Coordinate expression of activating Fcγ receptors I and III and inhibiting Fcγ 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 Fcγ receptor types I and III (FcγRI and FcγRIII, respectively) and the inhibiting Fcγ receptor II (FcγRII) 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 FcγRI‐, FcγRIII‐, and FcγRII‐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 FcγRIII‐deficient mice and enhanced in mice lacking FcγRII. Mild cartilage destruction reflected by loss of PGs was consistent with the degree of inflammation. Mice lacking FcγRIII showed almost no PG depletion, whereas in FcγRII^−/− 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 FcγRIII^−/− and FcγRI^−/− mice, directing the two different critical steps of cellular influx and subsequent activation. These aspects were enhanced in FcγRII^−/− mice. In FcγRI^−/− and FcγRIII^−/− mice, VDIPEN expression was 90–99% lower, whereas in FcγRII^−/− mice, VDIPEN expression was increased 4‐fold. Chondrocyte death was reduced in FcγRIII^−/− mice (68% lower) and enhanced in FcγRII^−/− mice (6–12‐fold higher). Progression of arthritis and erosion of the cartilage surface were markedly elevated in FcγRII^−/− arthritic joints.

Conclusion

During ICA, FcγRIII is the dominant activating receptor mediating joint inflammation, whereas both FcγRI and FcγRIII are involved in cartilage destruction. FcγRII inhibits both joint inflammation and severe cartilage destruction during ICA.

     

Joint inflammation and chondrocyte death become independent of Fcγ receptor type III by local overexpression of interferon‐γ 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 Fcγ receptor type III (FcγRIII). Local adenoviral overexpression of interferon‐γ (IFNγ) in the knee joint prior to onset of IC‐mediated arthritis aggravated severe cartilage destruction. In FcγRI^−/− mice, however, chondrocyte death was not enhanced by IFNγ, whereas matrix metalloproteinase (MMP)–mediated aggrecan breakdown was markedly elevated, suggesting a role for the activating FcγRIII in the latter process. We undertook this study to determine the role of FcγRIII in joint inflammation and severe cartilage destruction in IFNγ‐stimulated IC‐mediated arthritis, using FcγRIII^−/− mice.

Methods

FcγRIII^−/− and wild‐type (WT) mice were injected in the knee joint with recombinant adenovirus encoding murine IFNγ (AdIFNγ) 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 FcγR expression levels were determined in synovial washouts and synovium, respectively.

Results

Injection of AdIFNγ in naive knee joints markedly increased levels of messenger RNA for FcγRI, FcγRII, and FcγRIII. Upon IFNγ overexpression prior to induction of IC‐mediated arthritis, joint inflammation was similar in FcγRIII^−/− 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 1α. Furthermore, IFNγ induced 2‐fold and 3‐fold increases in chondrocyte death in WT controls and FcγRIII^−/− mice, respectively. Notably, VDIPEN expression also remained high in FcγRIII^−/− mice.

Conclusion

IFNγ bypasses the dependence on FcγRIII in the development of IC‐mediated arthritis. Furthermore, both FcγRI and FcγRIII can mediate MMP‐dependent cartilage matrix destruction.

     

Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice

Objective

To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice.

Methods

The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry.

Results

VDIPEN neoepitope was detected mainly in the pericellular matrix of deep‐zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper‐zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice.

Conclusion

MMP‐ and aggrecanase‐generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.

     

Pathogenesis of osteoarthritis‐like changes in the joints of mice deficient in type IX collagen

Objective

To examine the pathogenetic mechanisms of osteoarthritis (OA)–like changes in Col9a1^−/− mice, which are deficient in type IX collagen.

Methods

Knee joints and temporomandibular joints (TMJs) from Col9a1^−/− mice and their wild‐type (Col9a1^+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP‐3) and MMP‐13, degraded type II collagen, and the discoidin domain receptor 2 (DDR‐2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle.

Results

Histologic analysis showed age‐dependent OA‐like changes in the knee and TMJs of Col9a1^−/− mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP‐13 and DDR‐2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1^−/− mice than in their wild‐type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1^−/− mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1^−/− mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites.

Conclusion

Lack of type IX collagen in Col9a1^−/− mice results in age‐dependent OA‐like changes in the knee joints and TMJs.

     

Developmental and osteoarthritic changes in Col6a1‐knockout mice: Biomechanics of type VI collagen in the cartilage pericellular matrix

Objective

Chondrocytes, the sole cell type in articular cartilage, maintain the extracellular matrix (ECM) through a homeostatic balance of anabolic and catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes are encapsulated by a narrow tissue region termed the “pericellular matrix” (PCM), which in normal cartilage is defined by the exclusive presence of type VI collagen. Because the PCM completely surrounds each cell, it has been hypothesized that it serves as a filter or transducer for biochemical and/or biomechanical signals from the cartilage ECM. The present study was undertaken to investigate whether lack of type VI collagen may affect the development and biomechanical function of the PCM and alter the mechanical environment of chondrocytes during joint loading.

Methods

Col6a1^−/− mice, which lack type VI collagen in their organs, were generated for use in these studies. At ages 1, 3, 6, and 11 months, bone mineral density (BMD) was measured, and osteoarthritic (OA) and developmental changes in the femoral head were evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1‐month‐old Col6a1^−/−, Col6a1^+/−, and Col6a1^+/+ mice were assessed using an electromechanical test system, and mechanical properties of the PCM were measured using the micropipette aspiration technique.

Results

In Col6a1^−/− and Col6a1^+/− mice the PCM was structurally intact, but exhibited significantly reduced mechanical properties as compared with wild‐type controls. With age, Col6a1^−/− mice showed accelerated development of OA joint degeneration, as well as other musculoskeletal abnormalities such as delayed secondary ossification and reduced BMD.

Conclusion

These findings suggest that type VI collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or Col6a1^−/−‐derived joint laxity, can lead to progression of OA.

     

Gene deletion of either interleukin‐1β, interleukin‐1β–converting enzyme,inducible nitric oxide synthase,or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy

Objective

To investigate the development of osteoarthritis (OA) after transection of the medial collateral ligament and partial medial meniscectomy in mice in which genes encoding either interleukin‐1β (IL‐1β), IL‐1β–converting enzyme (ICE), stromelysin 1, or inducible nitric oxide synthase (iNOS) were deleted.

Methods

Sectioning of the medial collateral ligament and partial medial meniscectomy were performed on right knee joints of wild‐type and knockout mice. Left joints served as unoperated controls. Serial histologic sections were obtained from throughout the whole joint of both knees 4 days or 1, 2, 3, or 4 weeks after surgery. Sections were graded for OA lesions on a scale of 0–6 and were assessed for breakdown of tibial cartilage matrix proteoglycan (aggrecan) and type II collagen by matrix metalloproteinases (MMPs) and aggrecanases with immunohistochemistry studies using anti‐VDIPEN, anti‐NITEGE, and Col2‐3/4C_short neoepitope antibodies. Proteoglycan depletion was assessed by Alcian blue staining and chondrocyte cell death, with the TUNEL technique.

Results

All knockout mice showed accelerated development of OA lesions in the medial tibial cartilage after surgery, compared with wild‐type mice. ICE‐, iNOS‐, and particularly IL‐1β–knockout mice developed OA lesions in the lateral cartilage of unoperated limbs. Development of focal histopathologic lesions was accompanied by increased levels of MMP‐, aggrecanase‐, and collagenase‐generated cleavage neoepitopes in areas around lesions, while nonlesional areas showed no change in immunostaining. Extensive cell death was also detected by TUNEL staining in focal areas around lesions.

Conclusion

We postulate that deletion of each of these genes, which encode molecules capable of producing degenerative changes in cartilage, leads to changes in the homeostatic controls regulating the balance between anabolism and catabolism, favoring accelerated cartilage degeneration. These observations suggest that these genes may play important regulatory roles in maintaining normal homeostasis in articular cartilage matrix turnover.

     

Autoimmune regulator controls T cell help for pathogenetic autoantibody production in collagen‐induced arthritis

Objective

Autoimmune regulator (Aire) promotes the ectopic expression of tissue‐restricted antigens in medullary thymic epithelial cells (mTECs), leading to negative selection of autoreactive T cells. This study was undertaken to determine whether loss of central tolerance renders Aire‐deficient (Aire^−/−) mice more susceptible to the induction of autoimmune arthritis.

Methods

Medullary TECs were isolated from Aire^−/− and wild‐type C57BL/6 mice for gene expression analysis. Collagen‐induced arthritis (CIA) was elicited by injection of chick type II collagen (CII) in adjuvant. Cellular and humoral immune responses to CII were evaluated. Chimeric mice were created by reconstituting lymphocyte‐deficient mice with either Aire^−/− or wild‐type CD4 T cells and wild‐type B cells.

Results

Wild‐type, but not Aire^−/−, mTECs expressed the CII gene Col2a1. Aire^−/− mice developed more rapid and severe CIA, showing elevated serum anti‐CII IgG levels, with earlier switching to arthritogenic IgG subclasses. No evidence was found of enhanced T cell responsiveness to CII in Aire^−/− mice; however, Aire^−/− CD4 T cells were more efficient at stimulating wild‐type B cells to produce anti‐CII IgG following immunization of chimeric mice with CII.

Conclusion

Our findings indicate that Aire‐dependent expression of CII occurs in mTECs, implying that there is central tolerance to self antigens found in articular cartilage. Reduced central tolerance to CII in Aire^−/− mice manifests as increased CD4 T cell help to B cells for cross‐reactive autoantibody production and enhanced CIA. Aire and central tolerance help prevent cross‐reactive autoimmune responses to CII initiated by environmental stimuli and limit spontaneous autoimmunity.

     

Apolipoprotein E–deficient mice are resistant to the development of collagen‐induced arthritis

Objective

To determine whether elevated serum lipid levels resulting from feeding animals a high‐fat diet can affect the inflammatory process in C57BL/6 (B6) wild‐type (WT) and B6 ApoE^−/− mouse models of collagen‐induced arthritis (CIA).

Methods

Male B6 WT or ApoE^−/− mice were fed either a normal chow diet or a high‐fat diet. CIA was induced in mice at 12 weeks of age using type II chicken collagen, Freund's complete adjuvant, and, on occasion, a lipopolysaccharide boost. Expression levels of autoantibodies and cytokines were measured using enzyme‐linked immunosorbent assay and multiplex assay, respectively.

Results

Whereas B6 WT mice developed severe articular inflammation after collagen immunization, ApoE^−/− mice developed no clinical or histologic evidence of disease regardless of whether they had been fed a high‐fat diet or a normal chow diet. The fact that arthritis was not present in ApoE^−/− mice did not result from inadequate production of serum IgG2a collagen antibodies, since levels observed in ApoE^−/− mice were similar to those observed in arthritic B6 WT control mice. Critically, development of atherosclerosis in ApoE^−/− mice was not affected by the CIA protocol.

Conclusion

Our findings suggest that ApoE^−/− mice are resistant to the development of CIA. Intriguingly, induction of host autoimmunity in the absence of articular inflammation had no effect on atherosclerosis progression, suggesting that articular inflammatory load may be a critical risk factor in vascular pathology.

     

Articular cartilage and biomechanical properties of the long bones in Frzb‐knockout mice

Objective

Ligands and antagonists of the WNT pathway are linked to osteoporosis and osteoarthritis. In particular, polymorphisms in the FRZB gene, a secreted WNT antagonist, have been associated with osteoarthritis. The aim of this study was to examine cartilage and bone in Frzb^−/− mice.

Methods

The Frzb gene in mice was inactivated using a Cre/loxP strategy. Three models of osteoarthritis were used: collagenase, papain, and methylated bovine serum albumin induced. Bone biology was studied using density measurements and microfocal computed tomography. Bone stiffness and mechanical loading–induced bone adaptation were studied by compression of the ulnae.

Results

Targeted deletion of the Frzb gene in mice increased articular cartilage loss during arthritis triggered by instability, enzymatic injury, or inflammation. Cartilage damage in Frzb^−/− mice was associated with increased WNT signaling and matrix metalloproteinase 3 (MMP‐3) expression and activity. Frzb^−/− mice had increased cortical bone thickness and density, resulting in stiffer bones, as demonstrated by stress–strain relationship analyses. Moreover, Frzb^−/− mice had an increased periosteal anabolic response to mechanical loading as compared with wild‐type mice.

Conclusion

The genetic association between osteoarthritis and FRZB polymorphisms is corroborated by increased cartilage proteoglycan loss in 3 different models of arthritis in Frzb^−/− mice. Loss of Frzb may contribute to cartilage damage by increasing the expression and activity of MMPs, in a WNT‐dependent and WNT‐independent manner. FRZB deficiency also resulted in thicker cortical bone, with increased stiffness and higher cortical appositional bone formation after loading. This may contribute to the development of osteoarthritis by producing increased strain on the articular cartilage during normal locomotion but may protect against osteoporotic fractures.

     

Murine tumor necrosis factor α–induced adipose‐related protein (tumor necrosis factor α–induced protein 9) deficiency leads to arthritis via interleukin‐6 overproduction with enhanced NF‐κB,STAT‐3 signaling,and dysregulated apoptosis of macrophages

Objective

To elucidate the role of tumor necrosis factor α–induced adipose‐related protein (TIARP; or tumor necrosis factor α–induced protein 9 [TNFAIP‐9]) in the development and pathogenesis of arthritis.

Methods

We generated TIARP‐deficient (TIARP^−/−) mice and investigated several organs in aged mice. Peritoneal macrophages were collected and cultured with lipopolysaccharide (LPS) and TNFα, and then the production of cytokines and subsequent NF‐κB signal transduction were analyzed. We also examined the susceptibility of young TIARP^−/− mice to collagen‐induced arthritis (CIA). Draining lymph nodes and splenocytes were isolated and cultured, and serum levels of anti–type II collagen (anti‐CII) antibodies, interleukin‐6 (IL‐6), and TNFα on day 60 were measured. We further investigated the effects of anti–IL‐6 receptor monoclonal antibody (mAb) on the development of arthritis in TIARP^−/− mice. IL‐6/STAT‐3 signaling was also analyzed using TIARP^−/− macrophages.

Results

TIARP^−/− mice developed spontaneous enthesitis and synovitis, had high serum levels of IL‐6, had increased CD11b+ cell counts in the spleen, and showed enhanced LPS‐ and TNFα‐induced IL‐6 expression in macrophages. Sustained degradation of IκBα with dysregulated apoptosis was also noted in TIARP^−/− macrophages. CIA was clearly exacerbated in TIARP^−/− mice, accompanied by marked neutrophil and macrophage infiltration in joints. The levels of anti‐CII antibodies in serum were unchanged, whereas autoreactive Th1 cell and Th17 cell responses were higher in TIARP^−/− mice. Treatment with anti–IL‐6 receptor mAb prevented the development of CIA in TIARP^−/− mice, and TIARP^−/− macrophages showed increased IL‐6–induced STAT‐3 phosphorylation.

Conclusion

These findings suggest that TIARP acts as a negative regulator of arthritis by suppressing IL‐6 production, its signaling and TNFα‐induced NF‐κB signaling, resulting in enhanced apoptosis in macrophages.

     

Induction of cartilage damage by overexpression of T cell interleukin‐17A in experimental arthritis in mice deficient in interleukin‐1

Objective

To examine the capacity of T cell interleukin‐17A (IL‐17A; referred to hereinafter as IL‐17) to induce cartilage damage during experimental arthritis in the absence of IL‐1.

Methods

Local IL‐17 gene transfer was performed in the knee joint of IL‐1–deficient mice and wild‐type controls during streptococcal cell wall (SCW)–induced arthritis. Knee joints were isolated at various time points for histologic analysis of cartilage proteoglycan (PG) depletion. Expression of messenger RNA for inducible nitric oxide synthase, matrix metalloproteinases (MMPs) 3, 9, and 13, and ADAMTS‐4 was determined by quantitative polymerase chain reaction analysis. VDIPEN staining was analyzed to study MMP‐mediated cartilage damage. In addition, systemic anti–IL‐1α/β antibody treatment was performed in mice immunized with type II collagen and injected locally with an adenoviral vector expressing IL‐17 or with control adenovirus. Knee joints were isolated and analyzed for cartilage PG depletion, chondrocyte death, and cartilage surface erosion.

Results

During SCW‐induced arthritis, local T cell IL‐17 gene transfer turned this acute, macrophage‐driven joint inflammation into a severe, chronic arthritis accompanied by aggravated cartilage damage. Of high interest, the IL‐1 dependency of cartilage PG depletion was fully abrogated when IL‐17 was locally overexpressed in the joint. Moreover, local IL‐17 gene transfer increased MMP expression without the need for IL‐1, although IL‐1 remained essential for part of the cartilage VDIPEN expression. Furthermore, when IL‐17 was overexpressed in the knee joints of mice with collagen‐induced arthritis, anti–IL‐1 treatment did not reduce the degree of chondrocyte death or cartilage surface erosion.

Conclusion

These data show the capacity of IL‐17 to replace the catabolic function of IL‐1 in cartilage damage during experimental arthritis.

     

Dipeptidyl peptidase I regulates the development of collagen‐induced arthritis

Objective

To examine the role of dipeptidyl peptidase I (DPPI), a widely expressed lysosomal cysteine protease, in the development of collagen‐induced arthritis (CIA) in mice.

Methods

Wild‐type (WT) and DPPI‐deficient (DPPI^−/−) mice backcrossed to DBA/1J mice for 10 generations were immunized with bovine type II collagen (CII), and disease susceptibility and severity were assessed over time. Collagen‐specific B cell and T cell responses and the production of proinflammatory cytokines (tumor necrosis factor α, interleukin‐1, and interleukin‐6) were measured. In addition, adoptive transfer of splenocytes from WT, CII‐sensitized mice was performed to evaluate the specific role of DPPI^−/− T lymphocytes.

Results

The majority of DPPI^−/− mice were resistant to CIA induction, although clinical disease (i.e., evidence of inflammation and bone erosions) did develop in a small number of DPPI^−/− mice. The protection against disease development was not attributable to a defect in the B and T cell response to collagen immunization, because both anticollagen antibody production and T cell proliferation in response to CII were normal. Release of the proinflammatory cytokines was largely unaffected in CII‐stimulated DPPI^−/− splenocytes. In addition, when cells isolated from the joints of DPPI^−/− mice were stimulated in vitro, they had no intrinsic defect in their ability to release inflammatory cytokines. Last, adoptive transfer of splenocytes from WT, CII‐immunized mice into naive WT and DPPI^−/− mice led to development of arthritis in WT mice but not in DPPI^−/− mice.

Conclusion

These results indicate that DPPI regulates a critical step in the development of CIA that is independent of T cell and B cell functions.

     

Attenuation of osteoarthritis progression by reduction of discoidin domain receptor 2 in mice

Objective

To investigate whether the reduction of discoidin domain receptor 2 (DDR‐2), a cell membrane tyrosine kinase receptor for native type II collagen, attenuates the progression of articular cartilage degeneration in mouse models of osteoarthritis (OA).

Methods

Double‐heterozygous (type XI collagen–deficient [Col11a1^+/−] and Ddr2‐deficient [Ddr2^+/−]) mutant mice were generated. Knee joints of Ddr2^+/− mice were subjected to microsurgical destabilization of the medial meniscus. Conditions of the articular cartilage from the knee joints of the double‐heterozygous mutant and surgically treated mice were examined by histology, evaluated using a modified Mankin scoring system, and characterized by immunohistochemistry.

Results

The rate of progressive degeneration in knee joints was dramatically reduced in the double‐heterozygous mutant mice compared with that in the type XI collagen–deficient mice. The progression in the double‐heterozygous mutant mice was delayed by ∼6 months. Following surgical destabilization of the medial meniscus, the progressive degeneration toward OA was dramatically delayed in the Ddr2^+/− mice compared with that in their wild‐type littermates. The articular cartilage damage present in the knee joints of the mice was directly correlated with the expression profiles of DDR‐2 and matrix metalloproteinase 13.

Conclusion

Reduction of DDR‐2 expression attenuates the articular cartilage degeneration of knee joints induced either by type XI collagen deficiency or by surgical destabilization of the medial meniscus.

     

Suppression of autoimmune arthritis in interleukin‐1‐deficient mice in which T cell activation is impaired due to low levels of CD40 ligand and OX40 expression on T cells

Objective

To elucidate the roles of interleukin‐1 (IL‐1) in the development of 2 etiologically different rheumatoid arthritis (RA) models: the type II collagen (CII)–induced arthritis (CIA) model and the human T cell leukemia virus type I transgenic (HTLV‐I Tg) mouse model.

Methods

For the CIA model, DBA/1J‐background IL‐1α^−/−, IL‐1β^−/−, IL‐1α/β^−/−, and wild‐type littermate mice were immunized with CII. For the HTLV‐I Tg model, BALB/c IL‐1β^−/− or IL‐1α/β^−/− mice were crossed with HTLV‐I Tg mice. The effects of IL‐1 deficiency were assessed as follows: Development of arthritis was assessed both macroscopically and microscopically. Serum antibody titer was measured by enzyme‐linked immunosorbent assay. Proliferative response of lymph node cells was assayed by measurement of ³H‐thymidine incorporation. Expression of T cell surface molecule CD40 ligand (CD40L) and OX40 was determined by multicolor flow cytometric analysis.

Results

The development of arthritis was markedly suppressed in IL‐1α/β^−/− mice in both models, although the effect was less prominent in HTLV‐I Tg mice. Deficiency of only IL‐1α or only IL‐1β was also associated with disease suppression. Antibody production after immunization with CII was normal in IL‐1α/β^−/− mice, while autoantibody production was suppressed in IL‐1α/β^−/− HTLV‐I Tg mice. In IL‐1α/β^−/− mice, the T cell proliferative response against CII was greatly reduced in both the CIA and the HTLV‐I Tg models, suggesting inefficiency of T cell activation. Furthermore, expression of CD40L and OX40 on T cells was greatly reduced in IL‐1α/β^−/− mice.

Conclusion

These observations suggest that T cell activation by IL‐1 is important for the development of autoimmunity and arthritis in these mice.

     

Joint damage and inflammation in c‐Jun N‐terminal kinase 2 knockout mice with passive murine collagen‐induced arthritis

Objective

Previous studies have demonstrated that inhibition of c‐Jun N‐terminal kinase (JNK) decreases joint destruction in the rat adjuvant arthritis model. The present study was undertaken to investigate whether selective loss of JNK‐2 function decreases joint destruction in JNK‐2 knockout mice, in order to determine the role of this isoform in inflammatory arthritis.

Methods

Passive collagen‐induced arthritis (CIA) was induced in Jnk2^−/− and wild‐type mice by administering anti–type II collagen antibodies. Arthritis was assessed daily using a semiquantitative clinical scoring system. Fibroblast‐like synoviocytes (FLS) were prepared from Jnk2^−/− and wild‐type mice, and JNK protein expression was determined by Western blot analysis. Matrix metalloproteinase 13 (MMP‐13) expression was determined by Northern blot analysis, and activator protein 1 (AP‐1) binding activity by electromobility shift assay (EMSA).

Results

The JNK protein level in Jnk2^−/− mice with CIA was 22% of that in wild‐type mice with CIA (P < 0.001), and mainly the 46‐kd isoform was expressed in the former group. Surprisingly, clinical arthritis was slightly more severe in the Jnk2^−/− mice. Histologic scores for synovial inflammation were not significantly different. However, Safranin O–stained sections from the Jnk2^−/− mice exhibited significantly less joint damage. Although joint destruction was decreased in Jnk2^−/− mice with CIA, EMSA and Northern blot analysis of total joint extracts revealed similar levels of AP‐1 binding and MMP‐13 expression in Jnk2^−/− and wild‐type mice. The lack of correlation with AP‐1 activity and MMP expression was probably because non‐FLS cells in the joint may express more JNK‐1 than do FLS.

Conclusion

JNK‐2 is a determinant of matrix degradation, but it has little effect on inflammation in arthritis. Complete inhibition of MMP expression and joint destruction will likely require combined JNK‐1 and JNK‐2 inhibition.

     

Increased collagen and aggrecan degradation with age in the joints of Timp3(-/-) mice

OBJECTIVE: To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage. METHODS: Hind joints of Timp3(-/-) and wild-type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (... VDIPEN and ... NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme-linked immunosorbent assay. RESULTS: Articular cartilage from Timp3-knockout animals (ages > or =6 months) showed reduced Safranin O staining and an increase in ...VDIPEN content compared with cartilage from heterozygous and wild-type animals. There was also a slight increase in ... NVTEGE content in articular cartilage and menisci of Timp3(-/-) animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3(-/-) mice compared with wild-type animals. This increase was significant in 15-month-old animals. CONCLUSION: These results indicate that TIMP-3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP-3 may play a pathophysiologic role in the development of this disease.     

Fcγ receptors directly mediate cartilage,but not bone,destruction in murine antigen‐induced arthritis: Uncoupling of cartilage damage from bone erosion and joint inflammation

Objective

To determine the relationship between synovial inflammation and the concomitant occurrence of cartilage and bone erosion during conditions of variable inflammation using various Fcγ receptor knockout (FcγR^−/−) mice.

Methods

Antigen‐induced arthritis (AIA) was introduced in the knee joints of various FcγR^−/− mice and wild‐type controls. Joint inflammation and cartilage and bone destruction levels were determined by histologic analysis. Cathepsin K, RANKL, and osteoprotegerin (OPG) levels were detected by immunolocalization.

Results

In FcγRIIb^−/− mice, which lack the inhibiting Fcγ receptor IIb, levels of joint inflammation and cartilage and bone destruction were significantly higher (infiltrate 93%, exudate 200%, cartilage 100%, bone 156%). AIA in mice lacking activating FcγR types I, III, and IV, but not FcγRIIb (FcR γ‐chain^−/− mice), prevented cartilage destruction completely. In contrast, levels of bone erosion and joint inflammation were comparable with their wild‐type controls. Of great interest, in arthritic mice lacking activating FcγR types I, II, and III, but not IV (FcγRI/II/III^−/− mice), levels of joint inflammation were highly elevated (infiltrate and exudate, 100% and 188%, respectively). Cartilage destruction levels were decreased by 92%, whereas bone erosion was increased by 200%. Cathepsin K, a crucial mediator of osteoclasts, showed a strong correlation with the amount of inflammation but not with the amount of activating FcγR, which was low in osteoclasts. RANKL, but not OPG, levels were higher in the inflammatory cells of arthritic knee joints of FcγRI/II/III^−/− mice versus wild‐type mice.

Conclusion

Activating FcγR are crucial in mediating cartilage destruction independently of joint inflammation. In contrast, FcγR are not directly involved in bone erosion. Indirectly, FcγR drive bone destruction by regulating joint inflammation.