Synovial interleukin-1 receptor antagonist and interleukin-1 balance in rheumatoid arthritis.

OBJECTIVE: To quantify interleukin-1 receptor antagonist (IL-1ra) and IL-1 production and gene expression by rheumatoid arthritis (RA) synovial tissue (ST) cells. METHODS: IL-1 alpha, IL-1 beta, and IL-1ra protein levels were measured by enzyme-linked immunosorbent assay in fresh and cultured ST cells, purified synovial macrophages, and fibroblast-like synoviocytes (FLS). The relative expression of the secreted form of IL-1ra (sIL-1ra) and the alternatively spliced intracellular form (icIL-1ra) was determined by reverse transcription polymerase chain reaction (RT-PCR) techniques. RESULTS: IL-1 alpha, IL-1 beta, and IL-1ra were present in fresh and cultured ST cell samples of synovium from RA and osteoarthritis patients. IL-1ra:IL-1 ratios ranged from 1.2 to 3.6, which is below the 10-100-fold excess of IL-1ra needed to inhibit IL-1 bioactivity. Isolated CD14+ synovial macrophages secreted IL-1ra, but the amount was much less than that of alveolar or in vitro-derived macrophages. Cultured FLS contained intracellular IL-1ra but secreted little IL-1ra into the culture supernatants. RT-PCR showed that icIL-1ra mRNA was more abundant than sIL-1ra mRNA in FLS and unfractionated ST cells. CONCLUSION: IL-1ra production by RA ST cells is deficient relative to total production of IL-1.

Mechanisms of methotrexate action in rheumatoid arthritis. Selective decrease in synovial collagenase gene expression.

OBJECTIVE: To measure the effect of methotrexate (MTX) treatment in rheumatoid arthritis (RA) on the expression of synovial collagenase, stromelysin, and tissue inhibitor of metalloproteinase 1 (TIMP-1) gene expression in a prospective study. METHODS: Serial percutaneous synovial biopsies (pretreatment and after 3-4 months) were performed on the knees of 8 patients (7 with RA, 1 with seronegative arthritis) who were beginning oral MTX therapy. Synovial gene expression was determined by quantitative in situ hybridization using computer-assisted image analysis. RESULTS: After therapy, patients had decreased joint counts, morning stiffness, and erythrocyte sedimentation rates. Synovial inflammation in the biopsy tissues was slightly decreased after therapy. In situ hybridization on pretreatment and posttreatment frozen sections was performed to quantify synovial messenger RNA (mRNA) levels. Collagenase gene expression significantly decreased after MTX therapy (P = 0.006) even though cell density in the region was unchanged. TIMP-1 and stromelysin mRNA levels were not changed by MTX therapy. To study the mechanism of MTX action in vitro, MTX-treated and control fibroblast-like synoviocytes were stimulated with interleukin-1 beta (IL-1 beta). MTX did not alter collagenase or TIMP-1 mRNA levels after IL-1 exposure. CONCLUSION: MTX therapy decreases collagenase gene expression but not TIMP-1 or stromelysin gene expression in the synovium. This action is probably an indirect effect due to an alteration in the synovial cytokine milieu, rather than a direct effect on gene expression.

Eosinophils express interleukin 5 and granulocyte macrophage-colony-stimulating factor mRNA at sites of allergic inflammation in asthmatics.

IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF) are important regulators of eosinophil survival, proliferation, and effector function. To determine whether IL-5 and/or GM-CSF are generated by eosinophils at sites of allergic inflammation, we have used in situ hybridization with 35S-labeled RNA probes to study the expression of IL-5 and GM-CSF mRNA in bronchoalveolar lavage (BAL) eosinophils derived from asthmatics (n = 5) before and after endobronchial allergen challenge. Endobronchial allergen challenge induced a significant airway eosinophilia (pre-allergen challenge 0.6 +/- 0.5% eosinophilia vs post-allergen challenge 48.2 +/- 25.6% eosinophilia). Post-allergen challenge eosinophils expressed IL-5 and GM-CSF mRNA, but did not express IL-1 beta or IL-2 mRNA. To determine whether the IL-5 mRNA-positive cells coexpressed GM-CSF mRNA, double mRNA labeling experiments with a digoxigenin-11-UTP nonradioactive labeled IL-5 RNA probe and a GM-CSF 35S-labeled RNA probe were performed. These studies demonstrated that individual eosinophils expressed one of four cytokine mRNA profiles (IL-5+, GM-CSF+, 34 +/- 13%; IL-5+, GM-CSF-, 34 +/- 5%; IL-5-, GM-CSF+, 11 +/- 9%; IL-5-, GM-CSF-, 21 +/- 25%). The expression of IL-5 and GM-CSF by eosinophils at sites of allergic inflammation in asthmatics may provide an important autocrine pathway, maintaining the viability and effector function of the recruited eosinophils.

IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium.

IL-1 can participate in the perpetuation of arthritis through direct stimulation of synoviocytes and augmentation of matrix degradation. Hence, local production of the IL-1R antagonist protein (IRAP) might be an important negative feedback signal that regulates synovitis. We assessed synovial IRAP production in synovia from 30 individuals, by using a specific mAb and the immunoperoxidase staining method. IRAP was detected in 11 of 12 rheumatoid arthritis (RA) synovial tissues (ST) and was located primarily in the sublining, particularly in perivascular regions enriched for macrophages. Some staining was observed in the intimal lining of the synovium, although this was significantly less than in the sublining (p less than 0.05). Nine of 12 osteoarthritis (OA) tissues were positive for IRAP. In contrast to RA, the staining was observed primarily in the synovial lining in OA, with only minimal sublining IRAP being detected. Synovia from four patients without arthritis were negative (three autopsy specimens and one post-traumatic sample). Of the other two patients with miscellaneous diagnoses, one sample was negative (tenosynovitis) and one was positive (seronegative inflammatory arthritis) (sublining). Studies of serial sections and double-immunostaining experiments indicated that macrophages are the major cells containing immunoreactive IRAP. IRAP gene expression in vivo was determined by performing in situ hybridization on ST from 17 arthritis patients. RNA sense IRAP probes did not hybridize to any tissues. Anti-sense IRAP probes bound to two of nine RA tissues, two of six OA tissues, one of one seronegative inflammatory arthropathy tissue, and none of one flexor tenosynovitis tissue. As with immunoreactive protein, IRAP mRNA was primarily localized to cells in the synovial lining in OA but was more prominent in perivascular lymphoid aggregates in RA and seronegative inflammatory arthropathy. Northern blot analysis was performed on RNA isolated from nine ST. The appropriately sized IRAP band was identified in six of nine samples (five of six RA and one of three OA). Supernatants from cultured RA and OA ST cells contained immunoreactive and biologically active IRAP. Hence, IRAP gene expression and protein production occur in RA and OA synovium, albeit in different distributions.

Stromelysin and tissue inhibitor of metalloproteinases gene expression in rheumatoid arthritis synovium.

In situ hybridization was used to localize stromelysin mRNA in rheumatoid arthritis synovial tissue. Stromelysin antisense probes hybridized primarily to the intimal lining layer in frozen tissue sections, with little or no sublining signal. The expression of stromelysin correlated with cellularity as determined by hybridization with an actin probe. Double-label experiments were performed to detect tissue inhibitor of metalloproteinases (TIMP) and stromelysin mRNA simultaneously in synovial tissue. Coexpression of both mRNA species was identified in a subpopulation of intimal lining cells. In some highly inflammatory tissues, virtually all of the lining cells hybridized to both probes. However, in other tissues, expression of the two genes was discordant, with large numbers of TIMPpositive/stromelysin(negative) cells. Similar results were observed with late-passage cultured synoviocytes. Unstimulated cells did not express the stromelysin gene, whereas TIMP was constitutively produced. Addition of interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) to cultures induced the former but had little effect on the latter. Double-label experiments clearly showed discordant expression in individual cells. Stromelysin and TIMP genes likely have distinct transcriptional controls that provide precise control over the local environment and matrix turnover.

Gene expression (collagenase, tissue inhibitor of metalloproteinases, complement, and HLA-DR) in rheumatoid arthritis and osteoarthritis synovium. Quantitative analysis and effect of intraarticular corticosteroids.

In situ hybridization was used to localize and quantify gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue. Collagenase, tissue inhibitor of metalloproteinases (TIMP), HLA-DR, and complement (C2 and C3) gene expression was studied in synovial tissue from 23 patients with RA, OA, or other inflammatory arthropathies. Gene expression was highly compartmentalized: Collagenase, TIMP, and C2 messenger RNA (mRNA) were localized primarily to the synovial lining layer; HLA-DR mRNA was prominent in the lining and in some sublining lymphoid aggregates; the C3 probe hybridized only to sublining lymphoid aggregates. Relative mRNA levels were quantified using computer-assisted image analysis. There was significantly more collagenase, C2, C3, and HLA-DR mRNA in RA compared with OA patients. However, TIMP mRNA levels were similar in RA and OA. Expression of collagenase, TIMP, C2, C3, and HLA-DR genes correlated with the degree of synovial inflammation. The effect of intraarticular corticosteroid injection on synovial tissue gene expression was studied using serial percutaneous synovial biopsy samples from the knees of 3 RA patients. Joints were biopsied, injected with triamcinolone, and rebiopsied 1-2 weeks later. Histologic inflammation scores were lower in posttreatment synovia. Collagenase and TIMP mRNA, although abundant in presteroid samples, were nearly undetectable in post-steroid tissues. HLA-DR mRNA levels also were significantly decreased. C2 and C3 hybridization significantly decreased in 2 of 3 patients and 1 of 3 patients, respectively. Hence, clinical response to intraarticular steroid therapy was accompanied by histologic improvement and decreased expression of genes that play a role in articular destruction.(ABSTRACT TRUNCATED AT 250 WORDS)