1. Top of page
  2. Abstract
  6. Acknowledgements


To study the necessity for activating Fcγ receptor types I and III (FcγRI and FcγRIII) in proteoglycan-induced arthritis (PGIA), a murine model of rheumatoid arthritis, and to determine whether usage of FcγRI or FcγRIII correlates with the Th1 phenotype or the autoantibody isotype in PGIA.


PGIA was induced by immunizing FcγRI−/−, FcγRIII−/−, and wild-type (WT) littermate mice with human PG. The development and severity of arthritis were monitored over time. PG-specific T cell interleukin-2 (IL-2) production and B cell antibody responses were assessed. FcγRIII blocking antibodies were used to inhibit arthritis in an adoptive transfer system. Inflammation in the hind paws was evaluated by assessing cytokine and chemokine messenger RNA (mRNA) transcripts by real-time polymerase chain reaction.


FcγRI−/− mice developed arthritis with similar kinetics and severity as WT littermate controls, whereas FcγRIII−/− mice failed to develop the disease. Both FcγRI−/− and FcγRIII−/− mice produced similar amounts of PG-specific antibody and IL-2 as littermate controls. Transfer of arthritis was successfully blocked in mice treated with a blocking antibody against FcγRIII. FcγRIII−/− mice displayed a significant decrease in cytokine and chemokine mRNA transcripts obtained from the hind paws of immunized mice, whereas FcγRI−/− mice demonstrated a similar increase in cytokine and chemokine transcripts as controls.


These results demonstrate that FcγRIII expression is critical to the development of PGIA, and usage of FcγRIII correlates with the IgG1 isotype of the PG-specific antibody response. FcγRIII expression appears to be important in the effector phase of arthritis, possibly by activating cytokine- and chemokine-secreting cells in the joint.

Autoantibody production in rheumatoid arthritis and the formation of IgG immune complexes (ICs) in the synovium are thought to be involved in the activation and infiltration of hematopoietic cells (1, 2). The binding and crosslinking of ICs to Fc receptors specific for IgG (FcγR) on leukocytes triggers the activation and regulation of a variety of cellular responses, including the release of inflammatory cytokines and chemokines (3–7). Two activating FcγR have been described in the mouse: FcγRI and FcγRIII (8). The high-affinity FcγRI is capable of binding monomeric IgG2a and IgG2a ICs. The low-affinity FcγRIII binds polymeric IgG of different IgG isotypes with different affinities (9). Based on these differences, it has been proposed that FcγRI prefers the IgG2a IC, whereas FcγRIII favors IgG1 and IgG2b ICs.

In proteoglycan-induced arthritis (PGIA), immunization with human PG leads to the development of autoreactive T cells and autoantibodies to murine PG (10). These autoantibodies gain access to the joint and bind cartilage PGs, forming ICs. We have recently shown that FcγR are critically involved in the development of arthritis. In FcR γ-chain−/− mice, joint inflammation is completely suppressed despite the activation of autoreactive T cells and B cells (11). Similar to PGIA, in several other murine models of arthritis, FcγR γ-chain expression is critical to the development of disease (12–16). Although these models have demonstrated the importance of FcγR expression, there is no clear consensus on which one, FcγR, FcγRI, or FcγRIII, is critical. In antigen-induced arthritis (AIA), neither FcγRI nor FcγRIII is involved in joint swelling and leukocyte infiltration, but cartilage damage is reduced in FcγRI−/− mice (17). In IC-mediated arthritis (ICA), FcγRIII is the principal activating FcγR that mediates joint inflammation; however, both FcγRI and FcγRIII are involved in cartilage destruction (18). In contrast, in the K/BxN model and collagen-induced arthritis (CIA), a significant reduction in joint swelling is observed in FcγRIII−/− mice (14, 19). This disparity in the requirement for FcγR suggests that different mechanisms may be involved in the development of disease.

One of the major differences between PGIA and other models of arthritis is the dependence on interferon-γ (IFNγ). IFNγ is an important proinflammatory cytokine released by Th1 cells, and it is central to the development of PGIA (20). We found that arthritis is suppressed and disease onset is substantially delayed in mice treated with anti-IFNγ antibodies or in IFNγ−/− mice (21). One of the important functions of IFNγ is the induction of FcγRI expression and the enhancement of IgG2a secretion via class switch (17, 22). Increased expression of FcγRI could facilitate enhanced binding of IgG2a ICs. Based on this information, we would speculate that FcγRI might be involved in PGIA. However, despite the high levels of IFNγ in PGIA, the PG-specific autoantibody response is dominated by the IgG1 isotype (23). If the IgG1 ICs containing PG-specific autoantibodies are important for arthritis, then FcγRIII may be involved in PGIA. To distinguish between the requirements for FcγRI and FcγRIII expression in PGIA, we assessed the development of PGIA in FcγRI−/− and FcγRIII−/− mice.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Antigen preparation.

Human cartilage tissue was obtained at the time of joint replacement surgery. PG from adult cartilage was prepared as previously described (24). Briefly, cartilage pieces were pulverized in liquid nitrogen and then extracted at 4°C with 4M guanidinium chloride in 50 mM sodium acetate, pH 5.8, containing protease inhibitors. 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). Native murine PG was obtained from the cartilage of newborn mice and prepared in a manner similar to human PG except that murine PG was not deglycosylated.

Immunization and assessment of arthritis.

Female BALB/c wild-type (WT) and BALB/c SCID mice were obtained from the National Cancer Institute (Frederick, MD). FcγRI−/− (CD64−/−) and FcγRIII−/− (CD16−/−) mice were developed by Dr. J. S. Verbeek (Leiden University Medical Center), as previously described (17, 25). FcγRI and FcγRIII heterozygous mice were backcrossed 8 generations on BALB/c and then intercrossed, and littermate FcγRI−/− and FcγRI+/+ and FcγRIII−/− and FcγRIII+/+ mice were used in all experiments. Littermate controls were used because differences between FcγRI+/+ and FcγRIII+/+ mice may reflect small genetic differences. Female mice >12 weeks of age were immunized intraperitoneally (IP) with 150 μg of PG measured as protein in Freund's complete adjuvant, as previously described (11, 20, 24). Mice were boosted twice IP with 100 μg of PG in Freund's incomplete adjuvant at 3-week intervals. Animal protocols were approved by the Institutional Animal Care and Use Committee of Rush University Medical Center.

The development of arthritis was scored by a blinded observer (YC). 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 0–4 scale as follows: 0 = normal; 1 = mild erythema and swelling of the paw or 1 or 2 toes; 2 = moderate erythema and swelling of the paw; 3 = more diffuse erythema and swelling of the paw; 4 = severe erythema and swelling of the entire paw. Each paw was scored individually. The maximum cumulative score ranged from 0 to 16. Results are presented in relation to days after the initial immunization. Histologic 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 (20). Sagittal sections were stained with hematoxylin and eosin.

Adoptive transfer of PGIA into SCID mice.

Splenocytes (5 × 107 cells/mouse) from arthritic WT mice and 150 μg human PG measured as protein were mixed in saline and injected IP into female BALB/c SCID mice, as previously described (11, 26). SCID mice were injected IP with either 250 μg of anti-FcγRIII/FcγRIIb (2.4G2) or 250 μg of anti-human class II major histocompatibility complex (DR5) starting 1 day before spleen cell transfer and every third day until day 32 after spleen cell transfer. The SCID mice were monitored for disease onset and severity every other day.

Determination of serum antibody titers.

Mice were bled from the orbital plexus. Isotype-specific anti-PG antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Plates (96-well Nunc-Immuno; Fisher Scientific, Pittsburgh, PA) were coated with 1.0 μg chondroitinase ABC–digested human PG, 1.5 μg native murine PG, or titrated concentrations of myeloma protein IgG1 and IgG2 in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Sera were serially diluted in phosphate buffered saline in 0.5% Tween 20. Serum dilutions of 1:100, 1:500, and 1:2,500 were used to detect murine PG–specific antibodies, while dilutions of 1:2,500, 1:12,500, and 1:62,500 were used to detect human PG–specific antibodies. Isotypes were detected with isotype-specific antibodies horseradish peroxidase (HRP)–conjugated rabbit anti-mouse IgG1 and HRP-conjugated IgG2a (Zymed, San Francisco, CA), followed by the substrate orthophenylenediamine, and absorbance was measured spectrophotometrically at 490 nm. Myeloma IgG1 and IgG2a proteins were titrated to generate a standard curve from which the relative concentration of IgG1 and IgG2a in the sera was determined.

Assessment of interleukin-2 (IL-2) production.

Splenocytes (2 × 106 cells/ml) were incubated in a 24-well Falcon plate (Fisher Scientific) in serum-free HL-1 medium (Cambrex BioScience, Walkersville, MD) in the presence or absence of 20 μg/ml human PG. IL-2 from supernatants harvested at 24 hours was measured by ELISA, using the OPT EIA mouse set (Pharmingen, San Diego, CA).

Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR).

One hind paw from each mouse was homogenized with a Polytron homogenizer in TRI reagent (Molecular Research, Cincinnati, OH) on ice. Homogenate was centrifuged to remove large debris, and RNA was extracted. Reverse transcription was performed with random hexamers for priming and SuperScript II (Invitrogen, Carlsbad, CA). The optimum concentration of probe, primers, and MgCl2 was determined in preliminary experiments: the PCR volume was 25.5 μl, containing 0.3 μl of each primer and 0.3 μl of Taq (Invitrogen). The annealing temperature was 58°C for all the primers. Complementary DNA was analyzed for the expression of cytokine PCR products with the DNA-intercalating SYBR Green I fluorescent dye, using a GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA).

Using the Omiga program (Oxford Molecular, San Diego, CA), primers were constructed based on the gene sequence or on published sequences. The following primer sequences were used: for murine monocyte chemotactic protein 1 (MCP-1), forward 5′-CCC-AAT-GAG-TAG-GCT-GGA-GA-3′ and reverse 5′-TCT-GGA-CCC-ATT-CCT-TCT-TG-3′; for murine MCP-2, forward 5′-TAA-GGC-TCC-AGT-CAC-CTG-CT-3′ and reverse 5′-TCT-GGA-AAA-CCA-CAG-CTT-CC-3′; for murine macrophage inflammatory protein α (MIP-1α), forward 5′-ATG-AAG-GTC-TCC-ACC-ACT-GC-3′ and reverse 5′-GAT-GAA-TTG-GCG-TGG-AAT-CT-3′; for murine RANTES, forward 5′-ATA-TGG-CTC-GGA-CAC-CAC-TC-3′ and reverse 5′-TCC-TTC-GAG-TGA-CAA-ACA-CG-3′; for murine IL-1β, forward 5′-TTG-ACG-GAC-CCA-AAA-GAT-G-3′ and reverse 5′-AGA-AGG-TGC-TCA-TGT-CCT-CA-3′; for murine tumor necrosis factor α (TNFα), forward 5′-ACG-GCA-TGG-ATC-TCA-AAG-AC-3′ and reverse 5′-GTG-GGT-GAG-GAG-CAG-GTA-GT-3′; and for murine β-actin, forward 5′-CAC-TGT-CGA-GCT-GCG-TCC-AC-3′ and reverse 5′-GCG-AAG-CCG-GCT-TTG-CAC-AT-3′.

To verify that equivalent amounts of RNA were added to each PCR, PCR amplification of murine β-actin was performed for each sample. The relative differences among the samples at different time points were determined using the delta delta Ct (ΔΔCt) method, as outlined in the manufacturer's protocol for RT-PCR (Applied Biosystems). The ΔCt value was calculated for each sample using the Ct value of murine β-actin to account for loading differences in RT-PCR and the Ct value of the input DNA samples to normalize the cytokine and chemokine results. The ΔΔCt value was then calculated by subtracting the ΔCt of the control (paw RNA from nonimmune mice) for each time point ΔCt within an experiment. The ΔΔCt values were converted to fold differences compared with the control by raising 2 to the −ΔΔCt power. Data represent the mean ± SEM of 4–5 mice from each group.

Statistical analysis.

The Mann-Whitney U test was used to compare nonparametric data for statistical significance. P values less than 0.05 were considered significant.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Dependence of PGIA development on FcγRIII expression.

Studies in a variety of arthritis models have demonstrated that FcRγ−/− mice are resistant to disease development (11–16). However, these studies differ in terms of which FcγR is critical for the progression of arthritis. To distinguish between the requirement for FcγRI and the requirement for FcγRIII in PGIA, we immunized FcγRI−/− and FcγRIII−/− mice and monitored the onset and severity of disease. FcγRIII−/− mice failed to develop any signs of inflammation, whereas the littermate control mice developed arthritis beginning 20 days after the last immunization with PG, reaching a maximal severity score of 9.75 ± 1.75 (mean ± SEM) (Figures 1A and C). Conversely, FcγRI−/− mice developed arthritis with a severity similar to that in littermate controls. Littermate controls developed the disease starting approximately on day 15 and with maximal arthritis scores reaching 8.2 ± 1.4, and FcγRI−/− mice also developed arthritis approximately on day 15, reaching maximal scores of 9.0 ± 2.22 (Figures 1B and D). The incidence of arthritis in FcγRI−/− mice (70%) was somewhat reduced in comparison with controls (100%), but the difference was not statistically significant. These data suggest that FcγRIII expression is critical for the development of PGIA, whereas the expression of FcγRI has a minimal effect.

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Figure 1. Inhibition of arthritis development in mice deficient in Fcγ receptor type III (FcγRIII). Fifteen FcγRIII+/+, 16 FcγRIII−/−, 13 FcγRI+/+, and 12 FcγRI−/− mice were immunized with human proteoglycan, and disease incidence and onset (A and B) and severity (C and D) were monitored over time. Values are the percent incidence and the mean ± SEM arthritis score. ∗ = P < 0.05, FcγRIII+/+ versus FcγRIII−/− mice, by Mann-Whitney U test.

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Prevention of histopathologic changes characteristic of PGIA by loss of FcγRII.

The inflammation observed as erythema and swelling of the paws after immunization with PG was accompanied by histopathologic changes that led to extensive cartilage and bone destruction over time. We assessed the effects of FcγRI and FcγRIII deficiency on joint histology in the groups of animals (Figure 2). Histopathologic signs of disease characteristically displayed during PGIA development were completely absent in the joints of FcγRIII−/− mice. In these mice, the lack of paw erythema and swelling correlated with the absence of cellular infiltration and joint destruction (Figure 2A). Conversely, in littermate controls and FcγRI−/− mice there was severe arthritis in the ankle joint, with edema of the synovial and periarticular tissues accompanied by synovial hyperplasia. Mononuclear and polymorphonuclear cell infiltration was abundant in the tissues and joint spaces, and signs of cartilage damage and hyperplastic chondrocytes were observed (Figures 2B–D). These results demonstrate that a deficiency in FcγRIII blocks the development of paw swelling, cellular infiltration, and synovial hyperplasia.

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Figure 2. Fcγ receptor type III (FcγRIII) deficiency prevents histopathologic changes associated with development of proteoglycan-induced arthritis. Sections are from ankle joints of A, FcγRIII−/−, B, FcγRIII+/+, C, FcγRI−/−, and D, FcγRI+/+ mice. Sections were stained with hematoxylin and eosin. Arrowheads indicate areas of cartilage destruction; thick arrows indicate areas of bone erosion; thin arrows indicate areas of cellular infiltration.

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Lack of association between suppression of arthritis in FcγRIII−/− mice and reduction in PG-specific antibody production or T cell priming.

Since FcγR indirectly regulates antibody responses (27), we assessed whether resistance to PGIA in FcγRIII−/− mice was due to a reduction in development of antibodies to PG. Mice were bled at a time point when littermate control mice were maximally arthritic, and PG-specific IgG1 and IgG2a serum antibody titers were measured by ELISA. FcγRI−/− and FcγRIII−/− mice produced levels of PG-specific antibody equivalent to those in control mice (Figures 3A and B). These results demonstrate that neither FcγRI nor FcγRIII regulates production of antibodies to PG and that resistance to arthritis development in FcγRIII−/− mice was not due to suppression of antibody production.

FcγR-mediated uptake of antigen is an efficient mechanism for antigen presentation to T cells (28). Furthermore, in PGIA, activation of PG-specific T cells is essential for arthritis development. Therefore, it was possible that a loss of antigen presentation by cells that lack either FcγRI or FcγRIII could alter T cell priming to PG. To address whether T cells were sufficiently primed in FcγRIII−/− mice, we compared IL-2 production from splenocytes isolated from FcγRI+/+, FcγRI−/−, FcγRIII+/+, and FcγRIII−/− mice at the time of death. FcγRI−/− and FcγRIII−/− mice produced levels of IL-2 that were similar to those in littermate controls (Figures 3C and D), demonstrating that neither FcγRI nor FcγRIII was necessary for the initiation of T cell responses in PG-immunized mice. Taken together, these data indicate that B cell and T cell priming to PG was unaffected by the deletion of FcγRI or FcγRIII, and that the resistance to PGIA development in FcγRIII−/− mice was not due to inadequate activation of T or B cells necessary for the development of arthritis.

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Figure 3. Antibody production and T cell priming in Fcγ receptor I–deficient (FcγRI−/−) and FcγRIII−/− mice. A and B, Serum was obtained from FcγRIII+/+ and FcγRIII−/− mice and from FcγRI+/+ and FcγRI−/− mice. The serum anti–human proteoglycan (hPG) and anti–murine PG (mPG) antibody isotypes (IgG1 and IgG2a) were measured by enzyme-linked immunosorbent assay (ELISA). C and D, Spleens of FcγRIII+/+ and FcγRIII−/− mice and of FcγRI+/+ and FcγRI−/− mice were harvested at the time of death, and interleukin-2 (IL-2) levels in supernatants from spleen cells cultured for 24 hours were measured by ELISA. Values are the mean and SEM. Differences between FcγRIII+/+ and FcγRIII−/− mice and between FcγRI+/+ and FcγRI−/− mice were not statistically significant.

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Inhibition of arthritis effector phase by blocking FcγRIII.

To further confirm that the deficiency in FcγRIII does not interfere with a systemic response to PG, we used an adoptive transfer system in which spleen cells from arthritic WT mice transferred arthritis into SCID recipient mice. Prior to the transfer of spleen cells, SCID mice were treated with anti-FcγRIII/FcγRIIb (2.4G2) or anti-DR5 as a control. SCID mice were treated 1 day before cell transfer and every third day until day 32. If FcγRIII were responsible for controlling inflammation in the effector phase, when T cells and B cells are adequately primed for the induction of PGIA, then anti-FcγRIII/FcγRIIb treatment should block arthritis development. Upon transfer of splenocytes, PGIA was effectively suppressed in SCID mice treated with anti-FcγRIII/FcγRIIb in comparison with animals treated with anti-DR5. Forty percent of control mice exhibited signs of inflammation on day 22, and 100% of the mice were arthritic by day 26 (Figure 4A). In contrast, in 40% of the mice treated with anti-FcγRIII/FcγRIIb, arthritis did not develop until day 36, 4 days after treatment was halted. Thereafter, mice treated with anti-FcγRIII/FcγRIIb developed arthritis with similar severity to that in animals treated with the control (Figures 4A and B). These results demonstrate a direct correlation between blocking of FcγRIII and prevention of joint inflammation. Furthermore, these findings suggest that FcγRIII is involved in PGIA at a time point after the development of systemic immunity to PG.

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Figure 4. Inhibition of adoptive transfer of proteoglycan-induced arthritis into SCID mice by anti–Fcγ receptor III (FcγRIII)/RIIb antibody (2.4G2) treatment. Splenocytes were obtained from wild-type arthritic mice and adoptively transferred into SCID mice, followed by immunization with proteoglycan. SCID mice were treated with 250 μg 2.4G2 (n = 5) or with 250 μg anti-DR5 (n = 5) on days −1, 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, and 32. Mice were monitored for disease incidence and onset (A) and severity (B). Values are the percent incidence and the mean ± SEM arthritis score. ∗ = P < 0.05 versus anti-FcγRIII/RIIb–treated mice, by Mann-Whitney U test.

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Correlation between joint inflammation and enhanced cytokine and chemokine messenger RNA (mRNA) expression.

We have previously demonstrated that chemokine and cytokine mRNA transcripts are elevated in the paws of arthritic mice (11, 20, 21). To determine whether FcγRI and/or FcγRIII influence the expression of inflammation mediators in the joint, we assessed chemokine and cytokine mRNA transcripts in PG-immunized arthritic and nonarthritic mice in comparison with nonimmune mice. Hind paws were harvested from FcγRI+/+, FcγRI−/−, and FcγRIII+/+ mice when the arthritis score of the paw was ∼4. Hind paws were harvested from nonarthritic FcγRIII−/− mice at the same time as from arthritic FcγRIII+/+ mice. Similar levels of chemokine and cytokine mRNA expression were observed in the joints of arthritic FcγRI+/+ and FcγRI−/− mice (Figure 5A). However, when FcγRIII−/− mice were compared with FcγRIII+/+ mice, levels of chemokines MCP-1 and MIP-1α and cytokines TNFα and IL-1β were significantly reduced whereas MCP-2 and RANTES were diminished, but not to a degree that reached statistical significance (Figure 5B). These results suggest that in the absence of FcγRIII-expressing cells in the joint, IC binding is inhibited, which reduces expression of cytokines and chemokines.

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Figure 5. Dependence of cytokine and chemokine responses on Fcγ receptor III (FcγRIII) expression. Ankle joints were harvested from 4 normal (nonimmunized), 5 FcγR+/+, 5 FcγRI−/−, 5 FcγRIII+/+, and 5 FcγRIII−/− mice. Joints were homogenized and total RNA was extracted from A, FcγRI+/+ and FcγRI−/− and B, FcγRIII+/+ and FcγRIII−/− mice. Real-time polymerase chain reaction was performed to detect relative quantities of monocyte chemotactic protein 1 (MCP-1), MCP-2, macrophage inflammatory protein 1α (MIP-1α), RANTES, interleukin-1β (IL-1β), and tumor necrosis factor α (TNFα). Values are the mean and SEM. ∗ = P < 0.05 versus FcγRIII+/+ mice, by Mann-Whitney U test.

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Alternatively, these data may reflect inhibition of cells recruited to the joint that could be activated by IC crosslinking. Since the inhibition of cytokine and chemokine mRNA was only partially dependent on FcγRIII expression, other mechanisms are also important for cytokine and chemokine expression in the joint. The difference in the increase in expression of mRNA in the FcγRI+/+ and FcγRIII+/+ mice was substantial. This does not reflect a difference in the degree of inflammation because the arthritis score was similar for all arthritic paws. This variation may implicate some difference in the background genes of the different BALB/c mice.


  1. Top of page
  2. Abstract
  6. Acknowledgements

In previous studies we demonstrated the necessity for FcγR in PGIA (11). We have also shown that IFNγ is critically important for the induction of disease (21). Since IFNγ regulates FcγRI and IgG2a antibody isotype expression and FcγRI preferentially binds to IgG2a IC, we speculated that FcγRI might be the FcγR involved in PGIA. However, despite the high levels of IFNγ in PGIA, the dominant PG-specific antibody isotype is IgG1 (20). Since IgG1 IC preferentially binds to FcγRIII, these data also suggested the possibility that FcγRIII could be involved in PGIA (9). In this study, we demonstrated that FcγRIII expression is critical for the development of PGIA, whereas FcγRI expression is not necessary. Upon immunization with PG, FcγRI−/− mice developed disease with similar kinetics and severity as in littermate controls, whereas FcγRIII−/− mice failed to develop arthritis (Figures 1 and 2). Thus, the requirement for FcγRIII correlates with the dominant IgG1 autoantibody isotype produced in PGIA. In addition to antibody isotype, the cell population responsible for inflammation may determine the relevant FcγR. In PGIA, neutrophils are the dominant cell population in the inflamed joint, and FcγRIII is the only activating FcγR expressed on murine neutrophils (17). Thus, it is plausible that the loss of FcγRIII on neutrophils may inhibit neutrophil binding to IgG1 IC in the joint, thus preventing their retention and/or activation in the joint.

These findings corroborate the results of experiments in K/BxN CIA and ICA models, which also demonstrate a significant reduction in ankle swelling in FcγRIII−/− mice (14, 19). However, these data differ from findings in the AIA model, where neither FcγRI nor FcγRIII is involved in joint swelling and leukocyte infiltration, although reduced cartilage damage is observed in FcγRI−/− mice (17). The disparity in the requirement for FcγR in AIA suggests that other mechanisms are involved in the development of arthritis.

There are several explanations as to why inhibition of disease is dependent on FcγR expression. PGIA begins as a systemic immune response to human PG that proceeds to an autoreactive response to murine PG prior to any signs of joint involvement (29, 30). FcγR may contribute to systemic immunity by regulating B cell and/or T cell priming. With regard to FcγR control of B cell priming, evidence suggests that FcγR expression may facilitate antibody production (27, 31). However, we were unable to detect a deficiency in the secretion of PG-specific antibody in either FcγRI−/− or FcγRIII−/− mice (Figure 3), indicating that antibody production in PGIA is independent of FcγR expression. FcγR are also involved in T cell priming through the efficient uptake of antigen by antigen-presenting cells (27, 32–35). However, in FcγRI−/− and FcγRIII−/− mice, the PG-specific recall response was equivalent to that in WT mice, indicating that FcγR expression is not critical for T cell priming in this model. It is possible that FcγR expression may be important for T and B cell priming only under conditions in which the concentration of antigen is limiting. Since the development of PGIA requires multiple immunizations with PG, the increase in antigen concentration could overcome any requirement for FcγR in PG-specific T and B cell priming.

The absence of a requirement for FcγRIII in the initiation of an immune response to PG suggests that FcγRIII is important in the effector phase. To confirm the importance of FcγRIII in the effector phase, we successfully blocked transfer of arthritis with an antibody to FcγRIII (Figure 4). Since spleen cells from PG-immune mice contain T and B cells that can induce arthritis, the inhibition with anti-FcγRIII/FcγRII antibody suggests that FcγRIII expression is necessary for inflammation to be maintained in the joint.

In the absence of FcγRIII expression, there was a significant decrease in cytokine and chemokine RNA transcripts in the joint, whereas in FcγRI−/− mice levels similar to those in WT mice were detected (Figure 5). However, these transcripts are only partially reduced in FcγRIII−/− mice despite the fact that there was no evidence of inflammation in the paws. These data indicate that FcγRIII is only partially involved in activation of these proinflammatory mediators and that other mechanisms also contribute to cytokine and chemokine expression in the joint. The reduction in cytokine and chemokine mRNA corresponds to the absence of joint inflammation in FcγRIII−/− mice and suggests that FcγRIII may control activation of cells that are recruited to the joint.

We propose that in PGIA, joint inflammation is initiated by the activation of autoreactive B cells and the release of autoantibodies, followed by IC deposition on joint cartilage surfaces. These ICs instigate some degree of inflammatory cytokine and chemokine expression that occurs independent of FcγR expression (11). The release of chemotactic agents within the joint stimulates the influx of FcγRIII-bearing neutrophils capable of binding ICs that are either within the synovial fluid or bound to joint tissues. The crosslinking of FcγRIII leads to further expression of cytokines and chemokines within the joint, thereby maintaining or amplifying the response.


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  2. Abstract
  6. Acknowledgements

We thank Dr. T. T. Glant for providing human and murine PG, and Dr. K. Mikecz and P. Doodes for their helpful suggestions and critical comments during manuscript preparation.


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  2. Abstract
  6. Acknowledgements
  • 1
    Winchester RJ, Agnello V, Kunkel HG. Gamma globulin complexes in synovial fluids of patients with rheumatoid arthritis: partial characterization and relationship to lowered complement levels. Clin Exp Immunol 1970; 6: 689706.
  • 2
    Harris ED Jr. Pathogenesis of rheumatoid arthritis. Am J Med 1986; 80: 410.
  • 3
    Abrahams VM, Cambridge G, Lydyard PM, Edwards JC. Induction of tumor necrosis factor α production by adhered human monocytes: a key role for Fcγ receptor type IIIA in rheumatoid arthritis. Arthritis Rheum 2000; 43: 60816.
  • 4
    Marsh CB, Wewers MD, Tan LC, Rovin BH. Fcγ receptor cross-linking induces peripheral blood mononuclear cell monocyte chemoattractant protein-1 expression: role of lymphocyte FcγRIII. J Immunol 1997; 158: 107884.
  • 5
    Khalkhali-Ellis Z, Bulla GA, Schlesinger LS, Kirschmann DA, Moore TL, Hendrix MJ. C1q-containing immune complexes purified from sera of juvenile rheumatoid arthritis patients mediate IL-8 production by human synoviocytes: role of C1q receptors. J Immunol 1999; 163: 461220.
  • 6
    Jarvis JN, Wang W, Moore HT, Zhao L, Xu C. In vitro induction of proinflammatory cytokine secretion by juvenile rheumatoid arthritis synovial fluid immune complexes [published erratum appears in Arthritis Rheum 1998;41:377]. Arthritis Rheum 1997; 40: 203946.
  • 7
    Gallin JI. Inflammation. In: PaulWE, editor. Fundamental immunology. 3rd ed. New York: Raven Press; 1993.
  • 8
    Ravetch JV, Kinet JP. Fc receptors [review]. Annu Rev Immunol 1991; 9: 45792.
  • 9
    Hazenbos WL, Heijnen IA, Meyer D, Hofhuis FM, Renardel de Lavalette C, Schmidt RE, et al. Murine IgG1 complexes trigger immune effector functions predominantly via Fcγ RIII (CD16). J Immunol 1998; 161: 302632.
  • 10
    Glant TT, Finnegan A, Mikecz K. Proteoglycan-induced arthritis: immune regulation, cellular mechanisms, and genetics [review]. Crit Rev Immunol 2003; 23: 199250.
  • 11
    Kaplan CD, O'Neill SK, Koreny T, Czipri M, Finnegan A. Development of inflammation in proteoglycan-induced arthritis is dependent on FcγR regulation of the cytokine/chemokine environment. J Immunol 2002; 169: 58519.
  • 12
    Blom AB, van Lent PL, van Vuuren H, Holthuysen AE, Jacobs C, van de Putte LB, et al. FcγR expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA). Arthritis Res 2000; 2: 489503.
  • 13
    Kleinau S, Martinsson P, Heyman B. Induction and suppression of collagen-induced arthritis is dependent on distinct Fcγ receptors. J Exp Med 2000; 191: 16116.
  • 14
    Ji H, Ohmura K, Mahmood U, Lee DM, Hofhuis FM, Boackle SA, et al. Arthritis critically dependent on innate immune system players. Immunity 2002; 16: 15768.
  • 15
    Kagari T, Tanaka D, Doi H, Shimozato T. Essential role of Fcγ receptors in anti-type II collagen antibody-induced arthritis. J Immunol 2003; 170: 431824.
  • 16
    Van Lent PL, van Vuuren AJ, Blom AB, Holthuysen AE, van de Putte LB, van de Winkel JG, et al. Role of Fc receptor γ chain in inflammation and cartilage damage during experimental antigen-induced arthritis. Arthritis Rheum 2000; 43: 74052.
  • 17
    Ioan-Facsinay A, de Kimpe SJ, Hellwig SM, van Lent PL, Hofhuis FM, van Ojik HH, et al. FcγRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection. Immunity 2002; 16: 391402.
  • 18
    Nabbe KC, Blom AB, Holthuysen AE, Boross P, Roth J, Verbeek S, et al. Coordinate expression of activating Fcγ receptors I and III and inhibiting Fcγ receptor type II in the determination of joint inflammation and cartilage destruction during immune complex–mediated arthritis. Arthritis Rheum 2003; 48: 25565.
  • 19
    Diaz de Stahl T, Andren M, Martinsson P, Verbeek JS, Kleinau S. Expression of FcγRIII is required for development of collagen-induced arthritis. Eur J Immunol 2002; 32: 291522.
  • 20
    Finnegan A, Mikecz K, Tao P, Glant TT. Proteoglycan (aggrecan)-induced arthritis in BALB/c mice is a Th1-type disease regulated by Th2 cytokines. J Immunol 1999; 163: 538390.
  • 21
    Finnegan A, Grusby MJ, Kaplan CD, O'Neill SK, Eibel H, Koreny T, et al. IL-4 and IL-12 regulate proteoglycan-induced arthritis through Stat-dependent mechanisms. J Immunol 2002; 169: 334552.
  • 22
    Finkelman FD, Holmes J, Katona IM, Urban JF Jr, Beckmann MP, Park LS, et al. Lymphokine control of in vivo immunoglobulin isotype selection [review]. Annu Rev Immunol 1990; 8: 30333.
  • 23
    Kaplan C, Valdez JC, Chandrasekaran R, Eibel H, Mikecz K, Glant TT, et al. Th1 and Th2 cytokines regulate proteoglycan-specific autoantibody isotypes and arthritis. Arthritis Res 2002; 4: 548.
  • 24
    Glant TT, Mikecz K, Arzoumanian A, Poole AR. Proteoglycan-induced arthritis in BALB/c mice: clinical features and histopathology. Arthritis Rheum 1987; 30: 20112.
  • 25
    Hazenbos WL, Gessner JE, Hofhuis FM, Kuipers H, Meyer D, Heijnen IA, et al. Impaired IgG-dependent anaphylaxis and Arthus reaction in FcγRIII (CD16) deficient mice. Immunity 1996; 5: 1818.
  • 26
    Bardos T, Mikecz K, Finnegan A, Zhang J, Glant TT. T and B cell recovery in arthritis adoptively transferred to SCID mice: antigen-specific activation is required for restoration of autopathogenic CD4+ Th1 cells in a syngeneic system. J Immunol 2002; 168: 601321.
  • 27
    Heyman B. Regulation of antibody responses via antibodies, complement, and Fc receptors [review]. Annu Rev Immunol 2000; 18: 70937.
  • 28
    Ravetch JV, Bolland S. IgG Fc receptors [review]. Annu Rev Immunol 2001; 19: 27590.
  • 29
    Mikecz K, Glant TT. Immunoregulation of proteoglycan-induced arthritis in Balb/c mice. Am J Ther 1996; 3: 4251.
  • 30
    Buzas EI, Mikecz K, Brennan FR, Glant TT. Mediators and autopathogenic effector cells in proteoglycan-induced arthritic and clinically asymptomatic BALB/c mice. Cell Immunol 1994; 158: 292304.
  • 31
    Barnes N, Gavin AL, Tan PS, Mottram P, Koentgen F, Hogarth PM. FcγRI-deficient mice show multiple alterations to inflammatory and immune responses. Immunity 2002; 16: 37989.
  • 32
    Dhodapkar KM, Krasovsky J, Williamson B, Dhodapkar MV. Antitumor monoclonal antibodies enhance cross-presentation of cellular antigens and the generation of myeloma-specific killer T cells by dendritic cells. J Exp Med 2002; 195: 12533.
  • 33
    Reijonen H, Novak EJ, Kochik S, Heninger A, Liu AW, Kwok WW, et al. Detection of GAD65-specific T-cells by major histocompatibility complex class II tetramers in type 1 diabetic patients and at-risk subjects. Diabetes 2002; 51: 137582.
  • 34
    Rafiq K, Bergtold A, Clynes R. Immune complex-mediated antigen presentation induces tumor immunity. J Clin Invest 2002; 110: 719.
  • 35
    Hamano Y, Arase H, Saisho H, Saito T. Immune complex and Fc receptor-mediated augmentation of antigen presentation for in vivo Th cell responses. J Immunol 2000; 164: 61139.