Development of Inflammation in Proteoglycan-Induced Arthritis Is Dependent on FcγR Regulation of the Cytokine/Chemokine Environment

Charles D. Kaplan, Shannon K. O’Neill, Tamas Koreny, Matyas Czipri and Alison Finnegan
J Immunol November 15, 2002, 169 (10) 5851-5859; DOI: https://doi.org/10.4049/jimmunol.169.10.5851

Abstract

FcγRs are specialized cell surface receptors that coordinately regulate immune responses. Although FcγR expression is a prerequisite for the development of several immune complex-mediated diseases, the mechanism responsible for FcγR-dependent regulation in autoimmunity remains unclear. Therefore, we assessed FcγR-dependent regulation of inflammation in proteoglycan-induced arthritis (PGIA) using FcγR−/− mice. FcγRIIb−/− mice developed arthritis at an earlier time point and with a greater severity than wild-type (WT) mice. In γ-chain−/− (FcγRI−/− and FcγRIII−/−) mice, no clinical or histological evidence of inflammation was observed. Exacerbation of arthritis in FcγRIIb−/− mice correlated with enhanced PG-specific Ab production, but did not significantly affect PG-specific T cell priming. In γ-chain−/− mice, the absence of arthritis did not correlate with serum Ab responses, as PG-specific Ab production was normal. Although PG-specific T cell proliferation was diminished, spleen cells from γ-chain−/− mice successfully adoptively transferred arthritis into SCID mice. Our studies indicated that the mechanism responsible for FcγR regulation of PGIA development was at the level of inflammatory cytokine and β-chemokine expression within the joint. FcγRIIb regulated the development of PGIA by controlling the initiation of cytokine and chemokine expression within the joint before the onset of arthritis, whereas the expression of FcγRI and or FcγRIII controlled cytokine and chemokine expression late in the development of PGIA during the onset of disease. These results suggest that FcγRs are critical for the development of inflammation during PGIA, possibly by maintaining or enhancing inflammatory cytokine and β-chemokine production.

Rheumatoid arthritis is a chronic inflammatory autoimmune disease that manifests itself in the diarthrodial joints. This disease is characterized by leukocyte invasion of the synovial lining and hyperplasia of the resident synoviocytes with an ensuing overproduction of cytokines, chemokines, and other inflammatory mediators. Overproduction of these mediators ultimately results in cartilage destruction, bone erosion, and pathological remodeling of joint structures (1). Immune complexes are strongly implicated in creating inflammation in rheumatoid arthritis and other autoimmune diseases (2, 3, 4).

One of the main receptors responsible for immune complex activation of cells is the FcγR. The binding and cross-linking of immune complexes to FcRs specific for IgG (FcγRs) on leukocytes triggers the activation and regulation of a variety of cellular responses, including Ab production, Ab-dependant cellular cytotoxicity, phagocytosis, and release of inflammatory cytokines and chemokines (5, 6, 7, 8, 9). There are three structurally distinct types of murine FcγRs: FcγRI, FcγRIIb, and FcγRIII (10). FcγRI is a high affinity receptor exclusively expressed on monocytes and macrophages (11, 12). Although FcγRI can bind IgG-containing immune complexes, it is the only FcγR capable of binding monomeric IgG (8). FcγRIII is a low affinity receptor that is predominantly expressed on lymphoid and myeloid cells (10). Both FcγRI and FcγRIII are dual-chain receptors, with the signal transduction unit of both receptors being the γ-chain. The cytoplasmic domain of the γ-chain contains an immunotyrosine-based activation motif (13, 14), and cross-linking either FcγRI or FcγRIII in vitro leads to activation of cell functions such as inflammatory cytokine and β-chemokine production (5, 6, 15). FcγRIIb is the most widely expressed FcγR, expressed on virtually all hemopoietic cells except T cells, NK cells, and RBCs. FcγRIIb is a second low affinity receptor and, like FcγRIII, is only capable of binding IgG-containing immune complexes (10). FcγRIIb, unlike either FcγRI or FcγRIII, is a single-chain receptor that contains an immunotyrosine-based inhibition motif (16, 17, 18). Cross-linking FcγRIIb with either of the activation FcγRs, FcγRI or FcγRIII, or the B cell receptor inhibits cell function (19). By using FcγR−/− mice, these receptors have been demonstrated to play an important role in controlling immune complex-mediated diseases such as acute anemia, the Arthus reaction, glomerulonephritis, alveolitis, and arthritis (4, 20, 21, 22, 23, 24, 25, 26, 27). Although FcγRs have been shown to regulate the development of these immune complex-mediated diseases, the mechanism responsible for FcγR regulation remains unclear.

Arthritis can be induced in BALB/c mice immunized with human cartilage proteoglycan (PG).3 Clinical and histologic studies of the diarthrodial joints of these mice reveal that the inflammatory response that develops shares many similarities to rheumatoid arthritis (28, 29). During the development of PG-induced arthritis (PGIA), but before arthritis onset, a systemic IgG Ab response develops that first recognizes human PG, but later cross-reacts with native mouse PG (30, 31). These Abs gain access to the joint and bind cartilage PGs, forming immune complexes. Accumulation of PG-specific IgG in the cartilage is characteristic of inflamed joints and is accompanied by a loss of cartilage PGs (32). Synovium contains macrophages that express FcγRs capable of binding immune complexes. Since both FcγR-bearing cells and IgG-containing immune complexes are present in the joints of arthritic mice, it is possible that the interaction between immune complexes and FcγRs drives the inflammatory response associated with this arthritis model.

In this study we used two types of FcγR−/− mice, γ-chain −/− (FcγRI−/− and FcγRIII−/−) and FcγRIIb−/− mice, to examine the involvement of FcγRs in the development of inflammation in PGIA. The results provide direct evidence that the inhibitory FcγRIIb controls disease severity, while the activating γ-chain FcγRs are essential in the development of arthritis. Moreover, the use of FcγR−/− mice demonstrates that FcγR-dependent activation/inhibition regulates inflammatory cytokine and β-chemokine mRNA expression in the joint both before and after the onset of arthritis. These results suggest that FcγRs are vital for the development of arthritis by maintaining or propagating the cytokine and chemokine responses associated with the development of inflammation in the joint.

Materials and Methods

Ag preparation

Human cartilage tissue was obtained at the time of joint replacement surgery. PG from adult cartilage was prepared as previously described (28). Cartilage pieces were pulverized in liquid nitrogen and then extracted with 4 M guanidinium chloride in 50 mM sodium acetate, pH 5.8, containing protease inhibitors at 4°C. High buoyant density PG monomers (aggrecan) were purified by dissociative cesium chloride gradient centrifugation. PGs were sequentially digested with endo-β-galactosidase and protease-free chondroitinase ABC (Seikagaku America, Rockville, MD) overnight at 37°C and then further purified on a Sephacryl S-200 column (Pharmacia Biotech, Uppsala, Sweden). Murine PG was obtained from the cartilage of newborn mice and prepared in a similar manner as human cartilage PG, except that murine PG was not deglycosylated.

Induction and assessment of arthritis

Wild-type (WT), FcγRIIb−/−, and γ-chain−/− mice were on a BALB/c background. All mice were bred at Taconic Farms (Germantown, NY) and used at >3 mo age. Arthritis was induced by injecting female BALB/c mice with 150 μg/ml human PG (hPG) measured as protein in the presence of CFA as previously described (28, 29). Mice were boosted at 3 and 6 wk with 100 μg/ml of hPG. The first booster was in IFA, and the second booster was in CFA. Mice developed arthritis in the diarthrodial joints ∼2–4 wk after the third immunization. Paws were scored for erythema and swelling every third day after the second immunization to assess arthritis onset and severity. Paws were scored on a scale from one to four as follows: 0, normal; 1, mild erythema and swelling, usually one or two toes; 2, moderate erythema and swelling of the paw; 3, more diffuse erythema and swelling of the paw; and 4, severe erythema and swelling of the entire paw. Each paw was scored individually; therefore, the cumulative score ranged from 0–16. Histological studies were performed to determine the extent of joint inflammation and damage. Hind paws were dissected, decalcified, embedded in paraffin, and sectioned at 6 μm as previously described (28). Sagittal sections were stained with H&E. Sections were examined by a blinded histologist, and arthritic changes were scored as normal (none), mild, moderate, or severe.

Detection of serum Ab titers

Mice were bled from the orbital plexus, and isotype-specific serum anti-PG Abs were measured by ELISA. Plates (96-well Nunc-Immuno plates; Fisher Scientific, Pittsburgh, PA) were coated with 1.0 μg of chondroitinase ABC-digested hPG or 1.5 μg native mouse PG (mPG) in carbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6). Sera were serially diluted in 1× PBS in 0.5% Tween 20. Sera dilutions of 1/100, 1/500, and 1/2,500 were used for detection of mPG-specific Abs, while dilutions of 1/2,500, 1/12,500, and 1/62,500 were used to detect hPG-specific Abs. Isotypes were detected with isotype-specific Abs, rabbit anti-mouse IgG1-HRP and IgG2a-HRP (Zymed, San Francisco, CA), followed by the substrate orthophenylenediamine, and absorbance was measured on a ELISA reader at 490 nm. A standard curve of myeloma IgG1 and IgG2a proteins was titrated to determine the concentrations of IgG1 and IgG2a in the sera. IgG1 and IgG2a proteins were detected using the same isotype-specific Abs as those used to detect PG-specific Ab isotypes. Serum IgG2b and IgG3 titers were also assessed and were found to be nearly undetectable (data not shown).

T cell proliferation assay

Spleens were harvested 82 days after the initial immunization, and single-cell suspensions were prepared as previously described (33). T cells were purified by passage through nylon wool. T cells (1.25 × 106 cells/ml) were incubated with irradiated (2500 rad) spleen cells (1.25 × 106 cells/ml) from nonimmunized mice in a fixed volume of 200 μl in serum-free medium (HL-1 medium; Fisher), 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine in a 96-well plate. T cells were stimulated in the presence or the absence of PG (3.13 and 1.5 μg/ml). T cell cultures were incubated at 37°C in 5% CO2 for 5 days, the last 18 h pulsed with [3H]thymidine (0.5 μCi/well). The cells were harvested using a cell harvester (Tomtec, Orange, CT), and the amount of incorporated [3H]thymidine was measured using a scintillation counter (EG&G Wallac, Galesburg, MD).

Assessment of in vitro splenic cytokine production

Splenocytes (2 × 106 cells/ml) were incubated in a 24-well Falcon plate (Fisher) in serum-free HL-1 medium. IFN-γ and TNF-α were measured from supernatants harvested on day 5 by ELISA using the OPT EIA mouse IFN-γ set (BD PharMingen, San Diego, CA) and the mouse TNF-α DuoSet ELISA development kit (R&D Systems, Minneapolis, MN).

Adoptive transfer of PGIA into SICD mice

Splenocytes (5 × 107 cells/mouse) from either arthritic WT mice or immunized γ-chain−/− mice and 150 μg human PG measured as protein were mixed in saline and injected i.p. into female BALB/c SCID mice (National Cancer Institute, Frederick, MD). The SCID mice were monitored for disease onset and severity every other day after cell transfer.

RNase protection assay

One hind paw was homogenized with a Polytron homogenizer (KRI Works, Cincinnati, OH) on ice. Homogenate was centrifuged to remove large debris, and RNA was extracted with Tri-Reagent (Molecular Research, Cincinnati, OH). RNase protection assay was preformed on 20 μg of RNA using the Riboquant Multiprobe RNase Protection Assay System (BD PharMingen) according to the manufacturer’s directions. The mCK-2b, mCK-3b, and mcK-5b templates were used to detect sets of cytokines and chemokines (IL-12 p35, IL-12 p40, IL-10, IL-1α, IL-1β, IL-1R antagonist (IL-1Ra), IL-18/IFN-γ-inducing factor, TNF-β, lymphotoxin β, TNF-α, IL-6, IFN-γ, IFN-β, TGF-β1, TGF-β2, TGF-β3, macrophage inflammatory factor, lymphotactin, RANTES, eotaxin, macrophage inflammatory protein-1β (MIP-1β), MIP-1α, MIP-2, monocyte chemotactic protein-1 (MCP-1), and T cell-activated gene-3) as well as housekeeping genes L32 and GAPDH. Labeled ([α-32P]UTP) antisense RNA was synthesized by in vitro transcription from a cDNA template provided in the kit. Antisense RNA probe was purified by phenol/chloroform extraction and ethanol precipitation and was hybridized with the mRNA samples overnight at 56°C. RNase was used to digest ssRNA. Protected dsRNA was purified by phenol-chloroform extraction and ethanol precipitation. The samples were electrophoresed on a 5% denaturing polyacrylamide gel. The gel was dried and exposed to a phosphorimager screen. The radioactivity of the samples was measured and analyzed by scanning densitometry on a STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The level of cytokine and/or chemokine mRNA was expressed as the ratio of each mRNA species to GAPDH. Increases in inflammatory mediator mRNA expression were also reflected at the protein level, as measured by ELISA (data not shown).

Statistical analysis

Fisher’s exact t test was used to determine statistical significance in the incidence of arthritis. The Mann-Whitney U test was used to compare nonparametric data for statistical significance. A value of p < 0.05 was considered significant.

Results

FcγR deficiency leads to either the absence or exacerbation of arthritis

BALB/c mice immunized with hPG develop an Ab response to hPG, which over time develops into a cross-reactive response to mPG (30, 31). Abs specific for mPG appear before the development of arthritis and correlate with Ab deposition in the joint (in the form of immune complexes), the loss of cartilage PGs, and later the development of arthritis (28, 34). To determine whether FcγRs are involved in the development of inflammation, WT, FcγRIIb−/−, and γ-chain−/− mice were immunized with hPG, and the development of arthritis was monitored over time. Disease onset occurred in FcγRIIb−/− mice at an earlier time point (50% on day 34) compared with WT mice (0% on day 34; Fig. 1⇓A). Not only did FcγRIIb−/− mice develop arthritis earlier than WT mice, they also developed a more severe disease (13.6 ± 1.65) than WT mice (6.1 ± 3.88; Fig. 1⇓B). In contrast, γ-chain−/− mice never displayed any signs of inflammation (Fig. 1⇓, A and B, and Fig. 2⇓C). Over an extended period of time (162 days), inflammatory symptoms were still absent in γ-chain−/− mice (data not shown). These data suggest that FcγR expression is critical for controlling the development of PGIA.

FIGURE 1.
FIGURE 1.

Alterations in the development of arthritis in FcγR-deficient mice immunized with PG. FcγRIIb−/− (n = 7), γ-chain−/− (n = 5), and WT mice (n = 9) were immunized with hPG on days 0, 21, and 42. Mice were scored twice a week after day 34 to observe disease incidence and onset (A) and disease severity (B). Values represent the mean and SEM of the arthritic score. ∗, Data that are statistically significant (p < 0.05) compared with WT mice. The data presented are representative of two experiments performed.

FIGURE 2.
FIGURE 2.

γ-Chain deficiency prevented histopathology associated with the development of PGIA. Sections of hind paws were examined at ×12 magnification. Sections of representative ankle joints from WT (A), FcγRIIb−/− (B), and γ-chain−/− (C) mice. Sections were stained with H&E. T, tibia; Ta, talus; C, calcaneus. The boxed area is the area that is magnified on the right side of each figure

γ-Chain−/− mice are protected from the histopathological changes associated with arthritis

To determine the extent of the inflammation, we examined ankle joint histology in WT, FcγRIIb−/−, and γ-chain−/− mice on day 82. In WT mice we observed a histologic picture characteristic of acute arthritis. Mononuclear and polymorphonuclear cell infiltration was abundant in the tissues and joint spaces. There was edema of the synovial and periarticular tissues accompanied by synovial hyperplasia. Cartilage erosion and disintegrating chondrocytes were also seen in the remaining layer of the articular surface (Fig. 2⇑A). In comparison, FcγRIIb−/− mice displayed histopathologic signs of a more chronic disease. The ankle joints of FcγRIIb−/− mice exhibited less leukocyte infiltration and synovial lining proliferation, with a complete cartilage loss, severe bone erosion, abnormal remodeling of joint structures, and osteophyte formation (Fig. 2⇑B). These features correlated with the fact that arthritis was initiated in these mice at an earlier time point than in WT mice (Fig. 1⇑). Conversely, cellular infiltrate and/or histopathological signs of disease were completely absent in γ-chain−/− mice. In these mice, the lack of paw erythema and swelling correlated with the absence of cellular infiltration and joint destruction (Figs. 1⇑B and 2C).

Exacerbation of disease in FcγRIIb−/− mice is associated with early production of PG-specific Abs

It has been previously demonstrated that B cells are required for the development of PGIA (34, 35). FcγRs regulate B cell function by either directly suppressing Ab production through FcγRIIb or indirectly regulating Ab responses through γ-chain FcγRs (36). It is therefore possible that a deficiency in FcγR expression may augment B cell Ab production and possibly exacerbate or accelerate the disease. To assess the role of FcγRs in the development Ab responses in PGIA, we first analyzed PG-specific serum Ab production in WT and FcγRIIb−/− mice. Mice were bled at 4 wk after the initial immunization (∼1–2 wk before the onset of arthritis in FcγRIIb−/− mice) and after the development of arthritis at 7 and 11.5 wk, and PG-specific IgG1 and IgG2a Ab titers were assayed by ELISA. Correlating with the rapid progression of disease in FcγRIIb−/− mice was a significant enhancement of the PG-specific serum Ab response at wk 4 (Fig. 3⇓A). However, by as early as wk 7, WT and FcγRIIb−/− mice produced the same amount of PG-specific Ab (Fig. 3⇓B).

FIGURE 3.
FIGURE 3.

Changes in the development of arthritis are not due to changes in B cell function late. Serum was obtained from WT, FcγRIIb−/−, and γ-chain−/− mice at wk 4 (A), 7 (B), and 11.5 (C). ELISA was used to assay for serum IgG1 and IgG2a titers. Values represent the mean and SEM of IgG1 and IgG2a titers. ∗, Data that are statistically significant (p < 0.05) compared with WT mice.

To determine the role the γ-chain FcγRs play in the development of Ab responses in PGIA, we analyzed PG-specific serum Ab production in WT and γ-chain−/− mice. Mice were bled at 7 wk after the initial immunization (∼1–2 wk before the onset of arthritis in WT mice) and after the development of arthritis at 11.5 wk, and PG-specific IgG1 and IgG2a Ab titers were assayed by ELISA. γ-Chain−/− and WT mice produced equivalent levels of PG-specific Ab both before and after the onset of arthritis (Fig. 3⇑, B and C). These results suggest that while γ-chain FcγRs do not appear to regulate Ab production during the development of arthritis, FcγRIIb does exert control over Ab production at a point before the onset of arthritis, possibly during the initiation of inflammation.

Alterations in disease outcome are not associated with a change in T cell function

It has been previously shown that PG-specific T cells are critical for the development of arthritis (34, 37). Since the FcγR γ-chain is shared by the TCR, it may play an important role in TCR signaling (38, 39). In addition, FcγR-mediated uptake of Ag is an efficient mechanism for Ag presentation (40). Consequently, it is possible that the FcγRs directly and/or indirectly control T cell priming, and thus a deficiency in FcγR expression may alter disease outcome in a T cell-dependent manner. Therefore, we examined T cell proliferation in response to PG in WT, FcγRIIb−/−, and γ-chain−/− mice. T cells isolated from PG-immunized FcγRIIb−/− mice proliferate to a similar extent as T cells isolated from WT mice, indicating that FcγRIIb is not involved in T cell priming (Fig. 4⇓A). By comparison, proliferation of γ-chain−/− T cells was significantly reduced compared with that of WT T cells (Fig. 4⇓A), suggesting the possibility that γ-chain−/− mice do not succumb to disease because T cells were not sufficiently primed.

FIGURE 4.
FIGURE 4.

Changes in disease development are not due to alterations in T cell function. Splenocytes were obtained from WT, FcγRIIb−/−, and γ-chain−/− mice on day 82. T cell function was assayed by [3H]thymidine incorporation, expressed as overall T cell proliferation (A), and by SICD transfer (B and C). Splenocytes were also transferred from WT and γ-chain-deficient mice into SCID mice. Disease incidence and onset (B) and disease severity (C) were assayed by paw scoring every other day after the initial transfer. Values represent the mean and SEM of T cell proliferation and the SD of the mean arthritic score. ∗, Data that are statistically significant (p < 0.05) compared with WT mice.

To assess whether γ-chain−/− T cells were sufficiently primed to induce arthritis, we used an adoptive transfer model in which splenocytes from PG-immunized mice were transferred into SCID mice. The SCID mice were then immunized with hPG to activate the primed T cells. If γ-chain−/− T cells were fully capable of inducing arthritis, then adoptively transferring PG-immunized γ-chain−/− splenocytes into SCID mice should induce disease (Fig. 4⇑, B and C). We transferred splenocytes, from either arthritic WT mice or PG-immunized, nonarthritic γ-chain−/− mice and hPG into SCID mice and monitored the onset and severity of disease. SCID mice that received splenocytes from γ-chain−/− mice developed disease with similar kinetics and severity as SCID mice that received WT splenocytes. By 35 days after the initial transfer, 100% of SCID mice receiving either WT or γ-chain−/− splenocytes developed arthritis with a similar severity (10.1 ± 2.36 to 8.0 ± 3.08, respectively; Fig. 4⇑, B and C). These data conclusively demonstrate that T cells in γ-chain−/− mice were primed to PG and fully capable of inducing arthritis despite the reduced level of PG-specific T cell proliferation.

Disrupting the FcγR/immune complex interaction alters inflammatory cytokine and β-chemokine mRNA expression during the effector phase of inflammation

It has been previously demonstrated that immune complex stimulation through cross-linking of FcγRIII causes an increase in inflammatory cytokine and β-chemokine production in vitro (5, 6). Since immune complexes and FcγR-bearing cells are present in the joints of arthritic mice, we assessed whether FcγR-dependent signaling controls the expression of cytokines and chemokines during the development of inflammation. To this end, we quantitated cytokine and chemokine mRNA transcripts before erythema and swelling of the paws and once mice overtly displayed signs of arthritis (Figs. 5⇓ and 6⇓). Levels of inflammatory cytokine mRNA (TNF-α, IFN-γ, IL-6, IL-1β), IL-1Ra, and β-chemokine mRNA (MIP-1α, MIP-2, MCP-1) expression were significantly increased in FcγRIIb−/− mice during both the pre-arthritic, initiation phase (3.5 wk after the initial immunization; Fig. 5⇓, A and C) and the arthritic, effector phase (5 wk after the initial immunization; Fig. 5⇓, B and D) compared with levels expressed in WT mice at these same time points. Considering that both the initiation and effector phases occur in FcγRIIb−/− mice before the initiation phase in WT mice (7.5 wk after the initial immunization), it is not surprising that expression of cytokine and chemokine mRNA is elevated only in the FcγRIIb−/− mice. However, when we compared cytokine and chemokine mRNA expression levels during the arthritic, effector phase for WT and FcγRIIb−/− mice (11.5 and 5 wk after the initial immunization, respectively), we observed similar levels of expression in both mice (Fig. 5⇓, B and D, and Fig. 6⇓, B and D). These data indicate that FcγRIIb−/− mice express similar levels of cytokines and chemokines as WT mice, but express them at an earlier time point. These results suggest that increased expression of cytokine and chemokine mRNA transcripts correlates with the early development of disease in FcγRIIb−/− mice.

FIGURE 5.
FIGURE 5.

FcγRIIb deficiency alters early mRNA expression of inflammatory cytokines and β-chemokines. Contra lateral ankle joints were obtained from WT and FcγRIIb−/− mice sacrificed at wk 3.5 (A) and 5 (B). Joints were homogenized, and total RNA was extracted. An RNase protection assay was performed using the Riboquant Multiprobe RNase Protection Assay System to detect relative quantities of inflammatory cytokines and β-chemokines. Values represent the mean and SEM of mRNA levels from four mice from each group. ∗∗, Data that are statistically significant (p <0.05) compared with normal, nonimmunized and WT mice.

FIGURE 6.
FIGURE 6.

γ-Chain deficiency alters inflammatory cytokine and β-chemokine mRNA expression late in the development of inflammation. Contra lateral ankle joints were obtained from WT and γ-chain−/− mice sacrificed at wk 7.5 (A) and wk 11.5 (B). Joints were homogenized, and total RNA was extracted. An RNase protection assay was performed using the RiboQuant Multiprobe RNase Protection Assay System to detect relative quantities of inflammatory cytokines and β-chemokines. Spleens were harvested from WT and γ-chain−/− mice and cultured for 5 days. Supernatants were assayed for TNF-α and IFN-γ from mice sacrificed at wk 11.5 by ELISA (C). Values represent the mean and SEM of mRNA levels from four mice from each group and the mean and SEM of cytokine levels from five mice from each group. ∗, Data that are statistically significant (p < 0.05) compared with normal, nonimmunized mice; ∗∗, data that are statistically significant (p < 0.05) compared with normal, nonimmunized and γ-chain-deficient mice; ∗∗∗, data that are statistically significant (p < 0.05) compared with γ-chain-deficient mice.

When we examined cytokine and chemokine expression in γ-chain−/− mice, we observed that TNF-α, IFN-γ, IL-6, IL-1Ra, MIP-1β, MIP-1α, MIP-2, and MCP-1 mRNA transcripts were expressed at equivalent levels to those obtained in WT mice during the pre-arthritic, initiation phase (7.5 wk after the initial immunization; Fig. 6⇑, A and C). However, when we examined the joints of γ-chain−/− mice during the arthritic, effector phase for WT mice (11.5 wk after the initial immunization), γ-chain−/− mice expressed significantly lower amounts of inflammatory cytokines and β-chemokines than WT mice (Fig. 6⇑, B and D). Taken together, these data demonstrate that γ-chain FcγRs are not responsible for the cytokine and chemokine responses observed during the initiation phase of this disease. However, γ-chain FcγR expression is necessary for the maintenance or enhancement of the expression of cytokines and chemokines during the arthritic, effector phase.

Cellular infiltrate was absent in γ-chain−/− mice during the arthritic, effector phase of inflammation (Fig. 2⇑C); therefore, the reduction in inflammatory cytokine and β-chemokine mRNA expression could be attributed to the absence of infiltrating cells. However, a decrease in the expression of inflammatory mediator transcripts could also be due to an inability of γ-chain−/− mice to respond to immune complex stimulation. Consequently, we examined ex vivo production of TNF-α and IFN-γ from spleens dissected from WT and γ-chain−/− mice to evaluate the overall functionality of γ-chain−/− cells in terms of cytokine production. When we assessed production of these inflammatory cytokines 11.5 wk after the initial immunization, we found that splenocytes isolated from γ-chain−/− mice produced significantly less TNF-α and IFN-γ than those from WT mice (Fig. 6⇑E), suggesting that γ-chain−/− cells are dysfunctional in cytokine production. Taken together, these results suggest that γ-chain FcγR expression controls inflammatory cytokine production, and that the reduction in γ-chain FcγR-dependent cytokine and chemokine production might contribute to the suppression of disease in γ-chain−/− mice.

Discussion

To our knowledge this is the first study that demonstrates a mechanism by which coordinate expression of the stimulatory FcγRs (FcγRI and FcγRIII) and the inhibitory FcγR (FcγRIIb) regulates the inflammatory process that determines the susceptibility to PGIA. We show that FcγRs regulate the inflammatory response in PGIA by controlling cytokine and chemokine expression in the joint. Our first observation is that γ-chain−/− (FcγRI−/− and FcγRIII−/−) mice are completely protected from arthritis, demonstrating that FcγRI and/or FcγRIII are required for development of the disease. In contrast, FcγRIIb−/− mice develop augmented disease that begins at an earlier time and is more severe than that in WT mice, indicating that FcγRIIb is essential for controlling disease severity.

Protection from PGIA in γ-chain−/− mice confirms similar observations in collagen-induced arthritis (CIA), immune-complex-mediated arthritis (ICA), and the K/B×N model. However, there are unique differences between the PGIA model and these other models of arthritis. For example, in PGIA both joint swelling and histological signs of inflammation are completely absent in γ-chain−/− animals, even 162 days after immunization. Contrary to PGIA, in CIA a mild form of arthritis develops in some γ-chain−/− mice (25). Since anti-collagen type II Abs are a prerequisite for the development of CIA, other factors, such as complement, may contribute to a greater extent in the development of inflammation in CIA (41, 42). The requirement for FcγRI or FcγRIII differs between murine models of arthritis as well. In the ICA model, reduced cartilage damage is observed in FcγRI−/− mice, but not in FcγRIII−/− mice, whereas in the K/B×N model a significant reduction in ankle swelling is only observed in FcγRIII−/− mice (43, 44). This disparity in the requirement for γ-chain FcγRs suggests that different mechanisms are involved in the development of disease in these arthritis models.

There are several explanations for why inhibition or exacerbation of disease is dependent on FcγR expression. In PGIA, systemic immunity to the immunizing Ag develops before any signs of disease (30, 31, 45). Therefore, FcγRs may contribute to systemic immunity by regulating B cell or T cell priming. With regard to FcγR control of B cell priming, evidence suggests that γ-chain FcγRs may alter B cell Ab production (36, 46). However, a deficiency in γ-chain expression does not affect the secretion of PG-specific Abs, indicating that Ab production in this model is independent of the γ-chain FcγR (Fig. 3⇑). In contrast, FcγRIIb directly suppresses Ab production (36). Our data are consistent with this hypothesis, since PG-specific Ab levels in FcγRIIb−/− mice are elevated early in disease concomitant with the early onset of arthritis. This concept is also supported by the observation in the K/B×N model that Abs from arthritic mice can transfer disease into WT and FcγRIIb−/− mice with similar kinetics and severity (47). These data imply that by circumventing the development of an Ab response, one bypasses the requirement for FcγRIIb regulation of that response. Taken together, these data indicate that FcγRIIb controls the development of arthritis by regulating Ab production.

With regard to FcγR control of T cell priming, T cell priming might be altered in γ-chain−/− mice because the γ-chain is shared with the CD3 complex and/or because efficient Ag presentation occurs through FcγR uptake of Ag (38, 39, 47, 48). Despite a decrease in overall PG-specific T cell proliferation in cells isolated from PG-immunized γ-chain−/− mice, splenocytes from γ-chain−/− mice adoptively transferred into SCID mice induce arthritis with similar kinetics and severity as WT splenocytes. These results demonstrate that T cells and B cells in immunized γ-chain−/− and WT mice are primed to a similar extent. In addition, these experiments separate the initial development of autoreactive B cell and T cells from downstream effector functions mediated through stimulatory FcγRs.

Our second important observation in this study is that coordinate expression of FcγRs regulates disease severity by controlling cytokine and chemokine activation. Although FcγRIIb and the γ-chain FcγRs control the stimulation and inhibition of cytokines and chemokines, these receptors control function at different points in the development of PGIA. Development of arthritis in PGIA can be separated into an initiation phase and an effector phase. During the initiation phase, clinical and histologic signs of arthritis are absent; however, systemic PG-specific T cell and B cell responses are detected. Here we show that cytokine and chemokine transcripts are also expressed in the joint before the onset of arthritis (Fig. 6⇑, A and C) (30, 31). During the effector phase of PGIA, once erythema and swelling are observed, the expression of cytokine and chemokine transcripts is maintained and/or further elevated, followed by joint destruction (Fig. 6⇑, B and D) (33). The release of these inflammatory mediators can be mimicked in vitro by cross-linking FcγRs on monocytes (5, 6). Since FcγR-bearing macrophages are present in the synovial membrane, it is possible that the activation of cytokines and chemokines are FcγR dependent.

In WT mice, cytokine and chemokine expression in the joint remained stable at early time points after the initial immunization (3.5 and 5 wk) compared with that in naive mice. However, by 7.5 wk mRNA transcript expression was enhanced (Fig. 5⇑ and Fig. 6⇑, A and C), and this time point corresponds to the initiation phase in WT mice. Conversely, in FcγRIIb−/− mice, activation of cytokines and chemokines is enhanced as early as 3.5 wk (Fig. 5⇑, A and C), demonstrating that the initiation phase for FcγRIIb−/− mice begins much earlier than that for WT mice. These results correlate early, elevated levels of cytokine and chemokine expression with accelerated disease development in FcγRIIb-deficient mice.

In contrast to the expression patterns observed in FcgRIIb−/− and WT mice, equivalent levels of inflammatory cytokines and β-chemokines are expressed in the joints of γ-chain−/− and WT mice during the initiation phase of the disease (Fig. 6⇑, A and C). Although initiation of inflammatory mediator expression appears to be γ-chain FcγR independent, it is possible that cytokine and chemokine expression is driven by Ab deposition in the joint. In support of this hypothesis, in FcγRIIb−/− mice, enhanced levels of Abs coincide with the activation of inflammatory mediators. There are several possible explanations of how Abs might stimulate cytokine and chemokine release during the initiation phase independently of γ-chain FcγR stimulation. One possibility is the recent finding of a low level of expression of FcγRI due to the continual expression of FcγRI α-chain in γ-chain−/− mice. However, γ-chain−/− macrophages are unable to phagocytose or produce cytokines in response to FcγR stimulation (46). Therefore, the FcγRI that is expressed in the absence of γ-chain expression is unlikely to be functional. A second explanation is that immune complexes may trigger the activation of the complement system and the generation of pro-inflammatory mediators. In support of this concept, in CIA and the K/B×N model, complement plays a critical role in the development of inflammation (41, 43, 49). In ICA, Ab and complement deposition in the joint is observed before the onset of arthritis (50). In addition, complement components, such as active C1q, C3a, and C5a, have the ability to stimulate production of inflammatory mediators, such as IL-1β, IL-6, IL-8, TNF-α, MIP-1β, MIP-1α, MIP-2, and MCP-1 (51, 52, 53, 54, 55, 56). Together, these studies suggest that Ab deposition, followed by complement activation, may activate inflammation.

The levels of cytokines and chemokines expressed in the joints of FcγRIIb−/− and WT mice during the initiation phase are sustained or enhanced in FcγRIIb−/− and WT mice during the effector phase (5 and 11.5 wk, respectively) (Fig. 5⇑, B and D, and Fig. 6⇑, B and D). On the other hand, γ-chain−/− mice at 11.5 wk express reduced inflammatory cytokine and β-chemokine transcripts compared with arthritic WT mice (Fig. 6⇑, B and D). These data correlate the expression of inflammatory mediators with γ-chain FcγR expression as well as arthritis, and they also demonstrate that γ-chain FcγRs are necessary for the maintenance or enhancement of the cytokine and chemokine responses that began during the initiation phase. It is possible that the increases in cytokine and chemokine expression observed in arthritic WT mice are due to the influx of inflammatory leukocytes. Therefore, the absence of infiltrating cells in γ-chain−/− mice could lead to a reduction in inflammatory mediator expression. Our ex vivo studies also demonstrate an inability of γ-chain−/− splenocytes to produce similar levels of inflammatory cytokines as WT cells. These results suggest that γ-chain−/− cells infiltrating the joint would not be activated by immune complex cross-linking of the FcγRs, and thereby would not be able to maintain or enhance the production of inflammatory mediators that commenced during the initiation phase. In further support of this hypothesis, in ICA studies infiltration without enhancement of inflammatory mediator expression is observed in the joints of γ-chain−/− mice (50).

Taken together, these studies suggest a possible cooperative mechanism of inflammation between the complement system and FcγRs, in that both are necessary for full induction of arthritis. We propose a model of arthritis that begins with systemic T cell and B cell responses, followed by Ab deposition and the formation of immune complexes in the joints. Subsequent complement deposition in the joint initiates inflammatory cytokine and β-chemokine expression that is observed before the development of arthritis. The chemotaxtic properties of complement plus β-chemokines stimulate the influx of primed B and T cells, macrophages, and neutrophils into the joint. Infiltrating FcγRI/FcγRIII-bearing cells bind to immune complexes that are either within the synovial fluid or bound to the joint itself, thereby further activating the expression of cytokines and chemokines. The release of these newly expressed inflammatory mediators serves to maintain or amplify the response that was initiated by the activation of complement.

Our studies demonstrate that FcγRs play an important and vital role in the development of joint inflammation. The possible role of FcγRs in PGIA is to control both inflammatory cytokine and β-chemokine production. Further studies are necessary to conclusively determine whether complement or immune complex/FcγR interactions drive inflammatory cytokine and β-chemokine production.

Acknowledgments

We thank Dr. T. T. Glant for providing human and mouse PGs, and Drs. J. Zhang and K. Mikecz for their helpful suggestions and critical comments during manuscript preparation.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants AR45652 and AR47657 (to A.F.).

  • ↵2 Address correspondence and reprint requests to Dr. Charles D. Kaplan, Department of Immunology/Microbiology, Rush-Presbyterian-St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. E-mail address: charles_kaplan{at}rush.edu

  • ↵3 Abbreviations used in this paper: PG, proteoglycan; CIA, collagen-induced arthritis; γ-chain−/−, FcγRI−/− and FcγRIII−/−; ICA, immune-complex-mediated arthritis; PGIA, proteoglycan-induced arthritis; hPG, human PG; mPG, native mouse PG; IL-1Ra, IL-1R antagonist; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; WT, wild type.

  • Received July 3, 2002.
  • Accepted September 11, 2002.

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