Ian K. Campbell

ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro.

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more ‘aggrecanases’ from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.

The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement.

A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.

The colony‐stimulating factors and collagen‐induced arthritis: exacerbation of disease by M‐CSF and G‐CSF and requirement for endogenous M‐CSF

There is increasing evidence that the colony‐stimulating factors (CSFs) may play a part in chronic inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). We examined the involvement of macrophage CSF (M‐CSF or CSF‐1) and granulocyte CSF (G‐CSF) in collagen‐induced arthritis (CIA), a murine model of RA. Daily injections of M‐CSF or G‐CSF, 20–24 days postprimary immunization with type II collagen, exacerbated disease symptoms in suboptimally immunized DBA/1 mice. Support for the involvement of endogenous M‐CSF in CIA was obtained by studies in which neutralizing monoclonal antibody reduced the severity of established CIA and also by studies showing the resistance of M‐CSF‐deficient op/op mice to CIA induction. These studies show that M‐CSF and G‐CSF can be proinflammatory in CIA and provide evidence that macrophage‐ and granulocyte‐lineage cells can exacerbate CIA. Our results also show that M‐CSF‐dependent cells are essential for CIA development, suggesting M‐CSF may be a suitable target for therapeutic intervention in RA.

Collagen‐induced arthritis in C57BL/6 (H‐2b) mice: new insights into an important disease model of rheumatoid arthritis

Collagen‐induced arthritis (CIA) is a widely used model of rheumatoid arthritis (RA) and has been important for understanding autoimmunity. CIA is purportedly restricted to mice bearing the MHC class II H‐2q or H‐2r haplotypes. In this study, we re‐examined established concepts regarding susceptibility to CIA. We found mice derived from the C57BL/6 (B6) (H‐2b) background can develop CIA with high incidence (60–70%), and sustained severity by using an immunization procedure modified for optimum response in DBA/1 (D1) (H‐2q) mice. Clinically and histologically the B6 disease resembles that of D1 mice and is dependent on immunization with type II collagen, as well as on B and CD4+ T cells. In contrast, 129/Sv mice, which share H‐2b, are resistant to CIA. We conclude that susceptibility to CIA may reflect immunization conditions and/or important contributions from non‐MHC genes, revealed by different immunization protocols. A practical outcome is that CIA can be directly applied to gene knockout mice generated from B6 embryonic stem cells without need for backcross onto the D1 background. This model may lead to improved understanding of autoimmunity in CIA and RA and may provide a platform for analysis of the contribution of non‐MHC genes to CIA.

Distinct roles for the NF-κB1 (p50) and c-Rel transcription factors in inflammatory arthritis

Rheumatoid arthritis (RA) is a complex disease, with contributions from systemic autoimmunity and local inflammation. Persistent synovial joint inflammation and invasive synovial pannus tissue lead to joint destruction. RA is characterized by the production of inflammatory mediators, many of which are regulated by the Rel/NF-kappaB transcription factors. Although an attractive target for therapeutic intervention in inflammatory diseases, Rel/NF-kappaB is involved in normal physiology, thus global inhibition could be harmful. An alternate approach is to identify and target the Rel/NF-kappaB subunits critical for components of disease. To assess this, mice with null mutations in c-rel or nfkb1 were used to examine directly the roles of c-Rel and p50 in models of acute and chronic inflammatory arthritis. We found c-Rel-deficient mice were resistant to collagen-induced arthritis but had a normal response in an acute, destructive arthritis model (methylated BSA/IL-1 induced arthritis) suggesting c-Rel is required for systemic but not local joint disease. In contrast, p50-deficient mice were refractory to induction of both the chronic and acute arthritis models, showing this subunit is essential for local joint inflammation and destruction. Our data suggest Rel/NF-kappaB subunits play distinct roles in the pathogenesis of inflammatory arthritis and may provide a rationale for more specific therapeutic blockade of Rel/NF-kappaB in RA.

Severe inflammatory arthritis and lymphadenopathy in the absence of TNF.

It has been postulated that TNF has a pivotal role in a cytokine cascade that results in joint inflammation and destruction in rheumatoid arthritis (RA). To evaluate this, we examined the response of TNF-deficient (Tnf(-/-)) mice in two models of RA. Collagen-induced arthritis (CIA) was induced by injection of chick type II collagen (CII) in CFA. Tnf(-/-) mice had some reduction in the clinical parameters of CIA and, on histology, significantly more normal joints. However, severe disease was evident in 54% of arthritic Tnf(-/-) joints. Tnf(-/-) mice had impaired Ig class switching, but preserved T cell proliferative responses to CII and enhanced IFN-gamma production. Interestingly, CII-immunized Tnf(-/-) mice developed lymphadenopathy and splenomegaly associated with increased memory CD4(+) T cells and activated lymph node B cells. Acute inflammatory arthritis was also reduced in Tnf(-/-) mice, although again some mice exhibited severe disease. We conclude that TNF is important but not essential for inflammatory arthritis; in each model, severe arthritis could proceed even in the complete absence of TNF. These results call into doubt the concept that TNF is obligatory for chronic autoimmune and acute inflammatory arthritis and provide a rationale for further studies into TNF-independent cytokine pathways in arthritis.

Granulocyte-macrophage colony stimulating factor exacerbates collagen induced arthritis in mice.

OBJECTIVE To examine the effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on disease progression in the collagen induced arthritis (CIA) model in mice. METHODS DBA/1 mice were primed for a suboptimal CIA response by intradermal injection of chick type II collagen without a secondary immunisation. Three weeks after immunisation the mice were given four to five consecutive daily intraperitoneal injections of recombinant murine GM-CSF (15 μg; 5 × 105 U), or vehicle, and arthritis development was monitored by clinical scoring of paws and calliper measurements of footpad swelling. At approximately six to eight weeks after immunisation mice were killed, their limbs removed and processed for histological analyses of joint pathology. RESULTS Control animals receiving a single immunisation with collagen exhibited a varied CIA response both in terms of incidence and severity. Mice treated with GM-CSF at 20 to 25 days after immunisation with collagen had a consistently greater incidence and more rapid onset of disease than the vehicle treated control mice, based on clinical assessment. GM-CSF treated mice showed higher average clinical scores and greater paw swelling than controls. Histological analyses of joints reflected the clinical scores with GM-CSF treated mice displaying more pronounced pathology (synovitis, pannus formation, cartilage and bone damage) than control mice. CONCLUSION GM-CSF is a potent accelerator of the pathological events leading to chronic inflammatory polyarthritis in murine CIA supporting the notion that GM-CSF may play a part in inflammatory polyarthritis, such as rheumatoid arthritis.

A key role for G-CSF–induced neutrophil production and trafficking during inflammatory arthritis

We have previously shown that G-CSF-deficient (G-CSF(-/-)) mice are markedly protected from collagen-induced arthritis (CIA), which is the major murine model of rheumatoid arthritis, and now investigate the mechanisms by which G-CSF can promote inflammatory disease. Serum G-CSF levels were significantly elevated during CIA. Reciprocal bone marrow chimeras using G-CSF(-/-), G-CSFR(-/-), and wild-type (WT) mice identified nonhematopoietic cells as the major producers of G-CSF and hematopoietic cells as the major responders to G-CSF during CIA. Protection against CIA was associated with relative neutropenia. Depletion of neutrophils or blockade of the neutrophil adhesion molecule, Mac-1, dramatically attenuated the progression of established CIA in WT mice. Intravital microscopy of the microcirculation showed that both local and systemic administration of G-CSF significantly increased leukocyte trafficking into tissues in vivo. G-CSF-induced trafficking was Mac-1 dependent, and G-CSF up-regulated CD11b expression on neutrophils. Multiphoton microscopy of synovial vessels in the knee joint during CIA revealed significantly fewer adherent Gr-1(+) neutrophils in G-CSF(-/-) mice compared with WT mice. These data confirm a central proinflammatory role for G-CSF in the pathogenesis of inflammatory arthritis, which may be due to the promotion of neutrophil trafficking into inflamed joints, in addition to G-CSF-induced neutrophil production.

G-CSF and GM-CSF as therapeutic targets in rheumatoid arthritis

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are well-recognized regulators of hematopoiesis and have an established role as growth factors in clinical practice. G-CSF and GM-CSF regulate myeloid cell production, differentiation and activation, and might also be important for driving inflammatory responses. Inappropriate engagement of this pathway could be a critical amplification mechanism when maladaptive immune responses predispose to autoimmunity and sterile tissue inflammation. We postulate that antagonism of G-CSF or GM-CSF could represent a novel therapeutic approach for a variety of autoimmune-mediated inflammatory diseases, including rheumatoid arthritis.

Exacerbation of acute inflammatory arthritis by the colony‐stimulating factors CSF‐1 and granulocyte macrophage (GM)‐CSF: evidence of macrophage infiltration and local proliferation

CSF‐1 and GM‐CSF have been implicated in the pathogenesis of rheumatoid arthritis. We report the effects of CSF‐1 and GM‐CSF in the development of an acute methylated bovine serum albumin (mBSA)‐induced murine arthritis model. Examination of histopathological features revealed that the systemic administration of CSF‐1 or GM‐CSF following mBSA administration into the knee resulted in the exacerbation of arthritis. This included synovial hyperplasia and joint inflammation, most evident at 7 and 14 days post‐mBSA administration, and the appearance of erosive pannus tissue. The exacerbation by CSF‐1 and GM‐CSF was not sustained but declined in incidence and severity by 21 days post‐mBSA administration, similar to the effects of IL‐1β in this model, reported here and previously. Macrophages expressing Mac‐2 and F4/80 were a prominent feature of the pathology observed, particularly the infiltration of Mac‐2+ macrophages seen in all mice administered CSF‐1, GM‐CSF or IL‐1β. Present in inflamed knees was a locally dividing population of cells which included Mac‐2+ and F4/80+ macrophages. These studies demonstrate that CSF‐1 and GM‐CSF can exacerbate and prolong the histopathology of acute inflammatory arthritis and lend support to monocytes/macrophages being a driving influence in the pathogenesis of inflammatory arthritis.