Soluble recombinant neutral endopeptidase (CD10) as a potential antiinflammatory agent.

Several endogenous peptides, including bradykinin and substance P, have potent inflammatory effects in the joint. Levels of these peptides are regulated by plasma and cell-associated peptide degrading enzymes. One of these peptidases, neutral endopeptidase-24.11 (NEP-24.11), is expressed constitutively and in high density on human synovial cells and is presumed to play a critical role in local regulation of peptide levels in the joint. We examined the role of endogenous NEP-24.11 in regulating bradykinin-mediated effects in an articular model, and investigated the ability of soluble, recombinant human NEP-24.11 to augment the effects of the endogenous enzyme. Our studies demonstrate that endogenous synovial NEP-24.11 does not significantly modulate inflammatory peptide effects on cells when competing with colocalizing peptide receptors expressed in high density. Administration of excess, soluble recombinant NEP-24.11 can overcome this problem, however. Furthermore, the activity of the recombinant enzyme was not compromised in the presence of oxidants or inflammatory joint fluids. Recombinant NEP-24.11 holds promise as a novel therapeutic strategy for the treatment of inflammatory arthritis.

Mechanisms of prostanoid synthesis in human synovial cells: cytokine-peptide synergism.

Bradykinin (BK)2 and interleukin-1 (IL-1) interact synergistically to stimulate prostaglandin synthesis in human synovial fibroblast-like cells. The effect of BK is rapid and correlates with its capacity to elevate cytosolic levels of calcium ([Ca2+]i), while IL-1's effect is slow and s dependent upon de novo protein synthesis. The mechanism of this synergistic interaction was investigated. In the basal state, high levels of arachidonic acid (AA) were spontaneously released from synovial cells but near absent levels of cyclooxygenase activity prevented metabolism of AA to prostanoid. BK was a potent stimulus for elevating AA, but not prostaglandins, above basal levels. IL-1, in contrast, increased prostaglandins but not AA, above basal levels. IL-1 treatment was not associated with a loss or redistribution of AA among phospholipid classes. These results are consistent with high basal phospholipase activity in synovial cells and demonstrate the ability of BK, presumably via its ability to raise [Ca2+]i, to further elevate this activity(ies). Metabolism of AA to prostanoid is minimal in resting and BK-stimulated synovial cells, however, without the concomitant induction of cyclooxygenase activity by IL-1. These studies clarify the different, but synergistic, mechanisms of action of a peptide and cytokine in stimulating prostanoid synthesis in synovial cells. In addition, these data extend the results of previous investigations in demonstrating that basal phospholipase activity provides sufficient AA substrate for IL-1 induced prostanoid synthesis without invoking the concomitant induction of phospholipase activity by IL-1.

Type VI collagen-specific messenger RNA is expressed constitutively by cultured human synovial fibroblasts and is suppressed by interleukin-1.

OBJECTIVE: Type VI collagen is a prominent constituent of the synovial extracellular matrix. The cellular source of this matrix protein and the identity of local factor sin synovium that may regulate its expression have not been delineated, however. We examined the capacity of human fibroblast-like synovial cells to synthesize type VI collagen as well as the effect of interleukin-1 (IL-1) on this expression. METHODS: RNA was extracted from cultured human synovial cells derived from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Northern blots were analyzed using sequence-specific probes, and steady-state messenger RNA (mRNA) levels of the 3 alpha (VI) procollagen chains were measured. The effect of IL-1 treatment on these levels was determined. RESULTS: Abundant expression of 3 characteristic mRNA transcripts, corresponding to the alpha 1 (4.2-kb), alpha 2 (3.5-kb), and alpha 3 (8.5-kb) chains of type VI procollagen, was observed in untreated cells derived from RA and OA patients. IL-1 treatment consistently suppressed steady-state mRNA levels for all 3 alpha (VI) procollagen chains in a time- and dose-dependent manner. Tumor necrosis factor alpha induced a response similar to that of IL-1, while IL-2 was ineffective in this regard. Indomethacin partially restored alpha (VI) mRNA expression in IL-1--treated cells. CONCLUSION: These studies provide novel data demonstrating abundant steady-state levels of mRNA transcripts coding for all 3 type VI procollagen polypeptides in human synovial fibroblast-like cells, as well as coordinated down-regulation of these transcripts by IL-1. Local production of IL-1 may thus constitute an important means in vivo of regulating the production of type VI collagen.

Characterization of kinin receptors on human synovial cells and upregulation of receptor number by interleukin-1.

Bradykinin has been implicated in the pathogenesis of inflammatory arthritis by virtue of its potent proinflammatory properties. We have previously shown bradykinin to be a potent stimulus for the release of prostanoids from interleukin-1 (IL-1)-treated, but not untreated, human synovial cells. We hypothesize that one mechanism by which IL-1 induces responsiveness to bradykinin is by upregulation of number or affinity of kinin receptors on human synovial cells. We performed [3H]bradykinin binding studies in intact human synovial tissue and in cultured human synovial cells. Specific, saturable [3H]bradykinin binding sites in intact synovia were identified by autoradiographic localization and were present in much higher density in rheumatoid, than in osteoarthritis, synovia. In untreated human synovial cells in culture, a single (B2) class of kinin binding sites with a Kd of 2.3 nM and Bmax of 58 +/- 9 fmol/10(6) cells was demonstrated. In matched experiments, IL-1 treatment enhanced specific [3H]bradykinin binding 1.5- to 2.0-fold above that observed in untreated cells. This enhancement was attributable to an increase in Bmax (53 +/- 4 vs. 105 +/- 24 fmol/10(6) cells in untreated and IL-1-treated cells, respectively), rather than an alteration in Kd (1.7 and 1.4 nM, respectively). The potencies of a series of kinin analogs and antagonists and unrelated peptides in displacing [3H]bradykinin from IL-1-treated cells correlated well with their abilities to induce prostanoid release. These studies provide novel information regarding the nature of kinin receptors in intact human synovia and in cultured human synovial cells, their regulation by IL-1 and their role in IL-1-treated cells in kinin-mediated prostaglandin E2 production.