Nathan J. Zvaifler

Mesenchymal precursor cells in the blood of normal individuals

Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).

The Immunopathology of Joint Inflammation in Rheumatoid Arthritis

Publisher Summary This chapter focuses on the pathogenesis of rheumatoid arthritis. In an infectious etiology of rheumatoid arthritis, it is pertinent to consider the manner in which a pathogen might gain access to the synovial membrane. The joint seems to be uniquely predisposed to infection with some bacteria. Rheumatoid synovitis is characterized by a constellation of histological changes, which are characteristic but not pathognomonic of this disease. In hypertrophy of the synovial lining surface, the synovium appears edematous and inflamed and protrudes into the joint space as slender villous projections. In hyperplasia and hypertrophy of the synovial lining cells, the lining cells are multilayered, reaching to a depth of six to ten cells, as compared to the normal synovial lining that is only one to three cell layers. Vascular derangement—focal or segmental vascular changes—are a regular feature of rheumatoid synovitis. Cellular infiltrates—the connective tissue stronia of the synovial villus is packed with mononuclear cells—are collected into aggregates or follicles, particularly around small blood vessels, but true germinal centers are rarely seen.

Apoptosis in rheumatoid arthritis synovium.

RA synovial tissue (ST) was studied to determine if and where apoptosis occurs in situ. Genomic DNA was extracted from 5 RA and 1 osteoarthritis ST samples. Agarose gel electrophoresis demonstrated DNA ladders characteristic for apoptosis from each tissue. In situ and labeling (ISEL) was used to identify DNA strand breaks consistent with apoptosis in frozen sections. 12 RA and 4 osteoarthritis ST were studied by ISEL and all were positive, but only 2 of 4 normal tissues were positive. The primary location of apopotic cells was the synovial lining. Some sublining cells were also positive, but lymphoid aggregate staining was conspicuously absent. Immunohistochemistry and ISEL were combined and showed that the lining cells with DNA strand breaks were mainly macrophages, although some fibroblastlike cells were also labeled. Sublining cells with fragmented DNA included macrophages and fibroblasts, but T cells in lymphoid aggregates, which expressed large amounts of bcl-2, were spared. DNA strand breaks in cultured fibroblastlike synoviocytes was assessed using ISEL. Apoptosis could be induced by actinomycin D, anti-fas antibody, IL-1, and TNF-alpha but not by IFN-gamma. Fas expression was also detected on fibroblast-like synoviocytes using flow cytometry. Therefore, DNA strand breaks occur in synovium of patients with arthritis. Cytokines regulate this process, and the cytokine profile in RA (high IL-1/TNF; low IFN-gamma) along with local oxidant injury might favor induction of apoptosis.

Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases

Oxidative stress at sites of chronic inflammation can cause permanent genetic changes. The development of mutations in the p53 tumor suppressor gene and other key regulatory genes could help convert inflammation into chronic disease in rheumatoid arthritis and other inflammatory disorders.

Cytokines in chronic inflammatory arthritis. II. Granulocyte-macrophage colony-stimulating factor in rheumatoid synovial effusions.

A liquid culture technique was used to study 23 synovial fluids (SF) (21 from inflammatory joint diseases and 2 noninflammatory SF) and supernatants of two cultured rheumatoid arthritis (RA) synovial tissues for colony-stimulating factor (CSF). The proliferative responses of human peripheral blood macrophage-depleted non-T cells treated with synovial fluids, supernatants of synovial tissue explants, and recombinant granulocyte-macrophage (rGM)-CSF were compared. Aggregates of cells that formed in long-term cultures (15 d) were similar for each applied agent and consisted of macrophages, eosinophils, and large blasts. Tritiated thymidine incorporation was proportional to the concentration of rGM-CSF and was accompanied by an increase in number and size of cellular aggregates formed in the cultures. CSF activity was observed in inflammatory SF, with tritiated thymidine uptake of 3,501 +/- 1,140 cpm in the presence of RA samples (n = 15) compared to 1,985 +/- 628 for non-RA inflammatory SF (n = 7) (P less than 0.05) and 583 +/- 525 for medium (n = 6) (P less than 0.01). The proliferative response to RA SF was often more apparent when the samples were diluted, because at higher concentrations the RA SF was inhibitory. Two RA SF were fractionated by Sephadex G100 column chromatography; low levels of CSF activity were detected in fractions corresponding to Mr of 70-100 kD, but the major CSF activity was found in the 20-24-kD fractions. A polyclonal rabbit anti-GM-CSF antibody eliminated the stimulating activity from both rGM-CSF and RA SF. Finally, a specific RIA identified significant levels of GM-CSF (40-140 U/ml) in the culture supernatants of 3 additional RA synovial tissues. These data document the local production of GM-CSF in rheumatoid synovitis and are the first description of this cytokine at a site of disease activity.

Cytokines in chronic inflammatory arthritis. V. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes.

The effects of a broad array of cytokines, individually and in combination, were determined on separate functions (proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor [GM-CSF] production) and phenotype (expression of class II MHC antigens) of cultured fibroblast-like RA synoviocytes. The following recombinant cytokines were used: IL-1 beta, IL-2, IL-3, IL-4, IFN-gamma, tumor necrosis factor (TNF)-alpha, GM-CSF, and macrophage colony-stimulating factor (M-CSF). Only IFN-gamma induced HLA-DR (but not HLA-DQ) expression. TNF-alpha inhibited IFN-gamma-mediated HLA-DR expression (46.7 +/- 4.1% inhibition) and HLA-DR mRNA accumulation. This inhibitory effect was also observed in osteoarthritis synoviocytes. Only TNF-alpha and IL-1 increased synoviocyte proliferation (stimulation index 3.60 +/- 1.03 and 2.31 +/- 0.46, respectively). IFN-gamma (but none of the other cytokines) inhibited TNF-alpha-induced proliferation (70 +/- 14% inhibition) without affecting the activity of IL-1. Only IL-1 beta and TNF-alpha induced collagenase production (from less than 0.10 U/ml to 1.10 +/- 0.15 and 0.72 +/- 0.24, respectively). IFN-gamma decreased TNF-alpha-mediated collagenase production (69 +/- 19% inhibition) and GM-CSF production but had no effect on the action of IL-1. These data demonstrate mutual antagonism between IFN-gamma and TNF-alpha on fibroblast-like synoviocytes and suggest a novel homeostatic control mechanism that might be defective in RA where very little IFN-gamma is produced.

Fibroblast-like synoviocytes of mesenchymal origin express functional B cell-activating factor of the TNF family in response to proinflammatory cytokines.

Immunohistochemical analysis revealed that the intimal lining cells of synovial tissue of inflamed joints of patients with rheumatoid arthritis differed from that of normal joints or of diseased joints in osteoarthritis in that they stained with mAb specific for the B cell-activating factor of the TNF family (BAFF; also called BLyS). We generated fibroblast-like synoviocytes (FLS) cell lines that were bereft of myelomonocytic cells to examine whether mesenchymal-derived FLS could express this critical B cell survival factor. We found that FLS expressed low amounts of BAFF mRNA relative to that of myelomonocytic cells. However, when various cytokines/factors were added to such FLS cell lines, we found that IFN-γ or TNF-α were unique in that they could induce significant increases in BAFF mRNA and protein. Even minute amounts of IFN-γ primed FLS for TNF-α, allowing the latter to stimulate significantly higher levels of BAFF mRNA and protein than could TNF-α alone. Consistent with this, B cells cocultured with IFN-γ and/or TNF-α-treated FLS had a significantly greater viability than B cells cocultured with nontreated FLS. The enhanced protection of B cells afforded by IFN-γ/TNF-α-treated FLS was inhibited by the addition of BAFF-R:Fc fusion protein. We conclude that the proinflammatory cytokines IFN-γ and TNF-α can induce mesenchymal-derived FLS to express functional BAFF in vitro. The induced expression of BAFF on FLS by proinflammatory cytokines may enhance the capacity of such cells to protect B cells from apoptosis in inflammatory microenvironments in vivo.

Regional analysis of p53 mutations in rheumatoid arthritis synovium.

The p53 tumor suppressor protein plays a central role in cell cycle regulation, DNA repair, and apoptosis. Recent studies indicate that DNA damage and somatic mutations in the p53 gene can occur because of genotoxic stress in many tissues, including the skin, colon, and synovium. Although somatic mutations in the p53 gene have been demonstrated in rheumatoid arthritis (RA) synovial tissue and synoviocytes, no information is available on the location or extent of p53 mutations. Using microdissected RA synovial tissue sections, we observed abundant p53 transition mutations, which are characteristic DNA damage caused by oxidative stress. p53 mutations, as well as p53 mRNA expression, were located mainly in the synovial intimal lining rather than the sublining (P < 0.01). Clusters of p53 mutant subclones were observed in some microdissected regions, suggesting oligoclonal expansion. Because IL-6 gene expression is regulated by wild-type p53, IL-6 mRNA expression in microdissected tissues was quantified by using real-time PCR. The regions with high rates of p53 mutations contained significantly greater amounts of IL-6 mRNA compared with the low mutation samples (P < 0.02). The microdissection findings suggest that p53 mutations are induced in RA synovial tissues by inflammatory oxidative stress. This process, as in sun-exposed skin and inflamed colonic epithelium, provides some of the mutant clones with a selective growth advantage. A relatively low percentage of cells containing p53 mutations can potentially affect neighboring cells and enhance inflammation through the elaboration of proinflammatory cytokines.

Complement-derived leukotactic factors in inflammatory synovial fluids of humans

A large per cent of rheumatoid synovial fluids contain chemotactic activity for rabbit granulocytes (neutrophilic). The chemotactic activity is, in large part, related to the fifth (C5) and sixth (C6) components of human complement; a combination of physical-chemical techniques indicates the activity to be attributable to C567 and C5a, a cleavage product of C5. Many rheumatoid synovial fluids contain a C5-cleaving enzyme which, on the basis of substrate specificity and susceptibility to inhibitors, is very similar to an enzyme extractable from lysosomal granules of human and rabbit granulocytes. Inflammatory nonrheumatoid synovial fluids contain chemotactic activity that is related to cleavage products (C3a) of the third component of human complement (C3). Also found in these fluids is a C3-cleaving enzyme capable of producing C3a. Of the other synovial fluids examined, lupus fluids were remarkable by their total lack of chemotactic activity. These findings record for the first time the presence of complement-derived chemotactic factors in pathological human fluids.

Regulation of Joint Destruction and Inflammation by p53 in Collagen-Induced Arthritis

The role of the tumor suppressor p53 as a key regulator of inflammation was examined in murine collagen-induced arthritis (CIA), a model of rheumatoid arthritis. Wild-type DBA/1 mice develop progressive arthritis in this model, in which p53 expression and apoptosis are evident in the synovial cells. In contrast, the joints of p53(-/-) DBA/1 animals with CIA showed increased severity of arthritis using clinical and histological scoring methods with almost no apoptosis. Consistent with this, collagenase-3 expression and cytokine production (interleukin-1 and interleukin-6) in the joints of p53(-/-) mice with CIA were significantly greater than in wild-type mice. Anti-collagen antibody titers, however, were not different. Therefore, p53 expression occurs during inflammation and acts to suppress local inflammatory responses. Because mutations in p53 have been described in the synovial membrane of rheumatoid arthritis patients, the loss of p53 function in synoviocytes or other cells in the joint because of dominant-negative mutations might contribute to invasion and destruction of the joint in this disease.