1 Eotaxin is a novel C-C chemokine with selective chemoattractant activity for eosinophils. We determined whether eotaxin could be produced by human airway smooth muscle (HASM) cells in culture and examined its regulation by interleukin-10 (IL-10) and the corticosteroid, dexamethasone. 2 Stimulation of the cells with interleukin-1b (IL-1b) or tumour necrosis factor (TNFa) each at 10 ng ml 71 induced the release of eotaxin protein with maximal accumulation by 24 h. Interferon-g (IFNg) alone at 10 ng ml 71 had no e ect and there was no synergy between these cytokines on the release of eotaxin. 3 Reverse phase high performance liquid chromatographic (HPLC) analysis of supernatents from cells treated with TNFa (10 ng ml 71 for 96 h showed immunoreactivity to eotaxin which eluted with the expected retention time of 34.5 ± 35 min. 4 Both IL-1b and TNFa-induced release of eotaxin was not inhibited by dexamethasone (1 mM), however IL-10 (10 ng ml 71 ) had a signi®cant inhibitory e ect. Dexamethasone and IL-10 did not inhibit the induction of eotaxin mRNA induced by IL-1b or TNFa. 5 Thus, human airway smooth muscle cells can release eotaxin and could be an important source of chemokine production during airway in¯ammatory events.

Endothelin-1 is a potent bronchoconstrictor peptide with pro-inflammatory and growth-promoting properties. After exposure of sensitized Brown-Norway rats to six repeated ovalbumin exposures, there was an increase in pro-endothelin (ET)-1 messenger RNA compared with saline-exposed control rats 24 h after the final exposure (P < 0.01). ET-1 immunoreactivity was increased sixfold in the bronchial epithelium of the larger conducting airways in the repeated allergen-exposed rats (P < 0.001). After repeated allergen exposure, there were increased rates of DNA synthesis in the airway smooth muscle (ASM) cells (P < 0.001) and epithelial cells (P < 0. 001) compared with saline-exposed controls, as measured by bromodeoxyuridine incorporation. Treatment with a dual endothelin A and B (ET(A+B)) receptor antagonist caused a significant attenuation in both ASM (P < 0.001) and epithelial cell (P < 0.001) bromodeoxyuridine incorporation compared with the allergen-challenged and vehicle-treated group. The dual ET(A+B) antagonist attenuated eosinophil recruitment into the airways (P < 0. 05) but had no significant effect on increased bronchial reactivity to acetylcholine in allergen-exposed rats. Increased levels of ET-1 in the airways may contribute to inflammation and ASM and epithelial cell DNA synthesis after repeated allergen exposure. Such processes may underlie increased proliferation of resident cells leading to airway wall remodeling in asthmatics.

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Stimulation of HEL 299 cells with tumor necrosis factor ␣ (TNF-␣) or interleukin 1␤ (IL-1␤) had no effect on M 2 muscarinic receptor expression. However, the combination of these two cytokines markedly down-regulated muscarinic M 2 receptor protein and mRNA expression and uncoupled M 2 receptors from adenylyl cyclase. There was no effect of TNF-␣ and IL-1␤ on the m2 muscarinic receptor mRNA stability, and nuclear run-on assays showed reduced m2 receptor gene transcription. Sequential cytokine addition suggests that the synergy involves postreceptor events. Although the cAMPdependent protein kinase inhibitor H8 provided a significant protection against receptor down-regulation, the protein kinase C inhibitor GF109203X had no effect. The ceramide analog C 2 -ceramide (N-acetylsphingosine) was without effect on m2 receptor expression. However, a strong synergistic effect was demonstrated when cells were treated with the combination of C 2 -ceramide and TNF-␣ or IL-1␤. TNF-␣ and/or IL-1␤ combination also activated the 46-and 55-kDa c-Jun NH 2 -terminal protein kinases and to a lesser extent p42 and p44 mitogenactivated protein kinase isoforms. Cycloheximide abolished the TNF-␣ and IL-1␤ effect, suggesting that de novo protein synthesis is required for receptor down-regulation. These results suggest that the TNF-␣ and IL-1␤ synergize to induce transcriptional down-regulation of the M 2 muscarinic receptor, which seems to be mediated through activation of both ceramide and cAMP-dependent protein kinase pathways. Furthermore, these results suggest that M 2 receptor expression is under the control of a cytokine network.

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