Joint inflammation and chondrocyte death become independent of Fcgamma receptor type III by local overexpression of interferon-gamma during immune complex-mediated arthritis.

OBJECTIVE: It has previously been shown that the onset and the degree of joint inflammation during immune complex (IC)-mediated arthritis depend on Fcgamma receptor type III (FcgammaRIII). Local adenoviral overexpression of interferon-gamma (IFNgamma) in the knee joint prior to onset of IC-mediated arthritis aggravated severe cartilage destruction. In FcgammaRI(-/-) mice, however, chondrocyte death was not enhanced by IFNgamma, whereas matrix metalloproteinase (MMP)-mediated aggrecan breakdown was markedly elevated, suggesting a role for the activating FcgammaRIII in the latter process. We undertook this study to determine the role of FcgammaRIII in joint inflammation and severe cartilage destruction in IFNgamma-stimulated IC-mediated arthritis, using FcgammaRIII(-/-) mice. METHODS: FcgammaRIII(-/-) and wild-type (WT) mice were injected in the knee joint with recombinant adenovirus encoding murine IFNgamma (AdIFNgamma) or with adenovirus encoding enhanced green fluorescent protein 1 day prior to induction of IC-mediated arthritis. Histologic sections were obtained 3 days after arthritis onset to study inflammation and cartilage damage. MMP-mediated expression of the VDIPEN neoepitope was detected by immunolocalization. Chemokine and FcgammaR expression levels were determined in synovial washouts and synovium, respectively. RESULTS: Injection of AdIFNgamma in naive knee joints markedly increased levels of messenger RNA for FcgammaRI, FcgammaRII, and FcgammaRIII. Upon IFNgamma overexpression prior to induction of IC-mediated arthritis, joint inflammation was similar in FcgammaRIII(-/-) and WT mice. The percentage of macrophages in the knee joint was increased, which correlated with high concentrations of the macrophage attractant macrophage inflammatory protein 1alpha. Furthermore, IFNgamma induced 2-fold and 3-fold increases in chondrocyte death in WT controls and FcgammaRIII(-/-) mice, respectively. Notably, VDIPEN expression also remained high in FcgammaRIII(-/-) mice. CONCLUSION: IFNgamma bypasses the dependence on FcgammaRIII in the development of IC-mediated arthritis. Furthermore, both FcgammaRI and FcgammaRIII can mediate MMP-dependent cartilage matrix destruction.

Liposomal targeting of glucocorticoids to synovial lining cells strongly increases therapeutic benefit in collagen type II arthritis.

OBJECTIVE: To investigate the effect of a single intravenous treatment with glucocorticoids (GC) encapsulated in long-circulating PEG-liposomes on both joint inflammation and cartilage destruction and to investigate the phenomenon of selective homing of these liposomes in the inflamed synovium. METHODS: Mice with collagen type II-induced arthritis (CIA) were intravenously treated with liposomal and free prednisolone phosphate (PLP) a few days after the first signs of the disease. Joint inflammation was scored during 1 week after treatment, after which sections of the knee joints were prepared for assessment of cartilage damage. In addition, arthritic mice were treated with liposomes containing colloidal gold. 24 hours after injection, knee joint sections were prepared in which the location of liposomes was visualised. RESULTS: Treatment of CIA with 10 mg/kg liposomal PLP resulted in a strong and lasting resolution of joint inflammation. 10 mg/kg free PLP only became slightly effective after repeated daily injections. Although joint inflammation recurred 1 week after treatment with liposomal PLP, knee joint sections prepared at this time indicated that the cartilage damage was still reduced. Localisation of gold labelled liposomes in the inflamed joints was seen in the proximity of blood vessels, in the cellular infiltrate, but mainly in the synovial lining. Unaffected joints did not take up liposomes. CONCLUSIONS: By using the property of long-circulating liposomes to target the synovial lining selectively in inflamed joints, the anti-inflammatory activity of GC can be greatly increased, showing also the beneficial effect of reduced cartilage destruction.

Crucial role of synovial lining macrophages in the promotion of transforming growth factor beta-mediated osteophyte formation.

OBJECTIVE: To investigate in vivo and in vitro whether macrophages have an intermediate role in transforming growth factor beta (TGFbeta)-induced osteophyte formation. METHODS: In vivo, synovial lining macrophages were selectively depleted by injection of clodronate-laden liposomes 7 days prior to injection of 20 ng or 200 ng of TGFbeta into murine knee joints 3 times, on alternate days. Total knee joint sections were obtained on day 7 after the last injection and stained with Safranin O. Production of bone morphogenetic protein 2 (BMP-2) and BMP-4 was determined by immunolocalization. The interaction between murine macrophages and mesenchymal cells (precursors with chondrogenic potential) was studied in vitro using a Transwell system in which RAW macrophages were cocultured with C3H10T1/2 mesenchymal cells. Spheroid neocartilage formation was quantified microscopically after staining with May-Grünwald-Giemsa. RESULTS: Triple injections of 20 ng or 200 ng of TGFbeta into normal murine knee joints induced significant osteophyte formation at the lateral and medial sites of the patella and femur on day 7 after the last injection. Strikingly, removal of synovial lining macrophages prior to TGFbeta injection resulted in a drastic reduction of osteophyte formation (by 70% and 64% after injection of 20 ng and 200 ng of TGFbeta, respectively). Synovial lining cells produced BMP-2 and BMP-4 after TGFbeta stimulation, whereas BMP-2 and BMP-4 were absent in the synovial tissue after macrophage depletion. In vitro, clustering and spheroid formation of C3H10T1/2 was induced by TGFbeta concentrations of >1 ng/ml. However, in the Transwell system, in the presence of murine macrophages, 0.5 ng/ml of TGFbeta was very effective in generating large spheroids, suggestive of macrophage-derived (co)factors. In coculture supernatants, TGFbeta concentrations were not elevated in the presence of macrophages, indicating generation of other growth factors involved in spheroid formation. CONCLUSION: These findings indicate that macrophages are crucial intermediate factors in osteophyte formation induced by TGFbeta, probably by inducing other chondrogenic signals.

Skewed balance in basal expression and regulation of activating v inhibitory Fcgamma receptors in macrophages of collagen induced arthritis sensitive mice.

BACKGROUND: Recently, it has been found that collagen type II arthritis susceptible mouse strains are hyperreactive to immune complexes (ICs), locally deposited into their knee joints. OBJECTIVE: To investigate whether this strain specific knee joint hyperreactivity is related to a disturbed regulation of activatory and inhibitory FcgammaR on their macrophages before and after stimulation with ICs. METHODS: Macrophages from collagen induced arthritis susceptible strains (DBA/1 and B10.RIII) and non-susceptible strains (C57BL/6 and BALB/c) were compared. FcgammaR levels on macrophages were detected at protein level by flow cytometric analysis and at mRNA level by reverse transcriptase-polymerase chain reaction. Macrophages were stimulated with ICs, and production of cytokines and enzymes was measured at different times. RESULTS: On synovial and peritoneal macrophages of DBA/1 mice a higher basal FcgammaRII and III expression was found, which was skewed towards the activating FcgammaRIII. In B10.RIII macrophages, however, FcgammaRIII levels were much lower. Regulation of FcgammaR mRNA levels in macrophages was tested after stimulation with ICs for one and three days. DBA/1 and B10.RIII macrophages showed a prolonged up regulation of activating FcgammaRI and III, whereas the inhibiting FcgammaRII was significantly down regulated compared with non-susceptible strains. In line with this, DBA/1 and B10.RIII macrophages showed a higher interleukin 1 (IL1) and matrix metalloproteinase (MMP) production after IC exposure, whereas IL6 production was significantly reduced. CONCLUSIONS: This study indicates that macrophages derived from collagen type II arthritis susceptible mice show a disregulated FcgammaR expression before, and even more clearly, after activation by ICs involved in inflammation and cartilage degradation, resulting in prolonged expression of activatory FcgammaRI and III, down regulation of inhibitory FcgammaRII and increased release of IL1 and MMP.

Coordinate expression of activating Fc gamma receptors I and III and inhibiting Fc gamma receptor type II in the determination of joint inflammation and cartilage destruction during immune complex-mediated arthritis.

OBJECTIVE: To study the role of the activating Fc gamma receptor types I and III (Fc gamma RI and Fc gamma RIII, respectively) and the inhibiting Fc gamma receptor II (Fc gamma RII) in inflammation and in various aspects of cartilage destruction during arthritis that is solely induced by immune complexes. METHODS: Immune complex-mediated arthritis (ICA) was passively induced by lysozyme-antilysozyme complexes in Fc gamma RI-, Fc gamma RIII-, and Fc gamma RII-knockout mice and their wild-type controls. Total knee joints were isolated to study inflammation and cartilage destruction (loss of proteoglycans [PGs], chondrocyte death, matrix metalloproteinase [MMP]-mediated neoepitope [VDIPEN] expression, and erosion). The presence of an active phenotype of macrophages was studied by detection of myeloid-related proteins 8 and 14 (MRP8 and MRP14, respectively). RESULTS: Influx and activation of inflammatory cells (MRP expression) during ICA was decreased in Fc gamma RIII-deficient mice and enhanced in mice lacking Fc gamma RII. Mild cartilage destruction reflected by loss of PGs was consistent with the degree of inflammation. Mice lacking Fc gamma RIII showed almost no PG depletion, whereas in Fc gamma RII(-/-) mice, PG depletion was increased 3-7-fold in various cartilage areas. Initiation of erosive cartilage destruction, as reflected by MMP-mediated VDIPEN expression, was reduced in Fc gamma RIII(-/-) and Fc gamma RI(-/-) mice, directing the two different critical steps of cellular influx and subsequent activation. These aspects were enhanced in Fc gamma RII(-/-) mice. In Fc gamma RI(-/-) and Fc gamma RIII(-/-) mice, VDIPEN expression was 90-99% lower, whereas in Fc gamma RII(-/-) mice, VDIPEN expression was increased 4-fold. Chondrocyte death was reduced in Fc gamma RIII(-/-) mice (68% lower) and enhanced in Fc gamma RII(-/-) mice (6-12-fold higher). Progression of arthritis and erosion of the cartilage surface were markedly elevated in Fc gamma RII(-/-) arthritic joints. CONCLUSION: During ICA, Fc gamma RIII is the dominant activating receptor mediating joint inflammation, whereas both Fc gamma RI and Fc gamma RIII are involved in cartilage destruction. Fc gamma RII inhibits both joint inflammation and severe cartilage destruction during ICA.

Local overexpression of adeno-viral IL-4 protects cartilage from metallo proteinase-induced destruction during immune complex-mediated arthritis by preventing activation of pro-MMPs.

OBJECTIVE: To determine whether IL-4 protects against metalloproteinase-induced cartilage destruction during immune complex mediated arthritis and to elucidate its mechanism. METHODS: Experimental immune complex arthritis (ICA) was raised by injecting lysozyme into the knee joints of mice which previously were given anti-lysozyme antibodies. Three days before ICA induction, mice were injected into the right knee joint with either IL-4 expressing or empty control recombinant human type 5 adenovirus. Joint inflammation and cartilage destruction (PG depletion, erosion) was measured by histology of total knee joints. Aggrecan breakdown in cartilage caused by metalloproteinases (MMPs) was studied by immunolocalization using anti-VDIPEN antibodies. RESULTS: Four days after ICA induction, histological analysis showed comparable exudate and infiltrate in both groups. Depletion of proteoglycans as measured by loss of red staining was also comparable in both groups. IL-4 treatment inhibited MMP-mediated neoepitope expression by 90%. Moreover, cartilage matrix erosion was evident in all animals (10 out of 10 mice) in the control group and significantly diminished (only two out of ten mice) in the IL-4 treated group. Incubation of patellae with APMA, which activates latent MMPs resulted in VDIPEN expression which was not significantly different from control ICA indicating that comparable amounts of latent pro-MMPs are present in IL-4 treated arthritic knee joints. CONCLUSION: This study indicates that during ICA, IL-4 largely prevents MMP-mediated aggrecan breakdown and severe cartilage erosion. IL-4 does not inhibit production of latent MMPs by the chondrocyte but predominantly interferes with its activation.