Role of Activatory FcγRI and FcγRIII and Inhibitory FcγRII in Inflammation and Cartilage Destruction during Experimental Antigen-Induced Arthritis
2001; Elsevier BV; Volume: 159; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63081-7
ISSN1525-2191
AutoresPeter L. E. M. van Lent, Karin C. Nabbe, Arjen B. Blom, A. E. M. Holthuysen, A. Slöetjes, Leo B. A. Van De Putte, Sjef Verbeek, Wim B. van den Berg,
Tópico(s)T-cell and B-cell Immunology
ResumoIgG-containing immune complexes, which are found in most RA joints, communicate with hematopoietic cells using three classes of Fc receptors(FcγRI, -II, -III). In a previous study we found that if a chronic T-cell-mediated antigen-induced arthritis (AIA) was elicited in knee joints of FcR γ-chain-deficient mice that lack functional FcγRI and FcγRIII, joint inflammation was comparable but severe cartilage destruction was absent. We now examined the individual role of the stimulatory FcγRI and FcγRIII and inhibitory FcγRII in inflammation and functional cartilage damage in knee joints with AIA using FcγRI-, FcγRII-, and FcγRIII-deficient mice. Three weeks after immunization with the antigen-methylated bovine serum albumin (BSA), cellular (T-cell responses as measured by lymphocyte proliferation) immunity raised against mBSA was comparable in all groups examined. Humoral (total IgG, IgG1, IgG2a, and IgG2b levels) immunity against mBSA was comparable in FcγRI−/− and FcγRIII−/− but higher in FcγRII−/− if compared to controls. Joint swelling as measured by99mTc uptake at days 1, 3, and 7 was similar in FcγRI−/− and FcγRIII−/− mice and significantly higher in FcγRII−/−. Chronic inflammation and cartilage damage (depletion of proteoglycans, metalloproteinase (MMP)-induced neoepitopes, and matrix erosion) was studied histologically in total knee joint sections stained with hematoxylin or safranin-O. Histologically, at day 7 after AIA induction, exudate and infiltrate in the knee joint was similar in FcγRI−/− and FcγRIII−/− and significantly higher (230% and 340%) in FcγRII−/− mice if compared to controls. Aggrecan breakdown in cartilage caused by MMPs and, which is related to severe irreversible cartilage erosion, was further studied by immunolocalization of MMP-mediated neoepitopes (VDIPEN) and image analysis. MMP-induced neoepitopes determined in various cartilage layers (tibia and femur) were primarily inhibited in FcγRI−/− (79 to 87% and 87 to 88%, respectively) and comparable in FcγRIII−/−. VDIPEN neoepitopes were much higher (82 to 122% and 200 to 250%, respectively) in FcγRII−/− mice. Initial depletion of proteoglycans was similar (60 to 100%) in all groups. In the chronic phase, cartilage matrix erosion in the lateral and medial tibia was significantly elevated in FcγRII−/− (222% and 186%, respectively) but not in FcγRI−/− or FcγRIII−/− mice. These results suggest that during T-cell-mediated AIA, FcγRI and FcγRIII act in concert in acute and chronic inflammation whereas FcγRI is the dominant FcR involved in severe cartilage destruction. FcγRII is a crucial inhibiting factor in acute and chronic inflammation and cartilage erosion. IgG-containing immune complexes, which are found in most RA joints, communicate with hematopoietic cells using three classes of Fc receptors(FcγRI, -II, -III). In a previous study we found that if a chronic T-cell-mediated antigen-induced arthritis (AIA) was elicited in knee joints of FcR γ-chain-deficient mice that lack functional FcγRI and FcγRIII, joint inflammation was comparable but severe cartilage destruction was absent. We now examined the individual role of the stimulatory FcγRI and FcγRIII and inhibitory FcγRII in inflammation and functional cartilage damage in knee joints with AIA using FcγRI-, FcγRII-, and FcγRIII-deficient mice. Three weeks after immunization with the antigen-methylated bovine serum albumin (BSA), cellular (T-cell responses as measured by lymphocyte proliferation) immunity raised against mBSA was comparable in all groups examined. Humoral (total IgG, IgG1, IgG2a, and IgG2b levels) immunity against mBSA was comparable in FcγRI−/− and FcγRIII−/− but higher in FcγRII−/− if compared to controls. Joint swelling as measured by99mTc uptake at days 1, 3, and 7 was similar in FcγRI−/− and FcγRIII−/− mice and significantly higher in FcγRII−/−. Chronic inflammation and cartilage damage (depletion of proteoglycans, metalloproteinase (MMP)-induced neoepitopes, and matrix erosion) was studied histologically in total knee joint sections stained with hematoxylin or safranin-O. Histologically, at day 7 after AIA induction, exudate and infiltrate in the knee joint was similar in FcγRI−/− and FcγRIII−/− and significantly higher (230% and 340%) in FcγRII−/− mice if compared to controls. Aggrecan breakdown in cartilage caused by MMPs and, which is related to severe irreversible cartilage erosion, was further studied by immunolocalization of MMP-mediated neoepitopes (VDIPEN) and image analysis. MMP-induced neoepitopes determined in various cartilage layers (tibia and femur) were primarily inhibited in FcγRI−/− (79 to 87% and 87 to 88%, respectively) and comparable in FcγRIII−/−. VDIPEN neoepitopes were much higher (82 to 122% and 200 to 250%, respectively) in FcγRII−/− mice. Initial depletion of proteoglycans was similar (60 to 100%) in all groups. In the chronic phase, cartilage matrix erosion in the lateral and medial tibia was significantly elevated in FcγRII−/− (222% and 186%, respectively) but not in FcγRI−/− or FcγRIII−/− mice. These results suggest that during T-cell-mediated AIA, FcγRI and FcγRIII act in concert in acute and chronic inflammation whereas FcγRI is the dominant FcR involved in severe cartilage destruction. FcγRII is a crucial inhibiting factor in acute and chronic inflammation and cartilage erosion. Chronic inflammation and destruction of cartilage and bone are main characteristics of rheumatoid arthritis (RA).1Klippel JH Primer on the Rheumatic Diseases. Arthritis Foundation, Atlanta, Georgia1997Google Scholar IgG-containing immune complexes (ICs), present in large amounts in joints of most RA patients have been suggested to be major pathogenic factors in RA, responsible for initiation and persistence of the inflammatory cascade and its resulting destruction of the cartilage.2Cooke TDV Mechanism of cartilage destruction: relation to choice of therapeutic agent.Semin Arthritis Rheum. 1985; 51: S16-S23Abstract Full Text PDF Scopus (10) Google Scholar Apart from ICs T cells have shown to also be important in amplification of arthritis3Panayi GS Lanchbury JS Kingsley GH The importance of the T cell in initiating and maintaining the chronic synovitis of rheumatoid arthritis.Arthritis Rheum. 1992; 35: 729-735Crossref PubMed Scopus (477) Google Scholar, 4Zvaifler NJ Rheumatoid arthritis—the multiple pathways to chronic synovitis.Lab Invest. 1995; 73: 307-310PubMed Google Scholar and may enhance inflammatory reactions merely induced by ICs. Immune complexes containing IgG, the dominant immunoglobulin in the circulation, communicate with synovial cells via cellular receptors for IgG that belong to the IgG superfamily.5Ravetch JV Kinet JP Fc receptors.Annu Rev Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1267) Google Scholar, 6Fridman WH Bonnerot C Daeron M Amigorena S Teillaud JL Sautes C Structural bases of Fcγ receptor functions.Immunol Rev. 1992; 125: 49-76Crossref PubMed Scopus (125) Google Scholar, 7Van de Winkel JGJ Capel PJA Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications.Immunol Today. 1993; 14: 215-221Abstract Full Text PDF PubMed Scopus (628) Google Scholar Murine phagocytic effector cells express three different classes of IgG receptors (FcγRI, -II, -III).8Ravetch JV Fc receptors: rubor redux.Cell. 1994; 78: 553-560Abstract Full Text PDF PubMed Scopus (337) Google Scholar, 9Takai T, Ravetch JV: Fc receptor genetics and the manipulation of genes in the study of FcR biology. Immunoglobulin Receptors and Their Physiological and Pathological Roles in Immunity Edited by JGJ Van de Winkel, PM Hogarth. Dordrecht, The Netherlands, Kluwer Academic Publishers GroupGoogle Scholar FcγRI and FcγRIII are hetero-oligomeric complexes in which ligand-binding α chains are associated with the signal-transducing γ-chain. This γ-chain is required for their assembly and triggering of various effector functions including phagocytosis,10Simms HH Gaither TA Fries LF Frank MM Monokines released during short-term Fc gamma receptor phagocytosis up-regulate polymorphonuclear leucocytes and monocyte-phagocytic function.J Immunol. 1991; 147: 265-272PubMed Google Scholar antigen-presenting function,11Heijnen IAFM van Vugt MJ Fanger NA Graziano RF de Wit TPM Hofhuis FMA Guyre PM Capel PJA Verbeek JS van de Winkel JGJ Antigen targeting to myeloid-specific FcγRI/CD64 triggers enhanced antibody responses in transgenic mice.J Clin Invest. 1996; 97: 331-338Crossref PubMed Scopus (167) Google Scholar antibody-dependent cytotoxicity,12Huizinga TWJ Dolman KM van der Linden NJM Kleijer M Nuijens JH von dem Borne AEGKR Roos D Phosphatidylinositol-linked FcRIII mediates exocytosis of neutrophil granule proteins, but does not mediate initiation of the respiratory burst.J Immunol. 1990; 144: 1432-1437PubMed Google Scholar and the release of inflammatory mediators.13Huizinga TWJ van Kemenade F Koenderman L Dolman KM von dem Borne AEGKR Tetteroo PAT Roos D The 40 kDa Fc gamma receptor (FcRII) on human neutrophils is essential for the IgG-induced respiratory burst and IgG-induced phagocytosis.J Immunol. 1989; 142: 2365-2369PubMed Google Scholar These effector functions are regulated by an immunoreceptor tyrosine-based activation motif within the γ-chain.14Cambier JC Antigen and Fc receptor signaling. The awesome power of the immunoreceptor tyrosine-based activation motif (ITAM).J Immunol. 1995; 155: 3281-3285PubMed Google Scholar The third receptor class for IgG, FcγRII is a single α-chain receptor and contains an immunoreceptor tyrosine-based inhibitory motif-containing cytoplasmic domain that by co-ligation of the immunoreceptor tyrosine-based activation motif receptor, inhibits cellular activation signals through the recruitment of the inositol phosphatase SHIP.15Ono M Bolland S Tempst P Ravetch JV Role of the inositol phosphatase in negative regulation of the immune system by the receptor FcγRIIb.Nature. 1996; 383: 263-266Crossref PubMed Scopus (643) Google Scholar FcγRII has been shown to be a negative regulator of FcγRIII in IgG IC-triggered inflammation.16Schiller C Janssen-Graals I Baumann U Schwerter-Strumpf K Izui S Takai T Schmidt RE Gessner JE Mouse FcγRII is a negative regulator of FcγRIII in IgG immune complex-triggered inflammation but not in autoantibody-induced hemolysis.Eur J Immunol. 2000; 30: 481-490Crossref PubMed Scopus (66) Google Scholar Recently we found that activating FcγR (FcγRI and FcγRIII) were crucial in severe cartilage destruction during antigen-induced arthritis (AIA).17Van Lent PLEM van Vuuren AJ Blom AB Holthuysen AEM van de Putte LBA van de Winkel JGJ van den Berg WB Role of Fc receptor γ chain in inflammation and cartilage damage during experimental antigen-induced arthritis.Arthritis Rheumatism. 2000; 43: 740-752Crossref PubMed Scopus (65) Google Scholar Irreversible cartilage destruction within this model occurs through enzymatic cleavage by metalloproteinases (MMPs) of cartilage constituents. These Zn-dependent endopeptidases are capable of cleaving aggrecan and collagen type II, the main components of cartilage, which leads to severe cartilage erosion.18Hasty KA Reife RA Kang AH Stuart JM The role of stromelysin in the cartilage destruction that accompanies inflammatory arthritis.Arthritis Rheum. 1990; 33: 388-397Crossref PubMed Scopus (167) Google Scholar, 19Murphy G Cockett MI Ward RV Docherty AJ Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP).Biochem J. 1991; 277: 277-279Crossref PubMed Scopus (396) Google Scholar, 20Cawston T Matrix metalloproteinases and TIMPS: properties and implications for the rheumatic diseases.Mol Med Today. 1998; 4: 130-137Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar Various MMPs (MMP-1, -2, -3, -7, -8, -9, -13) have been found to cleave aggrecan between amino residues Asn341-Phe342 resulting in the neoepitope FVDIPEN that remains in the cartilage.21Flannery CR Lark MW Sandy JD Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan: evidence for proteolysis at this site in-vivo in human cartilage.J Biol Chem. 1992; 267: 1008-1014Abstract Full Text PDF PubMed Google Scholar AIA elicited in knee joints of FcR γ-chain −/− lacking functional FcγRI and FcγIII showed similar synovial inflammation if compared to controls at day 7 after arthritis induction. Nevertheless, severe cartilage destruction as MMP-mediated matrix destruction and erosion was fully absent in arthritic FcR γ-chain −/− knee joints. These results suggest that FcγRI and/or FcγRIII are of crucial importance in severe cartilage destruction within this model. FcγRIII has been suggested as the most likely candidate in IC-mediated joint inflammation.22Kleinau S Martinsson P Heyman B Induction and suppression of collagen-induced arthritis is dependent on distinct Fcγ receptors.J Exp Med. 2000; 191: 1611-1616Crossref PubMed Scopus (218) Google Scholar We now investigated the involvement of activating FcγRI and FcγRIII and the inhibitory FcγRII in severe cartilage destruction seen during AIA. Expression of MMP-induced aggrecan neoepitopes and erosion of the cartilage matrix was investigated in the knee joints of arthritic mice by histology and immunolocalization. Our findings indicate that FcγRI and not FcγRIII is the dominant activatory Fc receptor involved in severe cartilage destruction in a model in which T cells play a dominant role. In contrast, FcγRII is involved in inhibition of severe cartilage destruction within this model and may be a new therapeutic target to combat severe cartilage destruction. FcγRIII−/− mice were made deficient for the ligand-binding α-chain of FcγRIII (Dr. Verbeek) and were backcrossed to the C57BL/6 background for 12 generations.23Hazenbos WL Gessner JE Hofhuis FM Kuipers H Meyer D Heijnen IA Smidt RE Sandor M Capel PJ Daeron M van de Winkel JG Verbeek JS Impaired IgG-dependent anaphylaxis and arthus reaction in FcγRIII (CD16)-deficient mice.Immunity. 1996; 5: 181-188Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar FcγRI−/− were made deficient for the ligand-binding α-chain of FcγRI (Dr. Verbeek) and were backcrossed to BALB/c for six generations.24Fossati-Jimack L Ioan-Facsinay A Reininger L Chicheportiche Y Watanabe N Saito T Hoghuis FM Gessner JE Schiller C Schmidt RE Honjo T Verbeek JS Izui S Markedly different pathogenicity of four immunoglobulin G isotype-switch variants of an antierythrocyte autoantibody is based on their capacity to interact in vivo with the low affinity Fcgamma receptor III.J Exp Med. 2000; 191: 1293-1302Crossref PubMed Scopus (156) Google Scholar FcγRII−/− were developed by Dr. Takai (Sendai, Japan)25Takai T Ono M Hikida M Ohmori H Ravetch JV Augmented humoral and anaphylactic responses in Fc gamma RII-deficient mice.Nature. 1996; 379: 346-349Crossref PubMed Scopus (722) Google Scholar in the 129 SV (H-2b) and C57BL6 (H-2b) background. Control C57BL/6 and 129SV/C57BL/6 hybrids were derived from Jackson Laboratories (Bar Harbor, ME) and bred in our own facilities. Homozygous mutants and their wild-type controls, 10 to 12 weeks old, were used in the experiments. Methylated BSA-specific antibodies of various isotypes (total IgG, IgG1, IgG2a, IgG2b, IgG3) 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 twofold 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, USA). O-Phenylenediamine (1 mg/ml; Sigma, Zwijndrecht, The Netherlands) was used as substrate for peroxidase, and the antibody titer was determined by using 50% of the maximal extinction as an end point. Sera of FcγRI−/−, FcγRII−/−, and FcγRIII−/− mice were compared to sera of their wild-type controls. In each group at least 10 mice were tested. Mouse spleen cells were isolated and washed in RPMI supplemented with 10% fetal calf serum, glutamine (2 mmol/L), and pyruvate (1 mmol/L). Erythrocytes were lysed by treatment of the cells with a 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. One hundred μl of RPMI containing 1 × 105Ravetch JV Kinet JP Fc receptors.Annu Rev Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1267) Google Scholar 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 25, 12.5, and 6.25 μg/ml. Cultures were maintained at 37°C in a humidified atmosphere of 2% CO2 and 98% air for 4 days. Sixteen hours before harvesting, 1 μCi of [3H]-thymidine (6.7 Ci/mmol; New England Nuclear, Boston, MA) was added in 25 μl of RPMI. Cultures were harvested with a cell harvester (Tomtec) and [3H]-thymidine incorporation was determined. Mice were immunized with 100 μg of methylated BSA (mBSA, Sigma) emulsified in 100 μl of Freund's complete adjuvant. Injections were divided over both flanks and footpath of the forelegs. Heat-killedBordetella pertussis (RIVM, Bilthoven, The Netherlands) was administered intraperitoneally as an additional adjuvant. Two subcutaneous booster injections with 50 μg of mBSA/CFA were given in the neck region 1 week after the initial immunization.26Van den Berg WB Lessons from joint destruction from animal models.Curr Opin Rheumatol. 1997; 9: 221-228Crossref PubMed Scopus (42) Google Scholar Two weeks after these injections, arthritis was induced by intra-articular injection of either 15 μg (FcγRII−/−) or 60 μg (FcγRII−/−, FcγRI−/−, and FcγRIII−/−) of mBSA in 6 μl of saline into the right knee joint, resulting in chronic arthritis. The approval to induce arthritis in mice was given by the local ethical committee. Joint inflammation was measured by 99mTc pertechnetate uptake in the knee joint. This method has earlier been shown to correlate well with histological findings.27Lens JW van den Berg WB van de Putte LBA Quantitation of arthritis by 99mTc-uptake measurements in the mouse knee joint in inflammation scores.Agents Actions. 1984; 14: 723-728Crossref PubMed Scopus (35) Google Scholar Briefly, mice were injected intraperitoneally with 12 μCi of99mTc and subsequently sedated with chloralhydrate. Thirty minutes thereafter, γ-radiation was assessed by use of a collimated Na-I-scintillation crystal and the knee in a fixed position. Arthritis was scored as the ratio of the99mTc 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. 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 Fastgreen. Histological parameters (joint inflammation, proteoglycan depletion, and erosion) were scored by two independent observers in a blinded manner. Total knee joint sections were stained with Safranin-O and Fastgreen. Loss of red staining from various cartilage layers (femur and tibia), which is related to loss of proteoglycans, was determined using an arbitrary scale from 0 to 3. Normal cartilage and cartilage fully depleted of proteoglycans was taken as a 0 and 3 value, respectively. For immunohistochemical analysis, sections were deparaffinized, rehydrated, and digested with chondroitinase ABC (0.25 U/ml, 0.1 mol/L Tris-HCL, pH 8.0; Sigma) for 1 hour at 37°C, to remove chondroitin sulfate from the proteoglycans. Sections were then treated with 1% H2O2 in methanol for 20 minutes and subsequently 5 minutes with 0.1% (v/v) Triton X-100 in phosphate-buffered saline (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.28Singer II Kawka DW Bayne EK Donatelli SA Weidner JR Williams HR Ayala JM Mumford RA Lark MW Glant TT Nabozmy GH David CS VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalised in articular cartilage during inflammatory arthritis.J Clin Invest. 1995; 95: 2178-2186Crossref PubMed Scopus (112) Google Scholar, 29Mudget JS Hutchinson NI Chartrain NA Forsyth AJ McDonnell J Singer II Bayne EK Flanagan J Kawka D Shen CF Stevens K Chen M Trumbauer M Visco DM Susceptibility of stromelysin-1 deficient mice to collagen-induced arthritis and cartilage destruction.Arthritis Rheum. 1998; 41: 110-121Crossref PubMed Scopus (177) Google Scholar 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. Undecalcified cryosections were digested with proteinase-free chondroitinase ABC (0.25 U/ml Tris-HCl, pH 8.0) for 1 hour at 37°C to remove chondroitin sulfate from the PG. Subsequently, sections were fixed with periodate-lysine-paraformaldehyde fixative for 20 minutes. Sections were then treated with 1% H2O2 for 20 minutes followed by 5 minutes with 0.1% Triton X-100 in PBS. After incubation with 1.5% normal goat serum for 20 minutes, sections were incubated for 18 hours with the primary antibody recognizing the sequence NITEGE.30Arner EC Pratta MA Trzaskos JM Dedicco CP Tortorella MD Generation and characterization of aggrecanase. A soluble, cartilage-derived aggrecan-degrading activity.J Biol Chem. 1999; 274: 6594-6601Crossref PubMed Scopus (127) Google Scholar Then sections were incubated with biotinylated goat anti-rabbit IgG and were detected using avidin-peroxidase staining. Development of the peroxidase product was done using nickel enhancement. Counterstaining was done with orange G. Paraffin-embedded total knee joint sections were pretreated with chondroitinase ABC and additionally stained with goat anti-mouse IgG peroxidase overnight. Development of the peroxidase product was done using diaminobenzidine (0.5 mg/ml). Sections were counterstained with H&E. Erosion and chondrocyte death was determined in total knee joint sections stained with H&E. Erosion was detected as ruffling of the cartilage surface and was only mild at day 7 after AIA induction. Ruffling of the cartilage surface was determined using an arbitrary scale of 0 to 3. Normal cartilage surface and maximal ruffling within this experiment was taken as a 0 and 3 value, respectively. As the absence of a particular FcγR may alter the immunological response against methylated BSA during immunization, 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 proliferation against mBSA showed no significant differences between knockouts and their controls (Figure 1; B, D, and F). In addition, humoral immunity was measured by ELISA. Total IgG, IgG1, IgG2a, IgG2b, and IgG3 anti-mBSA levels were high but not significantly different in immunized FcγRI−/− and FcγRIII−/− if compared to their controls (Figure 1, A and C). In sera of FcγRII−/− immunized mice however, total IgG, IgG2a, and IgG3 anti-mBSA were fourfold higher and IgG1 was even eightfold higher. IgG2b anti-mBSA was not significantly different (Figure 1E). To investigate the role of a particular FcγR in joint inflammation, AIA was elicited by injection of 60 μg of mBSA directly into the knee joints of immunized FcγRI-, FcγRIII-, and FcγRII-deficient mice and their wild-type controls. Because FcγRIII−/− has been described as a main regulator of IC diseases, we expected a down-regulation of swelling in FcγRIII−/− mice but knee swelling was not significantly different from controls at all time points measured (Figure 2B). The course of knee joint swelling in FcγRI−/− was also comparable (Figure 2A) suggesting that FcγRI and FcγIII are redundant with respect to joint inflammation. Subsequently we investigated the role of FcγRII in joint inflammation. Because FcγRII−/− mice have been shown to be highly vulnerable to ICs, injection of 60 μg of mBSA into the knee joint may be too high and for that reason we also injected a lower (15 μg) mBSA dose. Injection of 15 μg of mBSA into knee joints of FcγRII−/− mice resulted in a significantly higher knee joint swelling if compared to controls (2.4versus 1.7) at day 1 but no difference was found anymore at day 4 or day 7 after AIA induction (Figure 2C). Injection of 60 μg of mBSA led to a much higher swelling at day 1 (3.0 versus 2.0) and compared to controls was still significantly higher at day 7 after AIA induction (2.0 versus 1.1) (Figure 2D). In addition, cellular infiltration and exudate in the knee joint was studied by histology. At day 7 after induction of AIA, total knee joint sections were made and stained with H&E. In FcγRI−/− and FcγRIII−/− arthritic knees, similar exudate and infiltrate was measured in all animals studied (Figure 3, A and B). In arthritic knees of FcγRII-deficient mice, both exudate and infiltrate were found to be significantly elevated (180% and 242% in the 60-μg group, respectively) (Figure 3D). This reached significance only in mice injected with the high dose of mBSA (Figure 3; D, E, and F). As the total number of inflammatory cells present in the synovium at day 7 after AIA was not different in FcγRI−/− and FcγRIII−/− and higher in FcγRII−/− mice, we further investigated whether the absence of a particular Fc receptor might influence the type of inflammatory cell present in the joint. The polymorphonuclear leukocyte/macrophage ratio was determined by immunolocalization using NIMP-R14 that stains polymorphonuclear leukocyte specifically. In arthritic knee joints of FcγRI−/−, FcγRIII−/−, and FcγRII−/− mice and their controls, no differences were found in the polymorphonuclear leukocyte/macrophage ratio that was 40:60 in the exudate and 25:75 in the infiltrate (data not shown). As Fc receptors have been shown to be involved in removal of ICs from various body compartments, the presence of IgG ICs localized in the arthritic joints at day 7 AIA was determined using rabbit anti-murine IgG antibodies. No significant differences in IC deposition was found in the knee joints of FcγRI−/−, FcγRIII−/−, and FcγRII−/− mice and their controls (data not shown). Subsequently, we studied the role of FcγRI, FcγRIII, and FcγRII in cartilage damage. The earliest cartilage damage seen during experimental arthritis is loss of proteoglycans from the cartilage matrix that is evident between 24 and 48 hours after AIA induction. Proteoglycan breakdown was measured by determining the loss of red staining in safranin O-stained knee joint sections using an arbitrary scale from 0 to 3. At day 7 after induction of AIA, loss of red staining in the cartilage layers of femur and tibia in control mice injected with 60 μg of mBSA reached almost maximal values (Figure 4; A, B, and D). In arthritic knee joints of FcγRI−/− (Figure 4A), FcγRIII−/− (Figure 4B), and FcγRII−/− (Figure 4D) mice, proteoglycan depletion was not significantly different from their arthritic control groups. Injection of the 15-μg mBSA dose showed lower PG depletion (Figure 4C). Although proteoglycan depletion was slightly higher in the FcγRII−/− mice, this difference did not reach significance. Within AIA, MMPs have been shown to be involved in degradation of aggrecan31Van Meurs J van Lent PLEM Holthuysen AEM Lambrou D Bayne E Singer I van den Berg WB Active matrix metalloproteinases are present in cartilage during immune complex-mediated arthritis: a pivotal role for stromelysin-1 in cartilage destruction.J Immunol. 1999; 163: 5633-5639PubMed Google Scholar, 32Van Meurs JBJ van Lent PLEM Holthuysen AEM Singer II Bayne EK van den Berg WB Kinetics of aggrecanase and metalloproteinase induced neoepitopes in various stages of cartilage destruction in murine arthritis.Arthritis Rheum. 1999; 42: 1128-1139Crossref PubMed Scopus (113) Google Scholar and collagen33Van Meurs JBJ van Lent PLEM Stoop R Holthuysen AEM Singer II Bayne EK Mudgett J Poole R Billinghurst C van der Kraan PK Buma P van den Berg WB Cleavage of aggrecan at Asn 341-Phe 342 site coincides with the initiation of collagen damage in murine antigen-induced arthritis: a pivotal role for stromelysin-1 in MMP activity during antigen-induced arthritis.Arthritis Rheum. 1999; 42: 2074-2084Crossref PubMed Scopus (100) Google Scholar leading to irreversible cartilage destruction. MMPs degrade aggrecan leaving the C terminal ending with the amino acid sequence VDIPEN that can be detected by specific antibodies around day 5 after induction of AIA.32Van Meurs JBJ van Lent PLEM Holthuysen AEM Singer II Bayne EK van den Berg WB Kinetics of aggrecanase and metalloproteinase induced neoepitopes in various stages of cartilage destruction in murine arthritis.Arthritis Rheum. 1999; 42: 1128-1139Crossref PubMed Scopus (113) Google Scholar For this reason, AIA day 7 was taken to detect VDIPEN expression in the cartilage matrix. The amount of VDIPEN was measured by de
Referência(s)