P.L.E.M. van Lent

FcγRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection

The high-affinity receptor for IgG, FcgammaRI, shares its capacity to bind IgG2a immune complexes (IgG2a-IC) with the low-affinity receptor FcgammaRIII and complement factors, hampering the definition of its biological role. Moreover, in vivo, FcgammaRI is occupied by monomeric IgG2a, reducing its accessibility to newly formed IgG2a-IC. By using a variety of FcgammaR(-/-) mice, we demonstrate that in the absence of FcgammaRI, the IgG2a-IC-induced cellular processes of phagocytosis, cytokine release, cellular cytotoxicity, and antigen presentation are impaired. FcgammaRI(-/-) mice showed impaired hypersensitivity responses, strongly reduced cartilage destruction in an arthritis model, and impaired protection from a bacterial infection. We conclude that FcgammaRI contributes substantially to a variety of IgG2a-IC-dependent immune functions and immunopathological responses.

Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis

Objective: To study the active involvement of Myeloid-related proteins S100A8 and S100A9 in joint inflammation and cartilage destruction during antigen-induced arthritis (AIA). Methods: Joint inflammation and cartilage destruction was measured with 99mTc uptake and histology. The role of S100A8/A9 was investigated by inducing AIA in S100A9–/– mice that also lack S100A8 at protein level, or after intra-articular injection of rS100A8 in mouse knee joints. Cartilage destruction was measured using immunolocalisation of the neoepitope VDIPEN or NITEGE. mRNA levels of matrix metalloproteinases (MMPs) and cytokines were measured using reverse transcriptase (RT)-PCR. Results: Immunisation of S100A9–/– mice with the antigen mBSA induced normal cellular and humoral responses, not different from wild type (WT) controls. However, joint swelling measured at day 3 and 7 after AIA induction was significantly lower (36 and 70%, respectively). Histologically, at day 7 AIA, cellular mass was much lower (63–80%) and proteoglycan depletion from cartilage layers was significantly reduced (between 50–95%). Cartilage destruction mediated by MMPs was absent in S100A9–/– mice but clearly present in controls. MMP3, 9 and 13 mRNA levels were significantly lowered in arthritic synovia of S100A9–/–. In vitro stimulation of macrophages by the heterodimer S100A8/A9 or S100A8 elevated mRNA levels of MMP3, 9 and in particular MMP13. Intra-articular injection of S100A8 caused prominent joint inflammation and depletion of proteoglycans at day 1. Significant upregulation of mRNA levels of S100A8/A9, cytokines (interleukin 1 (IL1)), MMPs (MMP3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4) was found in the synovium and correlated with strong upregulation of NITEGE neoepitopes within the cartilage layers. Conclusions: S100A8/A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis.

Oxidized LDL enhances pro-inflammatory responses of alternatively activated M2 macrophages: A crucial role for Krüppel-like factor 2

OBJECTIVE Macrophages are key players in atherogenesis because of their properties to form foam cells that produce a large variety of pro-inflammatory mediators. We addressed the potency of phenotypic different macrophages to accumulate oxidized LDL. METHODS AND RESULTS Surprisingly, anti-inflammatory M2 macrophages but not pro-inflammatory M1 macrophages rapidly accumulated oxidized LDL. Simultaneously, expression of Krüppel-like factor 2, a nuclear transcription factor known to suppress inflammation in endothelial cells and monocytes, decreased and the functional phenotype of M2 macrophages shifted towards a pro-inflammatory profile, characterized by higher production of IL-6, IL-8 and MCP-1 and lower expression of IL-10 upon stimulation with LPS. In contrast, Krüppel-like factor 2 expression and the phenotype of M1 macrophages remained largely unchanged upon oxidized LDL exposure. Downregulation of Krüppel-like factor 2 expression of M2 macrophages using siRNA technology led to a significant increase of LPS-induced MCP-1 secretion. CONCLUSIONS We show that (1) anti-inflammatory M2 macrophages are more susceptible to foam cell formation than pro-inflammatory M1 macrophages, (2) exposure to oxidized LDL renders M2 macrophages pro-inflammatory, and (3) Krüppel-like factor 2 is involved in the enhanced secretion of MCP-1 by M2 macrophages loaded with oxidized LDL. The phenotype switch of M2 macrophages from an anti- to a pro-inflammatory profile may play an important role in pathogenesis of atherosclerosis, and could represent a novel therapeutic target.

Role of Fc receptor γ chain in inflammation and cartilage damage during experimental antigen‐induced arthritis

OBJECTIVE To study the role of Fc receptor (FcR) gamma chain in inflammation and cartilage destruction during antigen-induced arthritis (AIA). METHODS FcR gamma-/- mice and controls were immunized with methylated bovine serum albumin (mBSA) in Freunds complete adjuvant, followed by induction of arthritis by local injection of mBSA into the right knee joint. Joint inflammation was studied by 99mTc uptake and by histology. Breakdown of proteoglycans from the cartilage matrix was determined by loss of red staining in Safranin O-stained knee joint sections, and matrix metalloproteinase (MMP)-mediated aggrecan degradation was determined by immunolocalization using anti-VDIPEN antibodies. Chondrocyte death was measured by determining empty lacunae in hematoxylin-stained sections and with the TUNEL assay in cryostat sections. Erosion was detected as ruffling of the cartilage surface. RESULTS Joint swelling, as measured by 99mTc uptake on days 1, 3, and 7, was significantly decreased in FcR gamma-/- mice compared with controls. On day 7 after AIA induction, sustained joint inflammation, as seen histologically, was not significantly lower in FcR gamma-/- deficient mice. In various cartilage layers (femur, tibia, patella) of central arthritic knee joints, marked depletion of proteoglycans (40-70%), chondrocyte death (25-50%), and mild surface erosion were found. In FcR gamma-/- knee joints, depletion of proteoglycans was comparable (40-70%). Strikingly, chondrocyte death and matrix erosion were absent. Furthermore, MMP-induced aggrecan neoepitopes, which were abundantly found in controls, were also absent in FcR gamma-/-. Nevertheless, latent MMPs were present in the cartilage matrix as seen in APMA-activated patellae. CONCLUSION FcR gamma chain is involved in the severity of acute and sustained inflammation and is a crucial factor in cartilage erosion during AIA, probably by regulating activation of latent MMPs present in the cartilage matrix.

Local removal of phagocytic synovial lining cells by clodronate-liposomes decreases cartilage destruction during collagen type II arthritis

OBJECTIVE To investigate whether local removal of phagocytic synovial lining cells (SLCs) from the knee joint before onset of collagen type II arthritis has an effect on development of cartilage destruction. METHODS Phagocytic SLCs were selectively depleted by a single injection of clodronate laden liposomes in the knee joint seven days before induction of collagen type II arthritis (CIA). Clodronate laden liposomes were given in one knee joint either alone or in combination with a short-term oral treatment of dexamethasone. Cartilage damage including proteoglycan depletion and chondrocyte death was measured in total knee joints sections stained with safranin-o or haematoxylin. RESULTS Local removal of phagocytic SLCs, seven days before arthritis onset, prevented cell influx for the larger part. Chondrocyte death was significantly decreased in the SLC depleted arthritic joint both at an early (6 days) and late (12 days) time point after CIA induction. However, depletion of proteoglycans from femoral and patellar cartilage layers was not prevented. If the mild acute inflammation caused by a single clodronate laden liposome injection in the left knee joint, was blocked by a short-term (on consecutive days 9, 8, 7, 6, 5 before CIA onset) oral treatment with dexamethasone, cell influx, but also proteoglycan depletion was almost completely blocked. In the contralateral control right knee joint prominent cell influx and severe cartilage damage was observed, indicating that there was no effect of dexamethasone anymore at the onset of CIA. CONCLUSIONS This study shows that removal of phagocytic lining cells before CIA induction, particularly in the presence of a short-term treatment with dexamethasone, decreases cartilage destruction.

Stimulation of chondrocyte‐mediated cartilage destruction by S100A8 in experimental murine arthritis

OBJECTIVE To investigate whether S100A8 is actively involved in matrix metalloproteinase (MMP)-mediated chondrocyte activation. METHODS S100A8 and S100A9 proteins were detected in inflamed knee joints from mice with various forms of murine arthritis, using immunolocalization. Murine chondrocyte cell line H4 was stimulated with proinflammatory cytokines or recombinant S100A8. Messenger RNA (mRNA) and protein levels were measured using reverse transcriptase-polymerase chain reaction and intracellular fluorescence-activated cell sorting (FACS). Breakdown of aggrecan on the pericellular surface of the chondrocytes was measured using VDIPEN and NITEGE antibodies and FACS, and breakdown in patellar cartilage was measured by immunolocalization. RESULTS S100A8 and S100A9 proteins were abundantly expressed in and around chondrocytes in inflamed knee joints after induction of antigen-induced arthritis or onset of spontaneous arthritis in interleukin-1 (IL-1) receptor antagonist-knockout mice. Stimulation of chondrocytes by the proinflammatory cytokines tumor necrosis factor alpha, IL-1beta, IL-17, and interferon-gamma caused strong up-regulation of S100A8 mRNA and protein levels and up-regulation to a lesser extent of S100A9 levels. Stimulation of chondrocytes with S100A8 induced significant up-regulation of MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 mRNA levels (up-regulated 4, 4, 3, 16, 8, and 4 times, respectively). VDIPEN and NITEGE neoepitopes were significantly elevated in a concentration-dependent manner in chondrocytes treated with 0.2, 1, or 5 microg/ml of S100A8. (VDIPEN levels were elevated 17%, 67%, and 108%, respectively, and NITEGE levels were elevated 8%, 33%, and 67%, respectively.) S100A8 significantly increased the effect of IL-1beta on MMP-3, MMP-13, and ADAMTS-5. Mouse patellae incubated with both IL-1beta and S100A8 had elevated levels of NITEGE within the cartilage matrix when compared with patellae incubated with IL-1beta or S100A8 alone. CONCLUSION These findings indicate that S100A8 and S100A9 are found in and around chondrocytes in experimental arthritis. S100A8 up-regulates and activates MMPs and aggrecanase-mediated pericellular matrix degradation.

Reversible depletion of synovial lining cells after intra-articular treatment with liposome-encapsulated dichloromethylene diphosphonate

SummaryWe studied the depletion and repopulation of synovial lining cells in mice. A single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP) in the mouse knee joint caused selective elimination of synovial lining cells. Depletion of cells occurred within a few days as evidenced by light microscopic, electronmicroscopic and immunohistochemical studies. Maximal depletion was seen on day 7. Repopulation was observed in the following weeks, starting at the bone side of the joint. Until day 30, full recovery (60% recovery) was not observed in the lining lying adjacent to the dermis. Side effects on cartilage metabolism, such as inhibition of proteoglycan synthesis or degradation of proteoglycans from the matrix was minor but significant, 1 and 2 days after liposome treatment but thereafter full recovery was observed. Selective elimination of lining cells from the joint enabled us to study the in vivo role of these cells in the onset and subsequent pathology of experimental arthritis. An immune-complex-mediated experimental arthritis elicited in lining cell depleted joints that had received CL2MDP-liposomes 7 days earlier prevented inflammation as compared to controls.

Quantification of mRNA levels in joint capsule and articular cartilage of the murine knee joint by RT-PCR: Kinetics of stromelysin and IL-1 mRNA levels during arthritis

We developed a method to isolate well defined joint specimens from different compartments of normal and arthritic murine knee joints in which mRNA levels of stromelysin and IL-1 were semiquantified using RT-PCR. Joint capsule specimens were isolated on medial and lateral sides of the patella with a biopsy punch. Cartilage layers were isolated from patellae after a mild decalcification with EDTA. EDTA treatment had no effect on the amount and efficiency of amplification of mRNA when tested on isolated chondrocytes. After induction of experimental arthritis, stromelysin mRNA was elevated approximately 50 times in both joint capsule and cartilage. IL-1 was elevated 100 times in joint capsule but only 10 times in cartilage. Kinetic analysis of mRNA levels in cartilage during arthritis showed a prolonged elevation of stromelysin mRNA compared to IL-1. The variation in mRNA levels between joints of individual mice proved to be low, showing that sampling of the specimens and subsequent RT-PCR can be performed reliably. The current method offers a valuable approach to study gene expression in knee joints during murine experimental arthritis.

THE ROLE OF MACROPHAGES IN CHRONIC ARTHRITIS

Abstract Rheumatoid arthritis is characterized by a mononuclear infiltrate in the synovial tissue of the affected joints, considerable thickening of the synovial lining layer and concomitant destruction of cartilage and bone. Macrophages probably play a central role and the contribution of the synovial lining macrophages is addressed in studies in experimental murine arthritis models. Emphasis is given to the involvement in arthritis expression and cartilage destruction. The role of TNF-α and IL-1, and the modulatory cytokines IL-4/ IL-10 is briefly discussed.

Toll-like receptor 4 induced FcγR expression potentiates early onset of joint inflammation and cartilage destruction during immune complex arthritis: Toll-like receptor 4 largely regulates FcγR expression by interleukin 10

Objective: To study the role of Toll-like receptor (TLR)2 and 4 in the onset of joint inflammation and cartilage destruction during immune complex-mediated arthritis (ICA), and its relationship with FcγR expression. Materials and methods: ICA was induced in knee joints of TLR2−/− and TLR4−/− mice and their wild-type controls. Joint inflammation and cartilage destruction were measured in the knee joint using histology. mRNA levels were determined in synovial specimens and macrophages using quantitative polymerase chain reaction and cytokine protein levels in synovial washouts using Bioplex. Results: Joint inflammation and cartilage destruction were not different in arthritic TLR2−/− and wild-type mice. By contrast, at day 1 after ICA induction, joint swelling and proteoglycan depletion in knee joints of TLR4−/− mice were considerably lower (inflammation 68–79% and proteoglycan depletion 27–76%) when compared with wild-type controls. Cytokine production at this time point was markedly reduced in TLR4−/− mice (interleukin (IL)1, IL6, macrophage inflammatory chemokine (MIP)-1α and keratinocyte-derived chemokine 49%, 72%, 68% and 84%, respectively). In arthritic synovia of TLR4−/− mice, and also after injection of the antigen poly-l-lysine (PLL) lysozyme alone, mRNA levels of FcγR, and the FcγR regulating cytokine IL10 were considerably lower. Stimulation of peritoneal macrophages with PLL lysozyme up regulated mRNA levels of FcγR and IL10, whereas neutralisation by anti-IL10 antibodies largely blocked FcγR up regulation. At day 4, joint inflammation and cartilage destruction were comparable in TLR4−/− mice and wild-type controls. Conclusion: TLR4 regulates early onset of joint inflammation and cartilage destruction during ICA arthritis by up regulation of FcγR expression and enhanced cytokine production. TLR4-mediated up regulation of FcγR is largely mediated by IL10.