Tumour necrosis factor-α (TNF-α) is implicated in the pathogenesis of many pulmonary and airway diseases. TNF-α stimulation may release interleukin-8 (IL-8) in airways mediated via an increase in intracellular oxidant stress. In the present study, we have assessed leukosequestration and IL-8 release in the airways in response to intratracheal administration of human recombinant TNF-α, and examined the modulatory role of endogenous NO by pretreatment with a NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME). 2. TNF-α (102-104 u) was administered intratracheally in male guinea-pigs which were anaesthetized with urethane and were ventilated artificially. TNF-α induced a time- and dose-related increase in neutrophil numbers and a concomitant increase in human IL-8 equivalent level retrieved from bronchoalveolar lavage (BAL) with the peak effect at 103 u at 6 h of TNF-α injection (late phase). Intratracheal administration of recombinant human (rh)IL-8 (0.025, 0.25, 2.5 ng) producing a similar range of human IL-8 equivalent levels in BAL as measured in our results induced neutrophil recovery in BAL fluid to a similar extent. Administration of anti-IL-8 antibody prevented the late phase of neutrophil recruitment induced by TNF-α or rhIL-8. 3. Pretreatment with L-NAME significantly enhanced the TNF-α (103 u)-induced neutrophil recruitment and human IL-8 equivalents production at 6 h, but not at 1 h of TNF-α administration (early phase). L-Arginine reversed the responses to L-NAME. Pretreatment with 0.2% DMSO (i.v.) significantly inhibited TNF-α-induced neutrophil recruitment and human IL-8 equivalents release both in the early and late phase of the responses. Pretreatment with DMSO also inhibited the enhancement effect of L-NAME on the late phase of TNF-α-induced responses. DMSO failed to modify exogenous rhIL-8-induced neutrophil recruitment. Neither L-NAME nor DMSO alone induced any significant change in neutrophil numbers or human IL-8 equivalent level in BAL fluid. 4. Neutrophil depletion by cyclophosphamide pretreatment failed to modify TNF-α-induced human IL-8 equivalent release. 5. The expression of β2-integrin, CD11b/CD18 on neutrophils was increased only in the late but not early phase of TNF-α stimulation. L-NAME failed to modify these responses. 6. In conclusion, we demonstrated that NO may be an important endogenous inhibitor of TNF-α-induced leukocyte chemotaxis via inhibition of IL-8 production. Thus, the production of NO in airway inflammatory diseases may play a negative feedback role in self-limiting the magnitude of inflammatory responses.
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