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RII Reduces Joint Inflammation and Destruction in Experimental Immune Complex-Mediated Arthritides Not Only by Inhibition of FcRI/III but Also by Efficient Clearance and Endocytosis of Immune Complexes
From the Department of Rheumatology,* University Medical Centre St. Radboud, Nijmegen, The Netherlands; the Department of Human Genetics, UMC, Leiden, The Netherlands; and the Institute of Experimental Dermatology, University of Munster, Germany
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Abstract |
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RII-/- mice identified the inhibitory function of this receptor in joint inflammation and cartilage destruction induced with immune complexes (ICs). To extend our insight in the role of FcRII in arthritis, we explored the role of FcRII in the absence of activating receptors I and III using FcRI/III-/- as well as FcRI/II/III-/- mice. When antigen-induced arthritis (AIA) was elicited, which is a mixture of T cell and IC-driven inflammation, arthritis was almost absent at day 7 in FcRI/III-/- mice. Remarkably, in FcRI/II/III-/- mice, this model induced a tremendously increased arthritis as compared to wild-type controls. This implies that FcRII regulates joint inflammation also in the absence of activating FcRI and III. To confirm the IC specificity of this finding, similar studies were done with ICs or zymosan as arthritogenic stimuli. Strongly elevated inflammation was found in FcRI/II/III-/- mice with IC but not with zymosan. Clearance studies identified accumulation of IgG in the knee joint in the absence of FcRII. Moreover, macrophages expressing only FcRII showed prominent endocytosis of preformed soluble ICs not different from controls. In total absence of FcR (FcRI/II/III-/-), macrophages completely failed to endocytose ICs. Although joint inflammation was much higher in AIA arthritic knee joints of FcRI/II/III-/- and the inflammatory cells still expressed an inflammatory phenotype, severe cartilage destruction (MMP-mediated neoepitopes in the matrix and chondrocyte death) was completely prevented in contrast to the marked destruction which was observed in the wild-type. Our study indicates that FcRII reduces joint inflammation in the absence of activating FcR by promoting endocytosis and clearance of ICs from the joint. Infiltrating cells, which fail to express activating FcR although they still become stimulated are no longer capable of inducing severe cartilage destruction.
The mechanism by which synovial intima macrophages become activated during RA is not known. One of the potential candidates are IgG-containing ICs. They are abundantly found in RA synovial fluid, synovium, and surface layers of the cartilage.6 In previous studies we have found that lining macrophages are of utmost importance in both onset and prolongation of experimental murine arthritis. When synovial intima macrophages were selectively depleted from the knee joint either before induction or during immune complex (IC)-mediated arthritides like collagen type II or antigen-induced arthritis (AIA), onset and course of arthritis was largely reduced.7-9
IgG-containing ICs communicate with lining macrophages using FcR.10
In the mouse, three classes of FcR have been described. FcRI and III are activating receptors and lead to elevation of intracellular signaling after binding of ICs.11-12
The third class is FcRII, which can co-ligate with either FcRI or FcRIII, resulting in inhibition of intracellular signaling.13
Coordinate expression of activating and inhibiting FcR on synovial lining cells has been shown to regulate both joint inflammation and severe cartilage destruction.10
The inhibiting FcRII exists as two isoforms, FcRIIb1 and FcRIIb2, differing by a 47-amino acid insertion in the intracytoplasmatic domain of FcRII encoded by the first exon of the FcRII gene.14
The in vivo role of FcRII was extensively studied using FcRII-deficient mice and it is generally agreed that inhibition occurs only when FcRII is co-clustered with ITAM-bearing receptors.15-16
The inhibitory function is mediated by the inositol phosphatase SHIP which associates with the phosphorylated ITIM of FcRII via the SHIP SH2 domain17
; however, in vitro studies also suggested other biological functions for FcRII. By transferring cDNA of both FcRII isoforms into fibroblastic cell lines which do not express FcR, it was found that FcRIIb2 is involved in endocytosis and enhancement of antigen presentation.18-20
FcRIIb1, which is preferentially expressed in B lymphocytes, lacks immune internalization properties, yet it inhibits B-cell activation and subsequent antibody production when cross-linked to membrane Ig. This suggests that FcRII, apart from inhibiting activating FcR, may also have other important functions in vivo.
In the present study, we investigated the in vivo role of FcRII, uncoupled from its function as inhibitor of activating FcR, in regulating joint inflammation and severe cartilage destruction in models of IC-mediated arthritis using mice which were made deficient for either both activating FcR (FcRI/III-/-) or all three FcR (FcRI/II/III-/-). We found that FcRII is a major regulator of joint inflammation by promoting clearance of ICs by synovial lining cells. Furthermore activating FcR on inflammatory cells appeared to be prerequisites for severe irreversible cartilage destruction.
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Materials and Methods |
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FcRI and FcRIII-/- were made deficient for the ligand-binding 
-chain of FcRI21
and FcRIII,22
respectively. FcRIII-/- were back-crossed to the C57BL/6 background for 12 generations. FcRIIb-/- were developed by Dr. Takai15
in the 129/Ola (H-2b) and C57BL/6 (H-2b) background. FcRI-/- was made in the 129Ola/C57BL/6 background. FcRII/III-/- and their controls (control 1) were developed in the 129Ola/C57BL/6 background. Intercrossing led to FcRI/II/III -/- in the 129Ola/C57BL/6/balb/c (enriched for C57BL/6), as were their triple controls (control 2). Control C57BL/6 and 129Ola/C57BL/6 were derived from Jackson laboratories (Bar Harbor, ME) and bred in our own facilities. Homozygous mutants and their wild-type (WT) controls, aged 10 to 12 weeks, were used in the experiments.
Humoral Immunity Against mBSA
Antibodies of various isotypes (IgG, IgG1, IgG2a, IgG2b, IgG3) directed against methylated bovine serum albumin (mBSA) were measured in sera of individual mice with an enzyme-linked immunosorbent assay (ELISA). Antigen was coated on microtiter plates (Greiner, Alphen a/d Rijn, The Netherlands) at a concentration of 100 µg/ml. Antibody titers were assessed by two-fold serial dilution of the sera followed by detection of bound mouse Ig with 1:500 diluted peroxidase-conjugated rabbit anti-mouse Ig (Miles Laboratories Inc., Elkhart, IN). O-Phenylenediamine (1 mg/ml; Sigma, St. Louis, MO) was used as substrate for peroxidase, and the antibody titer was determined by using 50% of the maximal extinction as an end-point.
Cellular Immunity Against mBSA
Mouse spleen cells were isolated and washed in RPMI supplemented with 10% fetal calf serum, glutamin (2 mmol/L), and pyruvate (1 mmol/L). Erythrocytes were lysed by treatment of the cells with an 0.16 mol/L NH4CL solution in 0.17 mol/L Tris, pH 7.2, for 5 minutes. After two washes in RPMI, the cells were plated on plastic T flasks (75 mm2) from Falcon Plastics (Oxnard, CA). After 60 minutes of incubation at 37°C, the nonadherent cells were harvested by aspiration and two 4- to 5-ml RPMI washes of the adherent cells. 100 µl of RPMI containing 1 x 105 T-cell-enriched spleen cells were placed in each well of a sterile, U-bottomed polystyrene microculture plate (Costar, Cambridge, MA). Antigens or mitogens were added in another 100 µl to give a total volume of 200 µl, and final concentrations of antigen of 50, 25, 12, 6, and 3 µg/ml. Cultures were maintained at 37°C in a humified atmosphere of 2% CO2 and 98% air for 4 days. Sixteen hours before harvesting, 1 µCi of [3H]-thymidine (6.7 Ci/mmol from New England Nuclear, Boston, MA) was added in 25 µl of RPMI. Cultures were harvested with a cell harvester (Tomtec, Hamden, CT) and [3H]-thymidine incorporation was determined.
Induction of Experimental Arthritis
AIA was induced by injecting 60 µg of mBSA in 6 µl PBS directly into the knee joints of mice that were previously immunized with that antigen. Mice were immunized with 100 µg of mBSA (Sigma), emulsified in 100 µl Freund’s complete adjuvant. Injections were divided over both flanks and footpath of the forelegs. Heat-killed Bordetella pertussis was administered intraperitoneally as an additional adjuvant. Two subcutaneous booster injections with 50 µg mBSA/CFA were given in the neck region 1 week after the initial immunization. Two weeks after these injections, arthritis was induced into the right knee joint, resulting in chronic arthritis.
Immune complex arthritis (ICA) was passively induced in knee joints of mice.23 Three micrograms of lysozyme in 6 µl were injected directly into the knee joint of mice that previously were given anti-lysozyme antibodies intravenously. An acute arthritis develops, which became maximal at day 3 and waned thereafter.
A non-IC-mediated zymosan-induced arthritis (ZIA) was induced by injecting 180 µg of sterilized zymosan in 6 µl PBS into the knee joint.
99mTc Uptake Measurements
Joint inflammation was measured by 99mTc pertechnetate uptake in the knee joint. This method was shown earlier to correlate well with histological findings.24 Briefly, mice were injected intraperitoneally with 12 µCi 99mTc and subsequently sedated with chloralhydrate. Thirty minutes thereafter, gamma radiation was assessed by use of a collimated Na-I-scintillation crystal with the knee in a fixed position. Arthritis was scored as the ratio of the 99mTc uptake in the right (R) and the left (L) knee joint. R:L ratios >1.1 were taken to indicate inflammation of the right knee joint.
Histology
Total knee joints were dissected, fixed in phosphate-buffered formalin (pH 7.4), decalcified in 5% buffered formic acid, and subsequently embedded in paraffin wax. Semiserial frontal whole knee joint sections (7 µm) were stained with hematoxylin and eosin (H&E) or safranin-O and Fast Green. The severity of joint inflammation was determined using an arbitrary score (0 to 3). Infiltrate and exudate were scored separately. Scoring was performed in a blinded manner by two independent observers: 0, no cells; 1, mild cellularity; 2, moderate cellularity; 3, maximal cellularity.
Endocytosis and Clearance of IgG Immune Complexes
Endocytosis and clearance of IgG ICs were studied both in vivo and in vitro. The clearance of IgG-containing ICs from arthritic knee joints of FcRI/II/III-/- and FcRI/III-/- mice was studied using anti-IgG immunolocalization. In one group, AIA was induced whereas in a second group knee joints were injected with 6 µg of heat-aggregated IgG. The latter was made by heating rabbit-IgG during 30 minutes at 61°C. Knee joints were isolated 7 days after AIA induction or 8 hours after injection of aggregated IgG. Paraffin-embedded total knee joint sections were pretreated with hyaluronidase ABC and additionally stained with either goat anti-mouse peroxidase or goat anti-rabbit peroxidase overnight. Development of the peroxidase product was done using diaminobenzidine (0.5 mg/ml). Sections were counterstained with H&E.
Isolation of Peritoneal Macrophages from Mice Previously Injected with Thioglycolate
ICs were preformed by incubating soluble fluorescein isothiocyanate (FITC)-labeled OVA (Molecular Probes, Leiden, The Netherlands) with 25 µg/ml polyclonal OVA-specific rabbit IgG (rIgG OVA; Sigma-Aldrich, Zwÿndrecht, The Netherlands) for 30 minutes at 37°C in polypropylene tubes. Fifty thousand peritoneal macrophages were added to FACS tubes containing OVA-ICs or soluble OVA and incubated for 15 minutes at 37°C. Cells were washed twice and resuspended in presence of 0.4% (w/v) trypan blue (Sigma-Aldrich), which quenches extracellular, but not intracellular, fluorescence. Flow cytometry was performed with FACScan. The mean fluorescence value of six measurements is shown.
Immunohistochemical Staining of Myeloid-Related Proteins MRP8 and 14
Rabbit anti-sera against recombinant murine MRP8 (-MRP8) and MRP14 (-MRP14) were produced as described earlier.25
Monospecificity of antibodies was analyzed by immunoreactivity against recombinant MRP8 and MRP14 and Western blot analysis of lysates of granulocytes.25
Formalin-fixed sections of knee joints were stained using a final antibody concentration of 1 µg/ml. Primary antibodies were detected using peroxidase-conjugated second-stage antibodies against rabbit IgG (Dianova). Finally, sections were counterstained with Mayer’s hematoxylin (Merck, Darmstadt, Germany). MRP8 and MRP14-positive cells present in the joint cavity and synovial lining were determined as expressed as percentage of the total cell population, using an arbitrary score (0:0%, 1:1 to 30%, 2:31 to 70%, 3:71 to 100%).
Immunolocalization of MMP-Induced Neoepitope (VDIPEN)
For immunohistochemical analysis, sections were deparaffinized, rehydrated, and digested with chondroitinase ABC (Sigma; 0.25 U/ml, 0.1 mol/L Tris-HCL, pH 8.0) for 1 hour at 37°C, to remove chondroitine sulfate from the proteoglycans. Sections were then treated with 1% H2O2 in methanol for 20 minutes and subsequently for 5 minutes with 0.1% (v/v) Triton X-100 in PBS. After incubation with 1.5% (v/v) normal goat serum for 20 minutes, sections were incubated with affinity-purified anti-VDIPEN IgG overnight at 4°C. These antibodies were kindly given by Irwin Singer and Ellen Bayne (Merck Research Laboratories, Rahway, NJ) and have been extensively characterized before.26,27 In addition, sections were incubated with biotinylated goat anti-rabbit IgG and binding-detected using avidin-peroxidase staining (Elite kit, Vector Laboratories, Inc., Burlingame, CA). Development of the peroxidase product was done using nickel enhancement and counterstaining was done with orange G (2%) for 5 minutes.
Measurement and Characterization of Chondrocyte Death
Chondrocyte death was determined at day 7 after AIA induction in total knee joint sections stained with H&E. Chondrocyte death was determined as percentage of the area of the cartilage containing empty lacunae in relation to the total area.
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Results |
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RII in the Absence of Activating FcR During Antigen-Induced Arthritis
To investigate the role of FcRII in the absence of activating FcR, we induced AIA in knee joints of FcRI/III-/- and FcRI/II/III-/- mice. As the absence of FcR may alter the immunological response against methylated BSA during immunization of these mice, thereby impairing the onset and course of arthritis, we first tested cellular and humoral immunity to mBSA, 3 weeks after immunization. Cellular immunity, as measured by spleen lymphocyte stimulation (LST) against various concentrations of mBSA, showed no significant differences between knockout (KO) and their controls (Table 1)
. In addition, humoral immunity was measured by ELISA. Total IgG, IgG1, IgG2a, and IgG3 anti-mBSA levels were two to four times higher, whereas IgG2b levels were not different in sera of both immunized FcRI/III-/- and FcRI/II/III-/- when compared to their WT controls (Table 1)
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RI/III-/- was significantly lower both at day 3 and day 7 when compared to arthritic controls (Figure 1A)
. Interestingly, inflammation in arthritic knee joints of FcRI/II/III-/- was not different from that seen in control knee joints both at day 3 and 7 after AIA induction (Figure 1B)
.
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RI/III -/- mice showed that although IgG2a antibody titers were much higher, exudate and infiltrate was significantly lower (90% and 87%, respectively) when compared to WT controls (Figure 2, A and D
, versus WT control, Figure 2E
). In contrast, at day 7 after AIA induction in knees of FcRI/II/III-/-, joint inflammation appeared to be markedly higher when compared to their controls (exudate and infiltrate were respectively 200% and 120% higher (Figure 2, B and F
, versus WT control, Figure 2E
) suggesting that FcRII is an important regulator of joint inflammation in the absence of activating FcR.
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RII Regulation of Joint Inflammation Is Specific for Immune Complexes
To further investigate whether FcRII regulation of joint inflammation in the absence of activating FcR is specific for ICs (and not, for example, by T cells also involved in AIA), we induced arthritis solely by ICs. ICA was passively induced by injecting lysozyme in knee joints of mice that were previously given anti-lysozyme antibodies. Histology taken at day 3 after arthritis induction showed that joint inflammation was almost completely prevented in FcRI/III-/-, whereas substantial arthritis was found in their WT controls (Figure 3A)
. When ICA was induced in knee joints of FcRI/II/III-/-, the inflammatory cell mass as measured at day 1 and 3 was in line with that found in AIA, again significantly higher when compared to WT controls. At day 1, exudate and infiltrate were 310% and 60%, respectively (Figure 3B)
, and at day 3, 2200% and 270% higher (Figure 3C)
.
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RI/II/III-/-. The inflammatory cell mass measured at day 3 after ZIA induction was not different from controls, suggesting that knee joints of these mice develop a normal inflammatory response after injection with zymosan (Figure 3D)
.
FcRII Is Involved in Efficient Endocytosis and Clearance of ICs
One of the reasons why joint inflammation is elevated in FcRI/II/III -/- may be an impaired endocytosis and clearance of ICs from the joint.
To investigate whether FcRII is involved in endocytosis of IgG containing ICs, we first investigated the presence of murine IgG, localized within the arthritic joints at day 7 after AIA induction using immunolocalization. No significant differences in IgG deposition was found in the knee joints of FcRI/III-/- and their WT controls, suggesting an effective clearance of IgG containing ICs in the presence of only FcRII (Figure 4B
versus WT controls, Figure 4A
). In contrast, arthritic knee joints of FcRI/II/III-/- contained large amounts of IgG, suggesting that removal of ICs is retarded (Figure 4D
versus WT controls, Figure 4C
). To confirm this finding, heat-aggregated IgG was injected in the knee joints of FcRI/III-/- and FcRI/II/III-/- mice and their WT controls. Immunolocalization of IgG showed that 8 hours after injection significantly more IgG was detected in FcRI/II/III-/- knee joints which was mainly bound to the synovial lining layer (Figure 5D
versus WT control, Figure 5E
). Similar intensity of staining was found when aggregated IgG was injected in knee joints of FcRI/III-/- and their controls (Figure 5B
versus WT control, Figure 5A
).
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RII function on macrophages, thioglycollate-induced peritoneal macrophages were isolated. When macrophages expressing only FcRII (FcRI/III-/-) were pre-incubated with pre-formed FITC-labeled OVA-IgG ICs, prominent endocytosis was found not different from control macrophages (Figure 6B
versus control, Figure 6A
). Interestingly when FcRII was also absent (FcRI/II/III-/-), endocytosis of ICs was completely prevented (Figure 6C
versus control, Figure 6A
).
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RI/II/III-/-
To further analyze the composition of the inflammatory cell mass within the arthritic FcRI/II/III-/- knee joint, we next investigated the type and activation state of the inflammatory cells using immunolocalization. PMN and monocyte/macrophage ratios were determined by immunolocalization using NIMP-R14, which stains PMN specifically. At day 7 after AIA induction in FcRI/II/III-/- knee joints, the majority of inflammatory cells appeared to be monocytes (ratio monocytes/PMN 60–40) and no differences were found between KO and their controls. In addition, we determined the pro-inflammatory phenotype of the infiltrated cells. In FcRI/II/III-/- mice, the infiltrated cells in the arthritic joint displayed an activated phenotype, according to high expression of MRP8 and 14 (respectively 47% and 92%: Figure 7, A and C
versus WT control, Figure 7B
), whereas infiltrated cells in the joints of FcRI/III-/- mice failed to express these activation markers (data not shown). In fact this implies that FcRII can prevent cellular activation even in the absence of FcRI/III.
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R Fail to Induce Severe Cartilage Destruction
As the majority of the infiltrated cells in the FcRI/II/III -/- knee joints were activated we additionally investigated whether these cells were capable of inducing severe cartilage destruction like metalloproteinase (MMP)-induced damage and chondrocyte death. MMPs are crucial in degradation of aggrecan and collagen, leading to irreversible cartilage destruction. MMPs degrade aggrecan leaving the C-terminal ending with the amino acid sequence VDIPEN which can be detected by specific antibodies around day 5 after induction of AIA.27
For this reason, AIA day 7 was taken to detect VDIPEN expression in the cartilage matrix.
The amount of VDIPEN was measured by determining the percentage of the area of cartilage expressing VDIPEN. In most investigated knee joints of WT mice injected with 60 µg of mBSA, VDIPEN staining was found particularly in the cartilage layers of tibia and femur (Figure 8,A, C, and E)
. In arthritic knees of FcRI/III-/-, VDIPEN expression was completely absent when comparable to WT controls (Figure 8, A and D
, versus WT control, Figure 8C
). Interestingly, in FcRI/II/III-/- arthritic joints, although much more joint inflammation was found which abundantly expressed the activation markers MRP8 and 14, VDIPEN was virtually absent when compared to arthritic controls (Figure 8, B and F
, versus WT control, Figure 8E
). This confirms again that only activating FcR mediates severe cartilage destruction, and that this prerequisite also holds in condition of abundant inflammatory cell influx showing an activated phenotype.
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. Chondrocyte death was absent in arthritic FcRI/III-/- knee joints (Figure 9, A and D
, versus WT control, Figure 9C
). Again in arthritic FcRI/II/III-/-, despite its high joint inflammation, chondrocyte death was completely absent (Figure 9, B and F
, versus WT control, Figure 9E
).
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Discussion |
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RII has been extensively studied using FcRII KO mice. Induction of IC-mediated inflammation within these mice caused a significantly elevated inflammation when compared to controls. In general, the function of FcRII as a major inhibitor of the activatory FcR is highlighted. In the present study we demonstrate that in the absence of activating FcR, the inhibiting FcRII still functions as an important down-regulator of synovial inflammation which might be related to IC clearance and complement activation.
FcRII, in the absence of activating FcR significantly reduced joint inflammation during T cell-mediated AIA. One of the explanations may be an altered T cell response. FcR are expressed on precursors of T cells28
and the absence of these receptors may have had an impact on the development of T cell reactivity and may explain the markedly elevated anti-mBSA IgG2a antibody responses in both immunized FcRI/III-/- as FcRI/II/III-/-. However anti-mBSA T cell responses were found not to be significantly different and this may indicate that FcRII present on resident synovial lining macrophages may be more important in regulating joint inflammation.
To further substantiate the involvement of FcRII on synovial macrophages, arthritis was induced by local deposition of IC within the joint. In that model, arthritis is regulated by lining macrophages and not by T cells.23
Comparable to that seen in AIA, FcRII, in the absence of activating FcR, again strongly reduced joint inflammation. The FcRII dependency appeared to be IC-specific since injection of zymosan directly into the knee joint of FcRI/II/III-/- caused similar joint inflammation than when injected in WT and thus indicates that the joints of these mice develop a normal inflammatory response on non-IC triggers.
The most plausible function of FcRII in joint inflammation in the absence of activating FcR is its role in clearance of IgG ICs from the joint. Clearance of IC is largely regulated by synovial lining cells and its efficiency is highly correlated to development of arthritis.29
IgG-containing ICs activate complement. In the mouse IgG2a and IgG3 mediate complement via the classical pathway,30
whereas IgG1, when attached to cartilage layers uses the alternative pathway.31
Co-dominance between complement and FcR has previously been described.32
Efficient removal of these ICs from the joint may lower the amount and course of complement activation within the joint, thereby lowering onset and/or prolongation of arthritis. This is in line with studies that show that complement is especially important in the onset, whereas at later time-points inflammation is more FcR-mediated.33
Within the joint, macrophages are crucial in clearance and endocytosis of ICs. In earlier in vitro studies using cDNA transfection, it was found that FcRII mediates internalization and lysosomal degradation of IgG-antigen complexes.34-35
In line with that, we now find that peritoneal macrophages from KO mice, which only express FcRII and no activating FcR, are still able to endocytose soluble ovalbumin-IgG complexes not different from controls, whereas in the absence of all FcR, endocytosis is almost completely blocked. Moreover when ICs were injected directly into the joint, clearance and endocytosis by lining cells were strongly retarded in the FcRI/II/III-/-. This suggests that in vivo FcRII is a major receptor for endocytosis. In vivo studies using FcRII-deficient mice have shown that FcRII inhibited phagocytosis and clearance, and this was explained by inhibiting activating FcR.36
We now clearly demonstrate for the first time that in vivo, FcRII also reduces inflammation by accelerating IC clearance and endocytosis. In contrast to our study, Mathis et al31
found no involvement of Fc II in the K/BXN serum transfer arthritis model. One explanation may be that arthritis within this model is regulated by anti-GPI antibodies of only the IgG1 isotype. These antibodies preferentially bind to FcRIII which may largely be responsible for IC removal within this model.
Removal of IC from the joint is a combined action of leakage through the pores of the lining layer into the draining lymph vessels and lymph nodes, and binding and endocytosis by synovial lining cells. Synovial intima macrophages first meet these ICs and have been shown to be crucial in both onset as well as propagation of synovial inflammation.8,37
This in contrast to synovial intima fibroblasts which fail to express FcR. Activation of lining cells forms one of the crucial events in arthritis development. Transfer of early activated lining cells appeared to be sufficient to induce arthritis in normal rats.38
It is generally accepted that FcRII acts by coligating with activatory FcRIII and probably also with FcRI eventually leading to inactivation of synovial macrophages and reduced production of cytokines and chemokines.16
In the present study we find that in the absence of all FcR, IgG-ICs when injected into the joint still bind to intimal synovial cells. Moreover these cells express abundant MRP8/14 indicating that they still become activated and likely produce sufficient pro-inflammatory factors leading to pronounced joint inflammation. As FcR are absent, IC may bind to other receptors. A good candidate may be the promiscuous complement receptor 3 (CR3).39
The complement splitting product C3bi tightly binds to various antibody isotypes involved in IC formation.40
C3bi may form the link between IC and binding to the CR3 receptor on macrophages not expressing FcR and may mediate intracellular signaling leading to activation of the intimal macrophage.
The amount of inflammatory cell mass within an inflamed joint is often related to severe cartilage destruction. In the present study we found a remarkable uncoupling between joint inflammation and severe cartilage destruction like MMP-mediated damage and chondrocyte death. Severe cartilage destruction seen during IC-mediated arthritides is mediated by metalloproteinases, which are released by chondrocytes in a latent pro-form within the cartilage matrix. Interleukin-1 appeared to be the master cytokine regulating MMP production by the chondrocyte.41 Large amounts of inactive MMPs accumulate within the cartilage matrix and on activation lead to destruction of the collagen type II network and the proteoglycans embedded within this matrix.42 The factors needed for this activation step are still unknown.
As inflammatory cells are capable of mediating activation of latent MMPs inside the cartilage matrix,43
the way in which these cells become activated within the joint seems crucial and recent studies by our lab suggest that activating FcR are of utmost importance.10,21,44
Binding of IC to activating FcR (especially FcRI) on macrophages may lead either to a higher production of MMP-activating factors or promote generation of mediators which inhibit these factors. MMP-activating factors may be other MMPs,45
enzymes like plasmin,46
EMPRINN,47
or oxygen radicals,48
which all have been shown to possess the capacity of activating latent MMPs. Oxygen radicals have been shown to be abundantly released by macrophages after IC binding to FcRI49
and may explain the clear chondrocyte death seen during IC-mediated arthritis. In the absence of FcRI, chondrocyte death was completely absent10,44
at day 7 after AIA induction. In contrast, FcR binding may also lead to a rise in inhibitors like TIMPs, which may efficiently reduce MMP-mediated cartilage destruction.
When infiltrating cells are activated by bacterial or yeast cell walls when injected into the knee joint of mice, although a pronounced inflammation developed, MMP-mediated cartilage destruction nor chondrocyte death was detected.27
Although these cells express an inflammatory phenotype, the released factors were incapable of activating latent MMP, which were found in large amounts within the cartilage layers of the joint.27
In line with this we now find that in knee joints of arthritic triple KO, despite abundant joint inflammation, no VDIPEN epitopes nor chondrocyte death was observed. Like in the non-IC arthritis, the infiltrated cells despite expressing an inflammatory phenotype were incapable to activate MMPs. This again confirms that FcR activation is a prerequisite for inducing irreversible cartilage destruction.
The present study underlines that activating FcR are crucial in induction of severe cartilage destruction and that FcRII is an important inhibiting receptor which regulates both chronic joint inflammation as well as cartilage destruction during arthritis. FcRII may be a powerful inhibitor to prevent both synovial inflammation and cartilage destruction and its overexpression may form a new therapeutic tool to combat the severe pathogenicity of ICs involved in RA.
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Footnotes |
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Supported by the Dutch Arthritis Association (grant 99-1-402).
Accepted for publication July 21, 2003.
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References |
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RI and FcRIII and inhibitory FcRII in inflammation and cartilage destruction during experimental antigen-induced arthritis. Am J Pathol 2001, 159:2309-2320. J Biol Chem 1991, 266:23477-23482
B pathway. J Biol Chem 2002, 277:30271-30282This article has been cited by other articles:
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  P L E M van Lent, L Grevers, A B Blom, A Sloetjes, J S Mort, T Vogl, W Nacken, W B van den Berg, and J Roth Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis Ann Rheum Dis, December 1, 2008; 67(12): 1750 - 1758. [Abstract] [Full Text] [PDF]
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 P. Boross, P. L. van Lent, J. Martin-Ramirez, J. van der Kaa, M. H. C. M. Mulder, J. W. C. Claassens, W. B. van den Berg, V. L. Arandhara, and J. S. Verbeek Destructive Arthritis in the Absence of Both Fc{gamma}RI and Fc{gamma}RIII J. Immunol., April 1, 2008; 180(7): 5083 - 5091. [Abstract] [Full Text] [PDF]
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M. Zhao, M. E. Trimbeger, N. Li, L. A. Diaz, S. D. Shapiro, and Z. Liu Role of FcRs in Animal Model of Autoimmune Bullous Pemphigoid. J. Immunol., September 1, 2006; 177(5): 3398 - 3405. [Abstract] [Full Text] [PDF]
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 E. van Mirre, W. B. Breunis, J. Geissler, C. E. Hack, M. de Boer, D. Roos, and T. W. Kuijpers Neutrophil responsiveness to IgG, as determined by fixed ratios of mRNA levels for activating and inhibitory Fc{gamma}RII (CD32), is stable over time and unaffected by cytokines Blood, July 15, 2006; 108(2): 584 - 590. [Abstract] [Full Text] [PDF]
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 J. C. Scatizzi, J. Hutcheson, E. Bickel, J. M. Woods, K. Klosowska, T. L. Moore, G. K. Haines III, and H. Perlman p21Cip1 Is Required for the Development of Monocytes and Their Response to Serum Transfer-induced Arthritis Am. J. Pathol., May 1, 2006; 168(5): 1531 - 1541. [Abstract] [Full Text] [PDF]
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T R D J Radstake, A W T van Lieshout, P L C M van Riel, W B van den Berg, and G J Adema Dendritic cells, Fc{gamma} receptors, and Toll-like receptors: potential allies in the battle against rheumatoid arthritis Ann Rheum Dis, November 1, 2005; 64(11): 1532 - 1538. [Abstract] [Full Text] [PDF]
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0.05). Note that swelling was almost absent in knee joints of arthritic Fc
