C/EBP beta in rheumatoid arthritis: correlation with inflammation, not disease specificity.

Rheumatoid arthritis synovial tissue was examined and compared with osteoarthritis tissue for the presence of the nuclear transcription factor C/EBP beta (NF-IL-6). The region (lining or sublining), cell type, and subcellular distribution (cytoplasmic or nuclear) of the expression of C/EBP beta was characterized. Rheumatoid arthritis synovial fluid and blood and normal peripheral blood were also examined. C/EBP beta was detected in the synovial lining and in sublining cells of synovial tissue from patients with both rheumatoid and osteoarthritis. A significant (P < 0.001 and < 0.05, respectively) increase in the percentage of cells with nuclear staining was seen in the lining layer, compared to cells in the sublining region, in rheumatoid and osteoarthritis. In both diseases a strong correlation (r = 0.79, P < 0.001) was observed between the percentage of cells in the synovial lining that were positive for nuclear C/EBP beta and lining cell depth. Two-color immunohistochemistry demonstrated that both macrophages and fibroblast-like synoviocytes were positive for nuclear C/EBP beta. The presence of C/EBP beta was confirmed by immunohistochemistry and Western blot analysis with isolated synovial fibroblasts. Nuclear C/EBP beta was also detected in rheumatoid synovial fluid monocytes/macrophages, but not in lymphocytes or neutrophils. Western blot analysis confirmed the presence of C/EBP beta in these cells. The intensity of C/EBP beta staining was greater (P < 0.001) in synovial fluid monocytes than in those from normal or rheumatoid peripheral blood. In conclusion, the enhanced nuclear staining for C/EBP beta in the synovial lining, compared to the sublining, suggesting activation in the lining, and the positive correlation of lining layer depth with the percentage of cells in the lining positive for nuclear C/EBP beta, suggest a potential role for C/EBP beta in chronic inflammation. The regulation of the production or activity of C/EBP beta, to inhibit inflammatory mediator expression by synovial macrophages and fibroblasts, offers a novel approach to therapeutic intervention.

Tumor necrosis factor alpha gene regulation: enhancement of C/EBPbeta-induced activation by c-Jun.

Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enriched nuclear transcription factor C/EBPbeta played an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPbeta and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPbeta, expression of c-Jun by itself had relatively little effect on TNF alpha promoter activity. However, the combination of C/EBPbeta and c-Jun was synergistic, resulting in greater than 130-fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNF alpha promoter. To determine if C/EBPbeta and c-Jun might cooperate to regulate the cellular TNF alpha gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPbeta and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperated with endogenous C/EBPbeta in the activation of the -120 TNF alpha promoter-reporter. DNA binding assays using oligonucleotides derived from the TNF alpha promoter suggested that C/EBPbeta and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPbeta and that this interaction contributes to the expression of the cellular TNF alpha gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNF alpha gene.

Recombinant human interleukin-1 receptor type I in the treatment of patients with active rheumatoid arthritis.

OBJECTIVE: To determine the safety and efficacy of recombinant soluble human interleukin-1 receptor type I (rHuIL-1RI) administered subcutaneously in patients with active rheumatoid arthritis (RA). METHODS: Twenty-three patients with active RA (>5 swollen joints) were enrolled into a randomized, double-blind, 2-center study. Patients received subcutaneous doses of rHuIL-1RI or placebo for 28 consecutive days. Patients were treated with 125, 250, 500, or 1,000 micrograms/m2/day of rHuIL-1RI. Physical examinations and laboratory assessments were performed at baseline (day 1), and 8, 15, 22, 29, 43, and 57 days after the start of the study. Analysis of peripheral blood by flow cytometry was performed on days 1 and 29 to determine the effects of rHuIL-1RI on the distribution and phenotypic characteristics of circulating inflammatory cells. RESULTS: Four of 8 patients who received rHuIL-1RI at 1,000 micrograms/m2/day demonstrated improvement in at least 1 of 8 individual measures of disease activity; however, only 1 of these 4 patients experienced clinically relevant improvement as defined by predetermined criteria. None of the patients treated with smaller doses of rHuIL-1RI, and none of the placebo-treated control patients, experienced any improvement as defined by the predetermined criteria. Monocyte cell surface IL-1alpha was significantly reduced following treatment with rHuIL-1RI at each dosage. Administration of rHuIL-1RI was stopped prematurely because of dose-limiting rashes in 2 patients treated with 1,000 micrograms/m2/day. No other adverse events prevented completion of the study. CONCLUSION: Only 1 patient, who was treated with the highest concentration of rHuIL-1RI employed (1,000 micrograms/m2/day), demonstrated clinically relevant improvement in this phase I study on this small group of patients with active RA. Dose-limiting toxicity was also observed in 2 patients treated with this highest concentration of rHuIL-1RI. Treatment with rHuIL-1RI did result in a reduction of monocyte cell surface IL-1alpha, which indicates that the dosages of rHuIL-1RI employed were functional.

Autocrine regulation of collagenase gene expression by TNF-alpha in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) has been shown to induce the production of interstitial collagenase by fibroblasts and chondrocytes. We investigated the role of TNF-alpha in collagenase gene expression by U937 monocyte/macrophage cells. Transcription of the TNF-alpha gene was observed after 0.5 h of phorbol myristate acetate (PMA) stimulation. Collagenase mRNA expression was not observed until 5-7 h of activation with PMA. TNF-alpha was detected in the culture supernatants 2-3 h before transcription of the collagenase gene. Neutralization of TNF-alpha protein with anti-TNF-alpha antibodies significantly reduced collagenase mRNA expression. Protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibition essentially abolished both PMA-induced TNF-alpha protein secretion and collagenase mRNA expression. Collagenase gene expression induced by exogenous TNF-alpha in U937 cells stimulated with a suboptimal concentration of PMA was suppressed by PTK, but not PKC, inhibition. The pyrrolidine derivative of dithiocarbamate, a potent inhibitor of nuclear factor-kappa B (NF-kappa B) activation, resulted in a marked reduction in collagenase gene transcription, however, no reduction of TNF-alpha secretion was noted. Anti-TNF-alpha antibodies inhibited PMA-induced NF-kappa B activation. These observations demonstrate an important role for TNF-alpha in the autocrine regulation of collagenase gene expression by U937 cells. Additionally, TNF-alpha-induced PTK and NF-kappa B activation were important in collagenase gene expression in this cell line.

Expression of functional B7 and CTLA4 on rheumatoid synovial T cells.

To assess the role of B7, CTLA4, and CD28 in the pathogenesis of chronic synovitis we analyzed the expression and function of these cell surface molecules in patients with rheumatoid arthritis, osteoarthritis, and psoriatic arthritis, and in normal controls. Immunoperoxidase staining of rheumatoid synovial membranes showed reactivity of 30% of T cells with anti-B7 mAb, in contrast to osteoarthritic and normal synovial membranes, in which no such staining was seen. In addition, rheumatoid synovial fluid T cells were positive by flow cytometric analysis for B7 (mean 20%, range 0 to 96%), as measured by staining with anti-B7 mAb or the CTLA4 Ig fusion protein, whereas no B7 expression was detected on peripheral blood T cells (mean 1%). To analyze the functional importance of B7 expressed on synovial fluid T cells, we used these cells as stimulator cells in primary allogeneic MLC. Purified synovial fluid T cells were far stronger stimulator cells compared with paired peripheral blood T cells and resulted in a fivefold greater increase in allogeneic T cell proliferation. Furthermore, the proliferation induced by purified synovial T cells was markedly inhibited by addition of the CTLA4 Ig fusion protein (77%). Moreover, anti-B7 mAb (37%), anti-CTLA4 mAb (33%), and Fab fragments of anti-CD28 mAb (52%) partially inhibited the primary MLC. The expression of functional B7, together with the increased expression of MHC class II molecules, indicates that synovial T cells may serve as functional APCs and may be capable of autocrine stimulation via the CD28 activation pathway.

Soluble intercellular adhesion molecule-1 in arthritis.

The objective of this study is to determine whether soluble intercellular adhesion molecule-1 (sICAM-1) is found in rheumatoid arthritis (RA) and other inflammatory disorders and to identify which cells are responsible for sICAM-1 production. Synovial fluid, blood and cells isolated from RA synovial fluids, and synovial tissues from 59 patients were studied. In addition, normal peripheral blood was obtained. sICAM-1 was assayed by an enzyme-linked immunoabsorbent assay. Synovial fluids from patients with RA and other inflammatory arthritides had significantly higher sICAM-1 levels than did osteoarthritis (OA) synovial fluids. Synovial fluid sICAM-1 levels were significantly positively correlated with synovial fluid leukocyte counts. RA synovial tissue fibroblasts released low levels of sICAM-1. Neutrophils (PMNs) isolated from synovial fluids of RA patients spontaneously released sICAM-1. However, mononuclear cells isolated from RA synovial fluid produced the largest quantities of sICAM-1. Phytohemagglutinin but not lipopolysaccharide enhanced mononuclear sICAM-1 release. sICAM-1 was increased in synovial fluids from RA compared to OA. This sICAM-1 may be important in modulating the trafficking of inflammatory leukocytes into diseased RA synovial tissue and fluid.

Ultrastructural characteristics of the spleen in hairy cell leukemia.

Eighteen spleens derived from patients with hairy cell leukemia (HCL) were analyzed by correlative scanning and transmission electron microscopy. In 15 of the cases, the white pulp areas were markedly decreased or absent when compared to normal spleens, although few hairy cells were observed within this region. In only one case did the white pulp appear normal. In all HCL cases, hairy cells were observed within normal, dilated, and abnormal sinuses. The abnormal sinuses contained hairy cells of typical morphology attached to other hairy cells, to endothelial lining, and to erythrocytes. The degree of sinus filling by hairy cells varied from loosely- to tightly-packed. Endothelial cells exhibiting degenerative changes, such as swelling with smooth surfaces and dilated intercellular spaces, were frequently seen. These results indicate that in addition to the previously described overcrowding of the spleen by hairy cells, the splenic tissue itself is considerably altered and sometimes severely damaged in patients with HCL.

Activation of synovial fluid T lymphocytes by 60-kd heat-shock proteins in patients with inflammatory synovitis.

OBJECTIVE: Synovial fluid lymphocytes from patients with rheumatoid arthritis and with other forms of inflammatory synovitis demonstrate enhanced proliferative responses to Mycobacterium tuberculosis antigens, in particular, the 65-kd heat-shock protein. There is a high degree of homology between the human and the mycobacterial 60-kd family of heat-shock proteins. These studies were performed to determine if the enhanced response to the mycobacterial 65-kd heat-shock protein was due to cross-reactivity of an immune response generated against the human homolog. METHODS: These studies were performed by in vitro culture of isolated synovial fluid mononuclear cells with crude and purified antigens. RESULTS: The synovial fluid lymphocytes of a majority of patients with rheumatoid arthritis recognized the mycobacterial 65-kd heat-shock protein, as evidenced by T cell proliferation. In contrast, only 18% of all samples tested responded to a highly purified recombinant human 60-kd heat-shock protein. With only one exception, proliferative responses to the mycobacterial antigen were stronger than those to the human homolog. The proliferative responses generated against mycobacterial 65-kd heat-shock proteins from different sources were highly correlated. CONCLUSION: The findings suggest that the enhanced proliferative response to the mycobacterial 65-kd heat-shock protein noted in most patients with rheumatoid arthritis and other forms of inflammatory synovitis is not due to cross-reactivity of an immune response directed against the human heat-shock protein.

HDS: Establishment of a New Cell Line from a Hairy Cell Leukemia Patient with Resistance to Alpha-Interferon Therapy.

A new cell line, designated "HDS", was established in a suspension culture derived from the peripheral blood of a patient with hairy cell leukemia (HCL) who developed clinical resistance to alpha-interferon (aIFN) therapy. The patient exhibited a clinical picture characteristic of HCL, including splenomegaly, cytopenias, and tartrate-resistant acid phosphatase (TRAP)-positive "hairy" cells in blood and marrow. Chromosomal studies revealed that the cultured cells possess the chromosomal abnormality +12. Cytochemical and immunologic studies show the HDS cell line had the phenotype of a B-lymphocyte. HDS cells expressed the HLA-DR and CD19 surface antigens, but were negative for early B (CD10) and T (CD2, CD3) cell markers. The cells are also negative for other T-cell, granulocytic and monocytic markers and for typical HCL markers such as CD11c and CD22. However, the expression of these antigens was induced by in-vitro treatment of the cells with the differentiation-inducing agent tetradecanoyl phorbol acetate (TPA). Ultrastructural analyses of the cultured cells revealed a display of surface microvilli mixed with ruffles in a classical hairy cell pattern. It is therefore highly likely that the HDS cells represent HCL cells in an atypical stage of differentiation.