Karin Polzer

Dickkopf-1 is a master regulator of joint remodeling

Degenerative and inflammatory joint diseases lead to a destruction of the joint architecture. Whereas degenerative osteoarthritis results in the formation of new bone, rheumatoid arthritis leads to bone resorption. The molecular basis of these different patterns of joint disease is unknown. By inhibiting Dickkopf-1 (DKK-1), a regulatory molecule of the Wnt pathway, we were able to reverse the bone-destructive pattern of a mouse model of rheumatoid arthritis to the bone-forming pattern of osteoarthritis. In this way, no overall bone erosion resulted, although bony nodules, so-called osteophytes, did form. We identified tumor necrosis factor-α (TNF) as a key inducer of DKK-1 in the mouse inflammatory arthritis model and in human rheumatoid arthritis. These results suggest that the Wnt pathway is a key regulator of joint remodeling.

CTLA-4 directly inhibits osteoclast formation

CTLA-4 is a regulator of co-stimulation and inhibits the activation of T cells through interfering with the interaction of CD80/86 on antigen-presenting cells with CD28 on T cells. CTLA-4 binds to the surface of antigen-presenting cells, such as dendritic cells and monocytes through CD80/86. Monocytes can differentiate in osteoclasts, the primary bone resorbing cells. Herein, we investigated whether the binding of CTLA-4 affects the differentiation of monocytes into osteoclasts in vitro and vivo. We show that CTLA-4 dose-dependently inhibits RANKL- as well as tumour necrosis factor (TNF)-mediated osteoclastogenesis in vitro without the presence of T cells. Furthermore, CTLA-4 was effective in inhibiting TNF-induced osteoclast formation in a non-T cell dependent TNF-induced model of arthritis as well as the formation of inflammatory bone erosion in vivo. These data suggest that CTLA-4 is an anti-osteoclastogenic molecule that directly binds osteoclast precursor cells and inhibits their differentiation. These findings are an attractive explanation for the anti-erosive effect of abatacept, a CTLA-4 immunoglobulin fusion protein used for the treatment of rheumatoid arthritis.

Tumour necrosis factor blockade increases lymphangiogenesis in murine and human arthritic joints

OBJECTIVE To investigate the presence and regulation of lymphatic vessels in inflamed joints of mice with experimental arthritis as well as patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA). METHODS Lymphatic vessels and blood vessels were assessed in synovial tissue of human tumour necrosis factor transgenic (TNFtg) mice and synovial biopsies from patients with RA and SpA by immunohistochemistry for podoplanin and CD31, respectively. Assessments were performed before and after TNF blockade in all biopsies. RESULTS Lymphatic vessels were abundantly present in the synovial tissue of hTNFtg mice as well as patients with RA and SpA. The number of lymphatic vessels was positively related to the severity of synovial inflammation. Treatment with infliximab led to an increase in the formation of lymphatic vessels in murine and human inflammatory tissue. CONCLUSIONS This study shows that TNF blockade promotes the proliferation of lymphatic vessels in the inflamed synovium of RA and SpA. This finding leads to the assumption that promotion of lymphangiogenesis may play an important part in efflux of cells and fluid out of the inflamed tissue.

Anti-inflammatory and cartilage-protecting effects of an intra-articularly injected anti-TNFα single-chain Fv antibody (ESBA105) designed for local therapeutic use

Objectives: (1) To show that a single-chain Fv antibody (scFv) against tumour necrosis factor α (TNFα) (ESBA105) has efficacy comparable to a full length anti-TNFα IgG (infliximab); (2) to evaluate whether ESBA105 has all the properties required for the local treatment of arthritis; and (3) to investigate its discriminative tissue penetration properties. Methods: In vivo efficacy was measured in arthritis of the knee joint induced by the intra-articular injection of recombinant human TNFα (rhTNFα) in Lewis rats. Cartilage penetration of scFv (ESBA105) and full length IgG (infliximab) were studied in bovine cartilage specimens ex vivo. Tissue penetration, biodistribution and pharmacokinetics of ESBA105 were followed and compared after intra-articular and intravenous administration. Results: In cell culture, ESBA105 showed similar TNFα inhibitory potency to infliximab. In vivo, ESBA105 inhibited rhTNFα-induced synovial inflammation in rats with efficacy again comparable to infliximab. An 11-fold molar excess of ESBA105 over rhTNFα resulted in 90% inhibition of knee joint swelling, inflammatory infiltrates and proteoglycan loss from cartilage. In ex vivo studies of bovine cartilage, ESBA105 penetrated well into the cartilage whereas infliximab remained on the surface. In vivo, rapid penetration into the synovial tissue, cartilage and surrounding tissues was observed following intra-articular injection of [125I]-ESBA105 into the knee joint of rabbits. Conclusions: ESBA105 potently inhibits inflammation and prevents cartilage damage triggered by TNFα. In contrast to a full length IgG, ESBA105 also penetrates into cartilage and can be expected to reverse the TNFα-induced catabolic state of articular cartilage in arthritides.

Antiinflammatory effects of tumor necrosis factor on hematopoietic cells in a murine model of erosive arthritis

OBJECTIVE To investigate the mechanisms leading to the influx of inflammatory hematopoietic cells into the synovial membrane and the role of tumor necrosis factor receptor I (TNFRI) and TNFRII in this process in an animal model of rheumatoid arthritis (RA). METHODS We performed bone marrow transplantations in human TNF-transgenic mice using hematopoietic cells from wild-type, TNFRI(-/-), TNFRII(-/-), and TNFRI/II(-/-) mice as donors and assessed the severity of arthritis histologically. Generation of osteoclasts from the different genotypes was analyzed in vitro and in vivo. Apoptosis was analyzed using annexin V staining as well as TUNEL assays. RESULTS Despite lacking responsiveness to TNF in their hematopoietic compartment, mice not only developed full-blown erosive arthritis but even showed increased joint destruction when compared with mice with a TNF-responsive hematopoietic compartment. We demonstrated different roles of the 2 different TNFRs in the regulation of these processes. The absence of TNFRI on hematopoietic cells did not affect joint inflammation but markedly attenuated erosive bone destruction via reduced synovial accumulation of osteoclast precursors. In contrast, the absence of TNFRII on hematopoietic cells increased joint inflammation as well as erosive bone destruction via the regulation of osteoclast precursor apoptosis. CONCLUSION Our findings indicate that selective blockade of TNFRI, leaving the antiinflammatory effects of TNFRII unaltered instead of unselectively blocking TNF, might be advantageous in patients with RA.

Anti-inflammatory and cartilage-protecting effects of an intra-articularly injected anti-TNF-α scFv (ESBA105) designed for local therapeutic use

Objectives: (1) To show that a single-chain Fv antibody (scFv) against tumour necrosis factor α (TNFα) (ESBA105) has efficacy comparable to a full length anti-TNFα IgG (infliximab); (2) to evaluate whether ESBA105 has all the properties required for the local treatment of arthritis; and (3) to investigate its discriminative tissue penetration properties. Methods: In vivo efficacy was measured in arthritis of the knee joint induced by the intra-articular injection of recombinant human TNFα (rhTNFα) in Lewis rats. Cartilage penetration of scFv (ESBA105) and full length IgG (infliximab) were studied in bovine cartilage specimens ex vivo. Tissue penetration, biodistribution and pharmacokinetics of ESBA105 were followed and compared after intra-articular and intravenous administration. Results: In cell culture, ESBA105 showed similar TNFα inhibitory potency to infliximab. In vivo, ESBA105 inhibited rhTNFα-induced synovial inflammation in rats with efficacy again comparable to infliximab. An 11-fold molar excess of ESBA105 over rhTNFα resulted in 90% inhibition of knee joint swelling, inflammatory infiltrates and proteoglycan loss from cartilage. In ex vivo studies of bovine cartilage, ESBA105 penetrated well into the cartilage whereas infliximab remained on the surface. In vivo, rapid penetration into the synovial tissue, cartilage and surrounding tissues was observed following intra-articular injection of [125I]-ESBA105 into the knee joint of rabbits. Conclusions: ESBA105 potently inhibits inflammation and prevents cartilage damage triggered by TNFα. In contrast to a full length IgG, ESBA105 also penetrates into cartilage and can be expected to reverse the TNFα-induced catabolic state of articular cartilage in arthritides.

The lonely death: Chondrocyte apoptosis in TNF-induced arthritis

Inflammatory joint disease typically provokes progressive cartilage damage. The proliferative synovial inflammatory tissue directly invades the cartilage and induces the expression and activation of degrading enzymes such as matrix metalloproteases (MMPs) and aggrecanases. However, also chondrocyte apoptosis has been observed in cartilage samples of inflamed joints. It remains unclear whether this is a secondary phenomenon due to cartilage damage or a primary event initiated by the synovial inflammation. To determine the presence or absence of chondrocyte death in experimental arthritis, we longitudinally assessed proteoglycan depletion and chondrocyte apoptosis in paw sections from human tumor necrosis factor transgenic (hTNFtg) mice and wild-type littermates. Whereas, wild-type mice showed no signs of cartilage damage, hTNFtg mice exhibited progressive proteoglycan loss starting at clinical onset of arthritis. However, we already found the first apoptotic chondrocytes well before cartilage matrix breakdown occurred indicating that chondrocyte death can be induced before matrix resorption. Chondrocyte death could constantly be observed until late stages of arthritis causing a continuous increase in the number of empty cartilage lacunae. As apoptotic cells in cartilage cannot be cleared by phagocytes due to their spatial isolation in the avascular lacunae of cartilage, having no contact to professional or amateur phagocytes. The dying cells are compelled to undergo a “lonely death” inevitable ending up in secondary necrosis giving rise to perpetuation of a pro-inflammatory cascade. These data indicate that chondrocyte death may play a primary role in inflammatory arthritis fuelling cartilage inflammation and damage due to secondary necrosis.

B-cell infiltrates induce endosteal bone formation in inflammatory arthritis.

The objective of this study was to investigate the function of inflammatory bone marrow infiltrates found in vicinity to joints affected by inflammatory arthritis. These bone marrow infiltrates are rich in B cells and emerge at the interphase between bone marrow and synovial inflammatory tissue, where cortical bone has been broken. We deleted an essential molecule of B‐cell development, Brutońans tyrosine kinase (Btk), in arthritic TNF‐transgenic mice and studied its effect on bone marrow inflammation. Although antigen responses, immunoglobulin levels, and autoantibody production were diminished in Btk−/−hTNFtg mice, synovial inflammation developed normally. However, bone marrow infiltrates were significantly diminished in Btk−/−hTNFtg mice, which lead to impaired bone formation at endosteal sites underneath bone erosions and an increased invasion of synovial inflammatory cells into the bone marrow. Expression of bone morphogenic protein‐7 was dramatically decreased in Btk−/−hTNFtg mice. These results do not only indicate that bone formation at endosteal regions next to bone marrow infiltrates is driven by B cells but also show that bone marrow aggregates in the vicinity of inflamed joint appear as an attempt to counter the invasion of inflammatory tissue into the bone marrow.

Pathogenesis of Churg–Strauss syndrome: Recent insights

Churg–Strauss syndrome (CSS) is a rare systemic necrotizing vasculitis associated with granuloma formation and severe blood and tissue eosinophilia. CSS occurs almost exclusively in patients with asthma. Its pathogenesis remains largely unknown, as triggering factors for CSS development have not been identified so far. AAb, such as anti-neutrophil cytoplasmic autoantibodies, are found in less than half of patients and possibly constitute a subtype of CSS with different clinical behaviour. On a cellular level, CSS is characterized by a strong Th2-type immune response. Th2-associated cytokines such as IL-4, IL-13 and IL-5 may precipitate the severe eosinophilia in CSS, while migration of Eos to inflammatory sites is possibly mediated by eotaxin-3. This review summarizes recent advances in the knowledge on epidemiology, clinical features, and pathogenesis of CSS.

Selective p38MAPK isoform expression and activation in antineutrophil cytoplasmatic antibody-associated crescentic glomerulonephritis: role of p38MAPKα

Objective: Crescentic glomerulonephritis (crGN) is a frequent and life-threatening manifestation of antineutrophil cytoplasmatic antibody-associated vasculitis. Up-regulation of proinflammatory cytokines contributes to renal damage by activation of p38 mitogen-activated protein kinases (MAPKs). However, it is unclear which of the four p38MAPK isoforms are expressed, activated and hence of major importance in crGN. Methods: Kidney biopsies of patients with antineutrophil cytoplasmatic antibody-positive crGN and control samples were investigated for the expression and phosphorylation of p38MAPK isoforms and downstream target kinase MAPKAP2 by immunohistochemistry. Expression and functional activation of p38MAPK isoforms by TNF was also assessed in a human podocyte cell line by reverse transcription–polymerase chain reaction, immunoblotting and kinase array. Results: Strong expression of p38MAPKα, β and γ isoforms was found in glomerular podocytes and crescents. Infiltrating leucocytes showed predominant p38MAPKα expression. Activation of p38MAPK and its downstream mediator MAPKAP2 was found in crGN confined to glomerular podocytes, crescents and inflammatory infiltrates. Interestingly, corticosteroid treatment before kidney biopsy diminished p38MAPK activation in crGN. Activated p38MAPK co-localised with α, β and γ isoforms in podocytes and crescents, while leucocytes showed mainly p38MAPKα activation. In a human podocyte cell line mRNA and protein of all four p38MAPK isoforms was expressed but only p38MAPKα was activated upon challenge with TNF. Conclusions: This study shows selective p38MAPK isoform expression and activation in crGN. Podocytes and podocyte-induce crescent formation is the main source of p38MAPK activation in crGN. TNF is a potent and selective activator of the α-isoform in podocytes, which therefore appears as a main contributor to proinflammatory signalling in the glomerulum of crGN.