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Diphenylamine-based nonsteroidal antiinflammatory drugs (NSAIDs) are able to cause in vitro the shedding of L-selectin. The aim of this work was to determine the physio-logic relevance of L-selectin shedding in the antiinflammatory effect exerted by NSAIDs in vivo. Chemical compounds structurally related to NSAIDs - including diphenyl-amine, N-phenylanthranilic acid (N-Ph), diphenylacetic acid - as well as the traditional NSAID indomethacin were studied using the zymosan air-pouch mouse model. Animals intramuscularly pretreated with indomethacin or N-Ph, but not with diphenyl-amine or diphenylacetic acid, showed a significant dose-dependent reduction in the number of neutrophils compared with untreated animals (N-Ph, IC50 = 6.7 mg/kg). Except for indomethacin, none of these compounds caused any significant reduction in cyclooxygenase-1 activity in vivo. In flow chamber experiments, N-Ph reduced the capability of human neutrophils to pass across the endothelial barrier by interfering with leukocyte rolling step on HUVEC. N-Ph, but not diphenylacetic acid, induced activation-independent L-selectin shedding in mouse neutrophils. Interestingly, N-Ph exerted an antiinflammatory effect similar to that of the anti-L-selectin blocking antibody Mel-14, although no additive action was observed when both compounds were combined. These data suggest that the L-selectin shedding induced by NSAIDs may be involved in the antiinflammatory action exerted by these compounds in clinical settings.

Objective To explore the potential involvement of the chemokine system in synoviocyte-mediated tissue destruction in rheumatoid arthritis (RA), we studied the expression profile of chemokine receptors and their function in the migration, proliferation, and matrix metalloproteinase (MMP) production of cultured fibroblast-like synoviocytes (FLS) from RA patients. Methods The presence of CC and CXC chemokine receptors on cultured FLS was studied at the messenger RNA (mRNA) level by reverse transcriptase–polymerase chain reaction and at the cell surface expression level by flow cytometry. Variations in cytosolic calcium influx induced by chemokine stimulation were assessed by flow cytometry on Fura Red–preloaded FLS. Two-compartment transwell chambers were used for FLS chemotaxis assays. Cell growth was measured by a fluorescence-based proliferation assay. Gelatinase and collagenase activities were determined by a fibril degradation assay and zymography. Results FLS constitutively expressed the receptors CCR2, CCR5, CXCR3, and CXCR4, both at the cell surface and mRNA levels, but failed to express CCR3 and CCR6. Significant intracytosolic calcium influx was observed on FLS challenged with monocyte chemotactic protein 1 (MCP-1), stromal cell–derived factor 1α (SDF-1α), and interferon-inducible protein 10 (IP-10). Stimulation with MCP-1, SDF-1α, IP-10, and monokine induced by interferon-γ enhanced the migration and proliferation of FLS. These chemokines, in addition to RANTES, increased in a dose- and time-dependent manner the gelatinase and collagenase activities in cell-free supernatants of cultured FLS. Interestingly, the chemokine-mediated up-regulation of MMP activities was significantly abrogated by the presence of anti–interleukin-1β, but not anti–tumor necrosis factor α, blocking antibodies. Conclusion These data suggest that through modulation of the migration, proliferation, and MMP production by FLS, the chemokine system may play a more direct role in the destructive phase of RA than is currently suspected, and thus emphasize the relevance of chemokines and their receptors as potential therapeutic targets in this disease.