained with hematoxylin and eosin for histological evaluation (25) or rat monoclonal NIMP-R14 (Abcam Inc. Cambridge, MA) for neutrophil quantitation (26). The total neutrophil number in the closed articular cavity were counted and was divided by the total area (mm2) of the articular cavity in each specimen measured by Image J software (NIH). For quantification of mast cell degranulation, mice given 100μl of normal mouse serum or K/BxN serum were euthanized 18hrs later and toluidine blue stain was applied to tissue specimens (24).
For complement depletion, 12.5 U/mouse CVF (Quidel, San Diego, California, USA) was injected i.p. on day -1, and 6.25 U/mouse on day 2, 5 and 10. C5a receptor antagonist (C5aRA, A8Δ71-73) (27) was injected intravenously (i.v.) 20 min before and 2 hrs after K/BxN serum injection, and then i.v. every 12 hrs up to day 4.
For blockade of LFA-1, 100μl of K/BxN serum was given at day 0 and 2 and 75μg functional blocking rat anti-CD11a IgG2a antibody (clone M17/4) (BD Pharmingen) or rat IgG isotype control were injected i.v. at day 0, 2 and 4 as previously described (22).
Mouse heat-aggregated IgG (mHAGG) was generated essentially as previously described (28). 100μl of monomeric mouse IgG (5mg/ml), mHAGG, K/BxN serum or normal mouse serum was diluted with 150μl of 0.3% Evans blue and 50μl PBS, and injected i.v. All four limbs per mouse were harvested and incubated with 600μl dimethylformamide at RT for 4 days and Evans blue in the supernatant was quantified by measuring theabsorbance at 595nm.
Peripheral blood sampled from the retroorbital plexus of mice, human peripheral neutrophils, bone marrow derived macrophages, peritoneal cells were collected as described (19, 29). For analysis of mast cells, peritoneal cells were harvested and used immediately. All antibodies were from BD Biosciences-Pharmingen unless otherwise indicated. FITC or PE-mouse anti-human CD18 (clone 6.7, mouse IgG1κ), FITC-rat anti-mouse CD11a (clone M17/4, rat IgG2aκ) and APC-rat anti-mouse CD11b (clone M1/70, rat IgG2b, eBioscicence). Cell populations were identified as follows: PE or APC-rat anti-Gr-1 (clone RB6-8C5, APC-Gr-1; BioLegend) for neutrophils, PE-rat anti-CD115 (clone AFS98; eBioscience) for monocytes, PE-Cy7-hamster anti-mouse CD3ε (clone 145-2C11) for T cells, APC-rat anti-F4/80 (clone A3-1; Caltag Laboratories) for macrophages and FITC-rat anti-mouse CD19 (clone 1D3) for peripheral B cells.
Mature neutrophils (>95% pure) were isolated from the bone marrow of femurs and tibias of FcγRIIA+/γ−/−or γ−/− mice as described (30). 2.0×107 neutrophilswere administered i.v. into recipient W/Wv mice on days 0, 1, 2, 3, and 200 μl K/BxN serum was given on day 0 and 2.
2×106 BMNs were stimulated with or without 100nM LTB4, 100nM human C5a, 100μg/ml fmlp (Sigma) for 20 min at 37°C, and washed twice at 4°C. Cells were incubated with mHAGG at 1:100 dilution on ice for 1hr, washed again, and stained with Alexa-conjugated donkey anti-mouse IgG (Invitrogen) and APC-anti Gr-1. Mean fluoroscent intensity (M.F.I.) in the Gr-1 positive cell population was evaluated by FACs to detect surface mHAGG.
Sheep RBC (Lampire) were labeled with fluorescence dye PKH67 according to manufacturer’s instructions (Sigma). For IgG-RBC, RBC were incubated with anti-sheep RBC IgG (Cedarlane) for 1h. For C3b-RBC, RBC were incubated with anti-sheep RBC IgM (Cedarlane) for 1h followed by C5 deficient serum (Sigma) for 1h. RBCs were coincubated with neutrophils at a 10:1 ratio in the presence or absence of C5a (5μg/ml), PMA (100ng/ml) or DMSO at 37° for 30 mins. External RBCs were lysed in ice-cold water, followed by the addition of trypan blue to quench fluorescence from remaining external RBC. Cells were visualized under a fluorescence microscope to assess the percentage of neutrophils with internalized RBCs. Normalized phagocytosis percentage was calculated by subtracting phagocytosis of control RBC from phagocytosis of IgG or C3b-coated RBC.
1×106 BMNs were suspended in PBS without Ca2+/Mg2+, and incubated with 10μg/ml mouse anti-hFcγRIIA (cloneIV.3, StemCell Technologies) on ice for 30 min. After washing in PBS twice, cells were incubated with or without 500ng/ml hC5a for 30 min. 50μM luminol in PBS with Ca2+/Mg2+ was added, followed immediately by goat anti-mouse F(ab′)2 (36μg/ml), (Jackson ImmunoResearch Laboratories). ROS generation was continuously monitored over time using a six-channel bioluminat LB-953 luminometer (Berthold).
All in vivo data are presented as mean±S.E.M. The in vitro data are presented as mean±S.D. Statistical differences were analyzed with the unpaired t-test. Significance was defined as p<0.05.
K/BxN serum-induced arthritis was evaluated in mice expressing the human FcγRIIA in γ−/−deficient mice (FcγRIIA+/γ−/−), mice lacking the common γ-chain (γ−/−) and wild-type mice. γ−/− mice transgenically expressing the other human neutrophil FcγR, FcγRIIIB+/γ−/− mice (19) were excluded from the analysis as hFcγRIIIB does not recognize mouse IgG (Supplemental S1). 100–150μl of K/BxN serum is the standard dose for inducing disease in wild-type mice. We used only 50μl of K/BxN as even this lower dose resulted in significant morbidity in FcγRIIA+/γ−/− mice as will become apparent below. As previously reported (12), γ−/− mice showed no evidence of disease as assessed by clinical and histological parameters. On the other hand, hFcγRIIA+/γ−/− mice demonstrated disease activity that significantly exceeded that observed in wild-type mice (Figure 1A) despite levels of FcγRIIA that are comparable to its murine counterpart FcγRIII on wild-type neutrophils (19). It is noteworthy that the inhibitory FcγRIIB is expressed in the FcγRIIA/γ−/− animals and is likely functional since administration of intravenous immunoglobulin (IVIG), which mediates protection in wild-type mice through its action on FcγRIIB (31), inhibits FcγRIIA mediated inflammation in the K/BxN model (data not shown). Histologically hFcγRIIA+/γ−/− mice exhibited severe synovial hyperplasia and bone erosion (Figure 1B) with robust neutrophil infiltration in the synovium and bone compared to γ−/− mice (Figure 1C,D). Thus activating human FcγRIIA on neutrophils is sufficient to restore neutrophil influx and susceptibility to arthritis in the absence of other activating FcγRs.
Mast cell deficient W/Wv and Sl/Sld mice fail to develop K/BxN serum-induced arthritis and degranulated mast cells are observed in joint tissue of wild-type animals within hours of K/BxN serum transfer (24). As reported, ankle specimens of wild-type mice after K/BxN serum-transfer showed significant mast cell degranulation while they were intact in γ−/− mice, which do not succumb to disease. However, ankle specimens of FcγRIIA+/γ−/− mice also exhibited virtually no mast cell degranulation despite significant synovitis (Figure 2A).
K/BxN serum induces joint localized vascular permeability within 5 mins of administration that depends on mast cells, neutrophils and FcγRs in C57Bl/6 mice (32). Heat aggregated, non-specific IgG also induces leakage indicating that IgG-ICs rather than other components potentially present in K/BxN serum, are sufficient to promote permeability(32). ICs (mouse heat-aggregated IgG or K/BxN serum) did not induce vascular permeability in FcγRIIA+/γ−/− mice and indeed was similar to that observed in the γ−/−and wild type animals treated with (control) monomeric IgG (Figure 2B). Permeability was also evaluated 18hrs after introduction of K/BxN serum. At this later time point, Evans blue leakage significantly increased in the FcγRIIA/γ−/− mice (data not shown), which is likely secondary to the robust inflammation developing in these animals. In summary, in the presence of hFcγRIIA on neutrophils, disease proceeds through mechanisms that are independent of mast cell degranulation and is not associated with early changes in vascular permeability.
To directly evaluate the contribution of mast cells to FcγRIIA-induced arthritis, bone marrow neutrophils (BMN) isolated from FcγRIIA+/γ−/− or γ−/− mice were i.v. injected into the W/Wv strain of mice for 4 consecutive days and K/BxN serum was delivered into reconstituted W/Wv mice at day 0 and 2. Absence of mast cells in the W/Wv animals was confirmed by immunostaining of ear skin (Figure 3A). W/Wv mice reconstituted with FcγRIIA+/γ−/− BMNs exhibited significantly more disease activity than mice given γ−/−BMNs (Figure 3B). Thus, in the context of human neutrophil FcγRIIA, mast cells are not essential for development of arthritis.
Mice lacking all CD18 integrins, or in which these integrins are functionally blocked, develop minimal arthritis (22). Here, the significance of CD18 integrins on neutrophils was evaluated by exploiting two complementary genetic approaches: The silencing of CD18 selectively in neutrophils of wild-type mice (miR-CD18) (20) and the expression of human CD18 selectively in neutrophils of mice lacking endogenous CD18 (hCD18+/mCD18−/−) (Supplemental S2). These two mouse models, together with wild-type mice and CD18-deficient mice were subjected to K/BxN serum arthritis. Neutrophil CD18 silencing impaired development of arthritis, CD18 deficient mice were resistant to disease, while hCD18+/mCD18−/− developed arthritis that was comparable to wild-type mice (Figure 4A). Together, these data provide compelling evidence that neutrophil CD18 is critical in the effector phase of arthritis.
Given the reliance of K/BxN arthritis on neutrophil CD18, we assessed the contribution of CD18 specifically in induction of arthritis in the hFcγRIIA mice. Functional blocking antibody to LFA-1 blocks K/BxN arthritis in wild-type mice (22). Here we show that this antibody completely suppressed clinical and histological features of arthritis in hFcγRIIA+/γ−/− mice (Figure 4C–D) that correlated with minimal joint neutrophil accumulation (Figure 4E).
Wild-type and FcγRIIA+/γ−/− mice were evaluated following depletion of C3 by systemic administration of cobra venom factor (CVF) (Figure 5A) or blockade of C5a receptor using a specific antagonist of the two C5a receptors, CD88 and C5L2 (27) (Figure 5B). Both mouse strains treated with CVF failed to develop arthritis (Figure 5A). However, C5aR blockade attenuated arthritis only in FcγRIIA+/γ−/− mice while the C5aR antagonist in wild-type mice did not, and indeed had a trend towards greater disease for reasons that are unclear (Figure 5B,C). Consistent with these findings, a significant reduction in neutrophil influx was observed only in FcγRIIA+/γ−/− mice treated with the C5aR antagonist (Figure 5D). In conclusion, C5aR regulates hFcγRIIA dependent neutrophil recruitment and joint injury.
Activation of the recruited neutrophils via FcγRs and complement receptor 3 (CR3) is essential for tissue damage in vivo (30, 33). Here we conducted studies in vitro to explore the possibility that in addition to effects on neutrophil recruitment, C5a regulates FcγRIIA-arthritis by increasing FcγRIIA-dependent neutrophil cytotoxicity. The bacterial peptide fmlp increases FcγRIIA-dependent ligand binding (34). Here FcγRIIA+/γ−/− neutrophils treated with C5a or other G-protein coupled receptor agonists fmlp and LTB4 exhibited enhanced binding of ICs although this was not statisically significant (p=0.07) (Fig 6A). Next, FcγRIIA phagocytic functions in the presence of C5a was examined. Phagocytosis of IgG-opsonized RBCs was undetectable in FcγRIIA expressing neutrophils, while pretreatment of cells for 30 min with C5a induced significant phagocytosis (Figure 6B). The enhancement was specific for FcγRs as phagocytosis of complement C3b-opsonized RBC (C3-RBC), mediated by CR3 (33) was largely unaffected by C5a, but was robust in the presence of PMA (Figure 6B) as described (33). To determine if C5a can regulate FcγR signaling downstream of receptor engagement we evaluated whether it enhanced FcγRIIA cross-linking induced reactive oxygen species (ROS) generation (Figure 6C). ROS was not detected in FcγRIIA+/γ−/− neutrophils pretreated with C5a for 30 min (Figure 6C) as expected since ROS returns to baseline within 5 min of C5a treatment (data not shown). FcγRIIA cross-linking alone produced minimal ROS. On the other hand, FcγRIIA+/γ−/− neutrophils pretreated with C5a prior to FcγRIIA cross-linking exhibited a significant and rapid enhancement of ROS production. C5a, under treatment conditions used in the above two assays did not increase surface FcγRIIA levels (data not shown). In summary, C5a is essential for FcγRIIA dependent pro-inflammatory effector functions.
There is considerable evidence in humans and animal models that ICs and innate immune mediators promote inflammation in joints and other tissues. However, the hierarchy of events and the downstream cellular and molecular effector mechanisms remain largely unknown. Furthermore, since there exists significant divergence in FcγR structure and function between mouse and man, the relevance of results obtained only with murine FcγRs for human IC-driven diseases is debatable. Previous mechanistic studies in K/BxN arthritis have shown that antigen:antibody IC deposition in the articular cavity leads to activation of tissue-resident FcγR-expressing cells that feed back to initiate recruitment of circulating leukocytes (35). Importantly, since FcγRIIA is found only in higher primates, the role of neutrophils activated by this pathway remains unexplored both in this model and in human disease. Herein we demonstrate that expression of human FcγRIIA in neutrophils alone confers significant susceptibility to K/BxN induced arthritis that in fact exceeds that observed in wild-type mice that express their whole complement of endogenous FcγRs. We further show that FcγRIIA on neutrophils is acutely regulated by complement C5a. The cross-regulation of C5aR and FcγRIIA provides an important link between the FcγRs and the complement network, both of which connect upstream initiating events in RA to downstream effector responses (12). C5a also contributes to neutrophil recruitment, but it is the integrin LFA-1 that is critical in this respect as its blockade ameliorates FcγRIIA-dependent neutrophil influx and the associated joint destruction. Thus human FcγRIIA, C5aR and LFA-1 represent an important regulatory complex on neutrophils that can coordinate neutrophil influx and cytotoxicity in IC-driven inflammation.
Engagement of human neutrophil FcγRIIA is well-known to trigger a cascade of signaling events leading to phagocytosis, ROS production, protease and leukotriene release and cytokine production in vitro (1). It is expressed on neutrophils, as well as a number of leukocyte subsets, platelets, mast cells, Langerhan and dendritic cells (36, 37). In previous studies, transgene expression of FcγRIIA primarily on platelets of wild-type mice increased susceptibility to collagen-induced arthritis (38). Here, the neutrophil-selective expression of FcγRIIA in the absence of other FcγRs demonstrates the potency of this molecular pathway in activating neutrophils in the context of inflammatory arthritis. We cannot formally rule out a role for the approximately 20% of monocytes expressing FcγRIIA (19) in development of arthritis. However, K/BxN induced RA proceeds normally in CSF-1 deficient mice (op/op) (31) which have a >90% reduction in monocytes and macrophages (39), and FcγRIIA is not detectable on macrophages of FcγRIIA/γ−/− mice (19). Therefore, the contribution of FcγRIIA on monocytes is likely not significant. Interestingly, the inflammatory pathway triggered by FcγRIIA does not proceed via increases in vascular permeability, a function previously ascribed to both neutrophils and mast cells (32). Further, the stimulation of neutrophils via FcγRIIA is not associated with mast cell degranulation and indeed proceeds in the absence of these cells. Thus human and murine activating FcγRs are not functionally equivalent, with neutrophil human FcγRIIA playing a primary and proximal role in the effector phase of arthritis. This might require a re-evaluation of what we have concluded about the pathogenesis of arthritis based on previous murine studies. Our work suggests that neutrophils were recruited through their own FcγRIIA. GPI expression has been observed on the surface of the synovial lining and on the endothelial cell surface (40). The latter observation suggests the possibility that FcγRIIA may directly interact with GPI/anti-GPI ICs within the joint vasculature in K/BxN serum transfer arthritis to promote neutrophil recruitment, similar to what we have shown by intravital microscopy to occur in the cremaster muscle following intravascular and tissue IC deposition (19).
Our analyses demonstrate a surprising requirement for the integrin LFA-1 in FcγRIIA-depenent neutrophil contributions to arthritis. Although neutrophil recruitment in joints of wild-type mice was previously reported to be dependent on neutrophil CD18 (22, 41), the LFA-1 requirement in our studies was somewhat unexpected as FcγRIIA can directly bind ICs both in vitro and in vivo (19). The absolute requirement for additional adhesion receptors was thus not apparent a priori. LFA-1 is known to co-cap with FcγRs (42) and cross-linking of FcγRs triggers proximity between the CD18 integrins and cortical microfilaments (43). We speculate that FcγRIIA engagement may lead to activation of LFA-1 and its subsequent interaction with its ligand ICAM-1 on the activated endothelium.
The role of complement in neutrophil FcγRIIA driven responses is also noteworthy. In wild-type mice, the alternate pathway of complement activation drives K/BxN arthritis (12). Here we show that C3 and C5a are required for FcγRIIA-induced disease pathogenesis. The effect of C5aR antagonism on neutrophil accumulation may have been expected as C5a is a powerful neutrophil chemoattractant. The additional information provided by our studies is that the effect of C5a appears to cooperate with FcγRIIA on neutrophils as wild-type mice were resistant to C5aR blockade with the antagonist. The reason for the discrepancy in results with the antagonist and previous work showing that C5aR (CD88) deficient are protected from K/BxN induced arthritis (12) is unclear. It is possible that antagonist penetration into joint tissues is inadequate to fully block C5aR. On the other hand, C5aRA also blocks the other C5aR, C5L2 which may act as a decoy receptor for C5a as has previously been demonstrated in some other models (13).
How does C5a regulate FcγRIIA dependent arthritis? Previous studies suggest that the regulatory impact of C5a on FcγRs on macrophages is mainly due to transcriptional downregulation of the α-chain of FcγRIIB (13) or upregulation of the γ-chain (44, 45), which is lacking in our system. Our studies in vitro suggest that C5a also has a more immediate effect by promoting FcγRIIA dependent phagocytosis and ROS generation in neutrophils. The absolute requirement for C5a in FcγRIIA-mediated phagocytosis of IgG-opsonized RBC suggests that FcγRIIA is relatively inactive in resting cells, which we anticipate may have evolved as a mechanism to maintain circulating neutrophils in a quiescent state much like leukocyte CD18 integrins that are activated to bind ligand only upon stimulation with inflammatory mediators (46). Consistent with this notion, under physiological flow conditions in vitro, FcγRIIA was insufficient to capture neutrophils on plate immobilized ICs (29) or IgG-coated endothelial cells (47) but in the presence of a GPCR agonist such as chemokines firmly adhered to ICs both in vitro (47, 48) and in vivo (19). We speculate that the C5a pathway may be particularly important under conditions of low avidity engagement of FcγRs where it may regulate the amplitude of the response. Interestingly, C5a appears to have some selectivity in its activity towards FcγR: The effect of C5a on complement C3/CR3 induced phagocytosis was marginal relative to its effects on FcγR mediated uptake. There is increasing appreciation of the cross-regulation between ITAM-associated FcγRs and Toll receptors (TLRs) and cytokine receptors (18, 49). However, unlike the TLRs and cytokine receptors, which indirectly link to ITAM-containing receptors, C5aR and FcγR cross-talk directly. The observation that C5a enhanced ROS generation upon direct clustering of FcγRIIA at the cell surface suggests a role for C5a/C5aR downstream of IgG-binding. C5aR may relocalize FcγRIIA to lipid rafts enriched in signaling molecules (50, 51) and/or engagement of C5aR and the ITAM-containing FcγRIIA may lead to convergent MAPK signaling (52).
In conclusion, our studies provide previously unappreciated insight into functional contributions from neutrophil FcγRIIA in joint inflammation in a humanized mouse model. Our studies focus attention on neutrophil FcγRIIA and by extension neutrophils, as critical links between antibody and immunological injury. This could not have been predicted from previous studies on the murine FcγRs. Translationally, since FcγRIIA bearing neutrophils comprise the predominant population in inflamed joint fluid in RA, with neutrophil influx estimated at greater than a billion cells/day in a single joint, it is likely that these insights bear significant relevance for our understanding of disease physiology and suggest that neutrophil FcγRIIA may represent a viable therapeutic target for treatment of RA. Further, there is growing evidence for the role of neutrophils and ICs in numerous other human diseases such as glomerulonephritis, IC-vasculitis, skin autoimmune diseases and others (1). Thus, it is highly likely that our finding of a FcγRIIA:C5aR:LFA-1 receptor triad that functions to provide tight regulation and fine tuning of neutrophil responses to ICs has relevance in other autoimmune diseases with evidence of IC pathophysiology.
The human MRP8 promoter construct was kindly provided by Dr. E. Lagasse (Univ. of Pittsburgh, PA), and I.L. Weissman (Stanford University, CA). We are grateful to Nick Calderone (Dept of Medicine, Brigham and Women’s Hospital. Boston, MA) for excellent technical assistance and Asuka Shimizu for generously helping N.T. with measurements in histological slides.
Grants and financial support: This work was supported by a Arthritis Foundation postdoctoral fellowship (N.T.), a scholarship from Novartis Foundation, (formerly Ciba-Geigy-Jubilee-Foundation) (T.E.), NIH RO1 HL065095 and AR050800 (T.N.M), KO1 AR054984 (X.C.), AI059745 (D.M.L) and AI059305 (J.K).
AUTHOR CONTRIBUTIONSNT, TE, XL and HN designed and performed the experiments and analyzed and interpreted the data. MH aided in obtaining the data. NT also contributed to writing the paper. XC, DM, DML and JK provided critical reagents and experimental protocols and gave conceptual advice. TNM contributed to the conception and design of the research, had full access to all the data and data analysis, and wrote the paper. All authors revised the paper for important intellectual content.