Interleukin (IL)-8 is a C-X-C chemokine that potently chemoattracts and activates neutrophils. We determined whether IL-8 could be produced by human airway smooth muscle cells in culture and examined its regulation. TNF-alpha stimulated IL-8 mRNA expression and protein release in a time- and dose-dependent manner, whereas IFN-gamma alone had no effect. Both cytokines together did not induce greater IL-8 release compared to TNF-alpha alone. IL-1beta was more potent in inducing IL-8 release and, together with TNF-alpha, there was a synergistic augmentation of IL-8 release. IL-8 release induced by TNF-alpha and IFN-gamma was partly inhibited by the Th-2-derived cytokines IL-4, IL-10, and IL-13, as well as by dexamethasone. In addition to its contractile responses, airway smooth muscle cells have synthetic and secretory potential with the release of IL-8 and subsequent recruitment and activation of neutrophils in the airways. Release of IL-8 can be modulated by Th-2-derived cytokines and corticosteroids.

The effects of tumor necrosis factor-alpha (TNF-alpha) on interleukin-8 (IL-8) expression and generation were examined in primary cultured human airway epithelial cells (HAEC) and a human lung epithelial cell line (A549). TNF-alpha increased IL-8 mRNA and protein expression in HAEC in a concentration- and time-dependent manner and these effects were inhibited by dexamethasone (1 microM). There was no change in the stability of IL-8 mRNA, and a nuclear run-on assay confirmed that TNF-alpha increased IL-8 gene transcription. TNF-alpha-induced IL-8 mRNA expression showed a biphasic response in HAEC, with an early increase at 2 h followed by a sustained increase from 8 h, which was abolished by the addition of cycloheximide, suggesting that the synthesis of another protein was involved. A549 cells also increased IL-8 secretion and mRNA after incubation of TNF-alpha, with inhibition by dexamethasone. However, A549 cells showed only an early single peak. A549 cells showed a 250-fold increase in the generation of IL-8 immunoreactivity, whereas primary cultured HAEC showed only a threefold increase, suggesting that HAEC and A549 cells may respond to TNF-alpha in different ways. The sustained increase in IL-8 secretion due to an increase in gene transcription in response to TNF-alpha may be an important amplification step in inflammatory diseases of the airways.

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Guinea pig peritoneal eosinophils stimulated by platelet-activating factor (PAF), leukotriene B4 (LTB4), and human recombinant C5a (C5a) undergo a rapid concentration-dependent and partially reversible homotypic aggregation as assessed by changes in light transmission. The phorbol ester phorbol myristate acetate similarly induces a concentration-dependent aggregation, which is, however, slower in onset, takes longer to reach maximal aggregation, and is irreversible. In addition, we confirmed, using light microscopy, that these agonist-induced changes in light transmission do indeed represent true homotypic aggregation. We further characterized the aggregation response and showed that there is homologous but little heterologous desensitization when PAF and LTB4 are used as stimuli. A requirement for both Ca2+ and Mg2+ for full manifestation of agonist-induced aggregation was observed. LTB4- and PAF-induced superoxide anion generation is enhanced by the diacyglycerol kinase inhibitor R59022, whereas aggregation induced by LTB4, but not PAF, is augmented. Lastly, we show that eosinophil aggregation is partially dependent on the adhesion glycoprotein CD18. In summary, therefore, we believe that eosinophil aggregation provides a useful and reliable measure of eosinophil activation.

1 The activation of neutrophils with particulate stimuli such as zymosan induces the generation of the C-X-C chemokine interleukin (IL)-8. There is evidence that neutrophil derived IL-8 plays an important role in human diseases such as the adult respiratory distress syndrome. In the present study, we examined the e ects of cyclic AMP elevating agents on the ability of human neutrophils to generate IL-8 in response to zymosan particles. 2 The PDE4 inhibitor rolipram had limited e ect on zymosan-induced IL-8 generation. In contrast, the PDE4 inhibitors RP 73401 and SB 207499 concentration-dependently suppressed IL-8 generation. The potency of these inhibitors was RP 73401 4 SB 207499 4 rolipram which is correlated with their rank order of potency at inhibiting the catalytic site of puri®ed neutrophil PDE4. Pretreatment of neutrophils with the PDE3 inhibitor ORG 9935 or the PDE5 inhibitor zaprinast had no e ect on IL-8 generation.3 The prostanoids prostaglandin E 1 (PGE 1 ) and PGE 2 inhibited zymosan-induced IL-8 release from neutrophils in a dose-dependent manner, in response to 10 75 M PGE 1 and PGE 2 inhibiting IL-8 generation by 89% and 75%, respectively. Similarly, the b 2 -adrenoceptor agonist salbutamol also inhibited IL-8 generation, but it was less e ective than the prostanoids. 4 Signi®cant synergism between prostanoids or salbutamol and the PDE4 inhibitors to inhibit IL-8 generation was observed. In contrast, there was no signi®cant synergism between PGE 2 and the PDE3 inhibitor ORG 9935 or the PDE5 inhibitor zaprinast. 5 In order to evaluate the potential role of protein kinase A in mediating the inhibitory e ects of cyclic AMP-elevating agents, we used the protein kinase A inhibitors, H 89 and KT 5720. Pretreatment of neutrophils with these drugs completely reversed the inhibitory e ects of a combination treatment with rolipram and PGE 2 on zymosan-induced IL-8 release. 6 Microscopic examination revealed that most neutrophils contained one or more zymosan particles and that combination treatment with rolipram and PGE 2 noticeably reduced the number of ingested particles. Moreover, there was a signi®cant reduction in the percentage of neutrophils which ingested three or more zymosan particles. 7 Thus, our results demonstrate that cyclic AMP-elevating agents modulate the ability of neutrophils to generate IL-8 in response to a particulate stimulus. However, these agents also modulate the ability of neutrophils to phagocytose zymosan particles. Whether this e ect will translate into inhibition of the ability of neutrophils to deal with infectious agents needs to be investigated further.