PMC:17821 / 30250-40652 JSONTXT

{"project":"2_test","denotations":[{"id":"11056679-4111070-4381695","span":{"begin":3247,"end":3249},"obj":"4111070"},{"id":"11056679-10623430-4381696","span":{"begin":6713,"end":6715},"obj":"10623430"}],"text":"Results\n\nFcγ receptors are essential for synovial inflammation during immune-complex-mediated arthritis\nTo investigate the involvement of Fcγ receptors in locally induced ICA, we used FcR γ-chain-/- C57BL/6 mice, which lack functional FcγRI and RIII. ICA was induced in knee joints of FcR γ-chain-/- mice and control C57BL/6 mice. Histology of total knee joints showed florid inflammation in C57BL/6 mice 3 days after induction of ICA (Fig. 1b). In knee joints of FcR γ-chain-/- mice, however, virtually no inflammatory cells were observed at day 3 of ICA (Fig. 1a). Joint inflammation, defined as cells present in the joint cavity (exudate) and synovium (infiltrate), was scored. Both exudate and infiltrate were substantial in C57BL/6 mice whereas, in FcR γ-chain-/- mice, cell influx was virtually absent (Table 1).\nTo investigate whether FcγRs are involved in the upregulation of inflammatory mediators seen during the first phase of this arthritis, RT-PCR was performed on synovial specimens from arthritic knee joints. Levels of mRNA of inflammatory mediators (IL-1β, IL-1Ra, MCP-1, MIP-2) were detected semiquantitatively in knockout and control mice, 6 and 24 h after ICA induction (Fig. 2). IL-1β and IL-1Ra mRNA levels were high in control arthritis and appeared to be decreased (down to 28) in FcR γ-chain-/- mice. MCP-1 and MIP-2 mRNA levels were also diminished in the early phase of ICA (6 h) relative to the levels of controls (respectively down to 26 and 24) suggesting downregula-tion of both polymorphonuclear neutrophils (PMNs) and monocyte-specific chemokines.\nSubsequently, intra-articular production of IL-1 and IL-1Ra protein during arthritis was measured. Washouts of synovial specimens from joints of control C57BL/6 mice contained considerable amounts of IL-1 (240 pg/ml) at 6 h after induction of arthritis; at 24 h, IL-1 levels were reduced to 70 pg/ml (Table 2). Washouts of arthritic FcR γ-chain-/- joints contained 200 ng/ml IL-1 at 6 h but no detected IL-1 at 24 h after induction, indicating that in the absence of functional FcγRI and RIII, a rapid downregulation of IL-1 production is found. IL-1Ra production was comparable at 6 h in the two strains (respectively 180 and 215 pg/ml) and below the detection limit at 24 h after ICA induction in both strains (Table 2).\nTo exclude the possibility that the strong reduction of inflammation during ICA in FcR γ-chain-/- mice is caused by a lower level of immune complexes which are formed or retained within the knee joint, deposition of IgG in the knee joint was detected using biotinylated goat anti-rabbit IgG. As Table 3 shows, at 6 and 24 h after ICA induction, no difference was found between FcR γ-chain-/- and C57BL/6 mice in the amount of immune complexes deposited within the knee joints. We compared deposition of complement (C3c) in the synovial tissue of both strains to rule out the possibility that a difference in complement deposition caused differences in inflammation. Control and knockout mice showed the same amounts of C3c in the tissue as was determined by immunohistochemistry (Table 3).\n\nExpression of FcγRII/III is elevated in resident macrophages of naïve CIA-sensitive mice that are hypersensitive to immune complexes\nIn a recent study [36], we noticed that certain mouse strains that are prone to develop collagen-type-II autoimmune arthritis are also particularly susceptible to immune-complex-induced inflammation. Induction of ICA in the DBA/1 knee joint resulted in a more severe arthritis than that in C57BL/6 mice, and this became chronic (Table 1). Local production of IL-1 was also significantly higher and more prolonged than in C57BL/6 mice, both at 6 and 24 h after ICA induction (Table 2). To investigate whether the enhanced inflammation in DBA/1 mice during ICA was still mediated by FcRs, FcR γ-chain-/- mice were backcrossed to DBA/1 (DBAγ-/-) mice. ICA was induced in these mice and the inflammation was compared with that in DBAγ+/+ littermates. We found that at day 3 after arthritis induction, arthritis was full-blown in DBAγ+/+mice (arbitrary scores of 1.0 ± 0.4 for exudate and 1.6 ± 0.3 for infiltrate) but was completely absent in γ-chain-deficient DBA/1 mice (0.1 ± 0.2 and 0.1 ± 0.1, respectively; not shown).\nTo examine whether the different reactions in knee joints of C57BL/6 and DBA/1 mice to immune complexes might be due to variable local expression of FcγR, expression in situ of FcγRs in naïve knee joints was determined immunohistochemically, using the monoclonal antibody (mAb) 2.4G2 (FcγRII/III). Synovial macrophage-like type A lining cells and deeper-lying synovial macrophages were the dominant cell types expressing these receptors, as was seen in double-labeling studies with F4/80 (data not shown). Interestingly, synovial macrophages of naïve DBA/1 mice stained more intensely than those of C57BL/6 mice (Fig. 3), indicating a higher constitutive expression of FcγRs in lining macrophages of these autoimmune-prone mice.\nTo quantify the difference in FcγR expression by macrophages of the two strains (DBA/1 and C57BL/6), we further investigated receptor expression on peritoneal macrophages, using FACS analysis. Macrophages of DBA/1 mice (mean fluorescence 440 ± 50 intensity per cell) displayed almost twice the mean fluorescence with mAb 2.4G2 as those of C57BL/6 mice (240 ± 30 intensity per cell) (Fig. 4). These findings also indicate that FcγRII/III is significantly upregulated on the membrane of macrophage populations present in various body compartments of naïve DBA/1 mice.\nTo further investigate whether a higher basal FcγR expression on macrophages has consequences for the activation of cells upon interaction with immune complexes, peritoneal macrophages of both strains were incubated with aggregated IgG and production of IL-1 was determined by bioassay. Incubation of C57BL/6 cells for 24 or 48 h caused the release of considerable amounts of bioactive IL-1 (45 pg/ml at 24 h and 50 pg/ml at 48 h) (Fig. 5). Interestingly, DBA/1 macrophages produced significantly higher amounts of IL-1 (120 and 135 pg/ml, respectively) than C57BL/6 mice.\n\nArthritogenic triggers other than immune complexes cause comparable inflammation in knee joints of mice that differ in FcγR expression levels\nTo substantiate the hypothesis that differences in FcR expression underlie the variation seen during immune-complex-induced joint inflammation, we injected various arthritogenic triggers (zymosan, SCWs) that do not act via FcγRs into the knee joints of three strains of mice (FcR γ-chain-/-, C57BL/6, and DBA/1). A previous study had shown that when these triggers were used, there was no difference between DBA/1 and C57BL/6 mice in joint inflammation [34]. In the present study, when zymosan was injected into knee joints of FcR γ-chain-/- mice, the inflammation was similar to that in control C57BL/6 mice. At day 3 after injection, inflammation in FcR γ-chain-/-mice was scored as 1.0 ± 0.4 for infiltrate and 0.9 ± 0.6 for exudate, versus 0.9 ± 0.3 and 0.9 ± 0.5, respectively, in control C57BL/6 mice. These findings indicate that knee joints of the three strains examined develop comparable, marked inflammation after injection of zymosan or SCWs. Moreover, IL-1β production in the knee joints during zymosan-induced arthritis was comparable in DBA/1 and C57BL/6 mice (Table 2).\n\nFcγR expression on macrophages is correlated with cartilage destruction during immune-complex-mediated arthritis\nApart from inflammation, absence or overexpression of FcγRs by local macrophages may also have serious consequences for cartilage destruction. We investigated several parameters of cartilage destruction (depletion of proteogly-cans [PGs], chondrocyte death, and erosion of the cartilage matrix). Arthritic knee joints of the three strains were compared at different time points after ICA induction. At day 3, control C57BL/6 mice showed marked PG depletion in various cartilage surfaces (patella, femur adjacent to patella, lateral and medial condyle of the femur, and lateral and medial condyle of the tibia). Strikingly, PGs were not depleted in arthritic knee joints of the FcR γ-chain-/- mice (Fig. 6a,b; Table 4). In contrast, arthritic DBA/1 knee joints showed markedly greater depletion of PGs than those of C57BL/6 mice. Moreover, PG depletion was still severe at days 7 and 14 in the DBA/1 mice, whereas in the C57BL/6 mice it was almost fully restored by day 14 (Fig. 6b-e; Table 4). When ICA was induced in FcR γ-/- mice with a DBA/1 background, PG depletion at day 3 was scored 2.6 ± 0.3 in the Fcγ-chain+/+ mice and was totally absent in the FcR γ-chain-/- ones.\nChondrocyte death was measured by determining empty lacunae in the cartilage matrix and breaks in DNA strands as evaluated using an in situ cell detection kit (TUNEL method; data not shown). In knee joints of C57BL/6 mice, chondrocyte death at day 3 was 5% in the patella and 30% in the adjacent femur. In the knee joints of knockout mice, chondrocyte death was completely absent (Table 4), whereas in the DBA/1 mice, it was strikingly higher in both the patella (50%) and the femur (90%) than in the control mice (C57BL/6).\nFinally, erosion of the cartilage matrix in knee joints was measured. Whereas no erosion was found in either FcR γ-chain-/- or C57BL/6 mice at day 3 after arthritis induction, in DBA/1 mice erosion was already considerable by day 3, and complete loss of the cartilage surface layer up to the tidemark was observed at days 7 and day 14 (Fig. 6d,e; Table 4).\n\nSevere cartilage destruction in DBA/1 mice is specific for immune-complex-mediated arthritis\nTo further substantiate the interpretation that immune complexes and FcγRs, rather than inflammatory cells, caused the observed severe joint cartilage destruction, various concentrations of zymosan were injected into the knee joints of all three mouse strains (FcR γ-chain-/-, C57BL/6, and DBA/1). Even large amounts of zymosan (up to 180 μg), although inducing a tremendous influx of inflammatory cells, failed to induce chondrocyte death or cartilage erosion. PG depletion at day 3, however, was scored as 3.6 ± 0.4 in FcR γ-/- mice, versus 0.6 ± 0.3 in C57BL/6 mice. At day 7, mean PG depletion was scored as 1.8 ± 0.5 in C57BL/6 mice and 1.7 ± 0.8 in DBA/1. PG depletion in the cartilage matrix in response to zymosan, therefore, was marked but did not differ between the three strains.\n"}

{"project":"Colil","denotations":[{"id":"T47","span":{"begin":6713,"end":6715},"obj":"10623430"},{"id":"T46","span":{"begin":3247,"end":3249},"obj":"4111070"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Results\n\nFcγ receptors are essential for synovial inflammation during immune-complex-mediated arthritis\nTo investigate the involvement of Fcγ receptors in locally induced ICA, we used FcR γ-chain-/- C57BL/6 mice, which lack functional FcγRI and RIII. ICA was induced in knee joints of FcR γ-chain-/- mice and control C57BL/6 mice. Histology of total knee joints showed florid inflammation in C57BL/6 mice 3 days after induction of ICA (Fig. 1b). In knee joints of FcR γ-chain-/- mice, however, virtually no inflammatory cells were observed at day 3 of ICA (Fig. 1a). Joint inflammation, defined as cells present in the joint cavity (exudate) and synovium (infiltrate), was scored. Both exudate and infiltrate were substantial in C57BL/6 mice whereas, in FcR γ-chain-/- mice, cell influx was virtually absent (Table 1).\nTo investigate whether FcγRs are involved in the upregulation of inflammatory mediators seen during the first phase of this arthritis, RT-PCR was performed on synovial specimens from arthritic knee joints. Levels of mRNA of inflammatory mediators (IL-1β, IL-1Ra, MCP-1, MIP-2) were detected semiquantitatively in knockout and control mice, 6 and 24 h after ICA induction (Fig. 2). IL-1β and IL-1Ra mRNA levels were high in control arthritis and appeared to be decreased (down to 28) in FcR γ-chain-/- mice. MCP-1 and MIP-2 mRNA levels were also diminished in the early phase of ICA (6 h) relative to the levels of controls (respectively down to 26 and 24) suggesting downregula-tion of both polymorphonuclear neutrophils (PMNs) and monocyte-specific chemokines.\nSubsequently, intra-articular production of IL-1 and IL-1Ra protein during arthritis was measured. Washouts of synovial specimens from joints of control C57BL/6 mice contained considerable amounts of IL-1 (240 pg/ml) at 6 h after induction of arthritis; at 24 h, IL-1 levels were reduced to 70 pg/ml (Table 2). Washouts of arthritic FcR γ-chain-/- joints contained 200 ng/ml IL-1 at 6 h but no detected IL-1 at 24 h after induction, indicating that in the absence of functional FcγRI and RIII, a rapid downregulation of IL-1 production is found. IL-1Ra production was comparable at 6 h in the two strains (respectively 180 and 215 pg/ml) and below the detection limit at 24 h after ICA induction in both strains (Table 2).\nTo exclude the possibility that the strong reduction of inflammation during ICA in FcR γ-chain-/- mice is caused by a lower level of immune complexes which are formed or retained within the knee joint, deposition of IgG in the knee joint was detected using biotinylated goat anti-rabbit IgG. As Table 3 shows, at 6 and 24 h after ICA induction, no difference was found between FcR γ-chain-/- and C57BL/6 mice in the amount of immune complexes deposited within the knee joints. We compared deposition of complement (C3c) in the synovial tissue of both strains to rule out the possibility that a difference in complement deposition caused differences in inflammation. Control and knockout mice showed the same amounts of C3c in the tissue as was determined by immunohistochemistry (Table 3).\n\nExpression of FcγRII/III is elevated in resident macrophages of naïve CIA-sensitive mice that are hypersensitive to immune complexes\nIn a recent study [36], we noticed that certain mouse strains that are prone to develop collagen-type-II autoimmune arthritis are also particularly susceptible to immune-complex-induced inflammation. Induction of ICA in the DBA/1 knee joint resulted in a more severe arthritis than that in C57BL/6 mice, and this became chronic (Table 1). Local production of IL-1 was also significantly higher and more prolonged than in C57BL/6 mice, both at 6 and 24 h after ICA induction (Table 2). To investigate whether the enhanced inflammation in DBA/1 mice during ICA was still mediated by FcRs, FcR γ-chain-/- mice were backcrossed to DBA/1 (DBAγ-/-) mice. ICA was induced in these mice and the inflammation was compared with that in DBAγ+/+ littermates. We found that at day 3 after arthritis induction, arthritis was full-blown in DBAγ+/+mice (arbitrary scores of 1.0 ± 0.4 for exudate and 1.6 ± 0.3 for infiltrate) but was completely absent in γ-chain-deficient DBA/1 mice (0.1 ± 0.2 and 0.1 ± 0.1, respectively; not shown).\nTo examine whether the different reactions in knee joints of C57BL/6 and DBA/1 mice to immune complexes might be due to variable local expression of FcγR, expression in situ of FcγRs in naïve knee joints was determined immunohistochemically, using the monoclonal antibody (mAb) 2.4G2 (FcγRII/III). Synovial macrophage-like type A lining cells and deeper-lying synovial macrophages were the dominant cell types expressing these receptors, as was seen in double-labeling studies with F4/80 (data not shown). Interestingly, synovial macrophages of naïve DBA/1 mice stained more intensely than those of C57BL/6 mice (Fig. 3), indicating a higher constitutive expression of FcγRs in lining macrophages of these autoimmune-prone mice.\nTo quantify the difference in FcγR expression by macrophages of the two strains (DBA/1 and C57BL/6), we further investigated receptor expression on peritoneal macrophages, using FACS analysis. Macrophages of DBA/1 mice (mean fluorescence 440 ± 50 intensity per cell) displayed almost twice the mean fluorescence with mAb 2.4G2 as those of C57BL/6 mice (240 ± 30 intensity per cell) (Fig. 4). These findings also indicate that FcγRII/III is significantly upregulated on the membrane of macrophage populations present in various body compartments of naïve DBA/1 mice.\nTo further investigate whether a higher basal FcγR expression on macrophages has consequences for the activation of cells upon interaction with immune complexes, peritoneal macrophages of both strains were incubated with aggregated IgG and production of IL-1 was determined by bioassay. Incubation of C57BL/6 cells for 24 or 48 h caused the release of considerable amounts of bioactive IL-1 (45 pg/ml at 24 h and 50 pg/ml at 48 h) (Fig. 5). Interestingly, DBA/1 macrophages produced significantly higher amounts of IL-1 (120 and 135 pg/ml, respectively) than C57BL/6 mice.\n\nArthritogenic triggers other than immune complexes cause comparable inflammation in knee joints of mice that differ in FcγR expression levels\nTo substantiate the hypothesis that differences in FcR expression underlie the variation seen during immune-complex-induced joint inflammation, we injected various arthritogenic triggers (zymosan, SCWs) that do not act via FcγRs into the knee joints of three strains of mice (FcR γ-chain-/-, C57BL/6, and DBA/1). A previous study had shown that when these triggers were used, there was no difference between DBA/1 and C57BL/6 mice in joint inflammation [34]. In the present study, when zymosan was injected into knee joints of FcR γ-chain-/- mice, the inflammation was similar to that in control C57BL/6 mice. At day 3 after injection, inflammation in FcR γ-chain-/-mice was scored as 1.0 ± 0.4 for infiltrate and 0.9 ± 0.6 for exudate, versus 0.9 ± 0.3 and 0.9 ± 0.5, respectively, in control C57BL/6 mice. These findings indicate that knee joints of the three strains examined develop comparable, marked inflammation after injection of zymosan or SCWs. Moreover, IL-1β production in the knee joints during zymosan-induced arthritis was comparable in DBA/1 and C57BL/6 mice (Table 2).\n\nFcγR expression on macrophages is correlated with cartilage destruction during immune-complex-mediated arthritis\nApart from inflammation, absence or overexpression of FcγRs by local macrophages may also have serious consequences for cartilage destruction. We investigated several parameters of cartilage destruction (depletion of proteogly-cans [PGs], chondrocyte death, and erosion of the cartilage matrix). Arthritic knee joints of the three strains were compared at different time points after ICA induction. At day 3, control C57BL/6 mice showed marked PG depletion in various cartilage surfaces (patella, femur adjacent to patella, lateral and medial condyle of the femur, and lateral and medial condyle of the tibia). Strikingly, PGs were not depleted in arthritic knee joints of the FcR γ-chain-/- mice (Fig. 6a,b; Table 4). In contrast, arthritic DBA/1 knee joints showed markedly greater depletion of PGs than those of C57BL/6 mice. Moreover, PG depletion was still severe at days 7 and 14 in the DBA/1 mice, whereas in the C57BL/6 mice it was almost fully restored by day 14 (Fig. 6b-e; Table 4). When ICA was induced in FcR γ-/- mice with a DBA/1 background, PG depletion at day 3 was scored 2.6 ± 0.3 in the Fcγ-chain+/+ mice and was totally absent in the FcR γ-chain-/- ones.\nChondrocyte death was measured by determining empty lacunae in the cartilage matrix and breaks in DNA strands as evaluated using an in situ cell detection kit (TUNEL method; data not shown). In knee joints of C57BL/6 mice, chondrocyte death at day 3 was 5% in the patella and 30% in the adjacent femur. In the knee joints of knockout mice, chondrocyte death was completely absent (Table 4), whereas in the DBA/1 mice, it was strikingly higher in both the patella (50%) and the femur (90%) than in the control mice (C57BL/6).\nFinally, erosion of the cartilage matrix in knee joints was measured. Whereas no erosion was found in either FcR γ-chain-/- or C57BL/6 mice at day 3 after arthritis induction, in DBA/1 mice erosion was already considerable by day 3, and complete loss of the cartilage surface layer up to the tidemark was observed at days 7 and day 14 (Fig. 6d,e; Table 4).\n\nSevere cartilage destruction in DBA/1 mice is specific for immune-complex-mediated arthritis\nTo further substantiate the interpretation that immune complexes and FcγRs, rather than inflammatory cells, caused the observed severe joint cartilage destruction, various concentrations of zymosan were injected into the knee joints of all three mouse strains (FcR γ-chain-/-, C57BL/6, and DBA/1). Even large amounts of zymosan (up to 180 μg), although inducing a tremendous influx of inflammatory cells, failed to induce chondrocyte death or cartilage erosion. PG depletion at day 3, however, was scored as 3.6 ± 0.4 in FcR γ-/- mice, versus 0.6 ± 0.3 in C57BL/6 mice. At day 7, mean PG depletion was scored as 1.8 ± 0.5 in C57BL/6 mice and 1.7 ± 0.8 in DBA/1. PG depletion in the cartilage matrix in response to zymosan, therefore, was marked but did not differ between the three strains.\n"}