The biologic activities of interleukin (IL)-13 and IL-4 often overlap, and evidence supports their importance in atopic disease and airways hyperresponsiveness. Here, their capacity to release eosinophil-activating cytokines was examined in cultured human airway smooth muscle. IL-13 and IL-4 induced selective release of eotaxin with no effect on granulocyte-macrophage colony-stimulating factor, regulated upon activation, normal T-cell expressed and secreted (RANTES), or IL-8. A profound synergistic increase in eotaxin release occurred when IL-13 or IL-4 was combined with IL-1beta that was abrogated by a neutralizing antibody to the IL-4 receptor alpha (IL-4Ralpha)-chain but not to the IL-2 receptor gamma (IL-2Rgamma)-chain. Expression of cell surface IL-4 receptors and IL-4Ralpha in lysates was constitutive and unchanged by treatment with IL-13 or IL-4 alone or in combination with IL-1beta. Activation of IL-4Ralpha by IL-13 or IL-4 induced signal transducer and activation of transcription-6 (STAT6), p42/ p44 ERK, p38, and to a lesser extent, SAPK/JNK mitogen-activated protein kinase phosphorylation. STAT6 and MAP kinase activation by IL-13 or IL-4 was not further potentiated after combined stimulation with IL-1beta. However, eotaxin release induced by IL-13 or IL-4 alone, and in combination with IL-1beta, was prevented by the MEK inhibitor U 0126 and by the p38 inhibitor SB 202190. Collectively, the data suggest that selective eotaxin release induced either by IL-13 and IL-4 or when combined with IL-1beta is mediated by a constitutive cell surface IL-4Ralpha and the activation of multiple intracellular pathways.

Airway smooth muscle (ASM) is a potential source of multiple proinflammatory cytokines during airway inflammation. In the present study, we examined a requirement for mitogen-activated protein (MAP) kinase activation for interleukin (IL)-1beta-stimulated GM-CSF, RANTES, and eotaxin release. IL-1beta induced concentration-dependent phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun amino-terminal kinase (SAPK/JNK). p42/p44 ERK and p38 MAP kinase phosphorylation peaked at 15 min and remained elevated up to 4 h. SAPK/JNK phosphorylation also peaked at 15 min but fell to baseline within 60 min. SB 203580 selectively inhibited IL-1beta-stimulated activation of p38 MAP kinase; U 0126 was selective against p42/p44 ERK activity. SB 202474, an inactive analog, had no effect on p42/p44 ERK, p38 MAP kinase, or SAPK/JNK activation, or on eotaxin or RANTES release. Eotaxin release was inhibited by SB 203580 and U 0126, whereas RANTES release was prevented by U 0126 only. GM-CSF release was inhibited by U 0126 but enhanced by SB 203580. These data indicate that RANTES release is dependent on p42/p44 ERK activation but occurs independently of p38 MAP kinase activity. Eotaxin release, however, is dependent on both p38 MAP kinase- and p42/p44 ERK-dependent mechanisms. GM-CSF release is p42/p44 ERK dependent and is tonically suppressed by a mechanism that is partially dependent on p38 MAP kinase, though direct inhibition of cyclooxygenase (COX) activity due to poor inhibitor selectivity may also contribute.

1 Airway smooth muscle (ASM) is a potential source of multiple pro-in¯ammatory cytokines during airway in¯ammation. b-Adrenoceptor agonist hyporesponsiveness is a characteristic feature of asthma, and interleukin (IL)-1b and tumour necrosis factor (TNF)-a are implicated in its cause. Here, the capacity of b-adrenoceptor agonists to prevent release of GM-CSF, RANTES, eotaxin and IL-8, elicited by IL-1b or TNFa, was examined in human ASM cells. 2 Isoprenaline (*EC 50 150 nM), a non-selective b-adrenoceptor agonist, and salbutamol (*EC 50 25 nM), a selective b 2 -adrenoceptor agonist, attenuated release of GM-CSF, RANTES and eotaxin, but not IL-8 (EC 50 41 mM). The maximum extent of attenuation was RANTES 5 eotaxin 4 GM-CSF 44 IL-8, and was prevented by either propranolol (1 mM), a non-selective b-adrenoceptor antagonist, or ICI 118511 (IC 50 15 nM), a selective b 2 -adrenoceptor antagonist. 3 The cyclic AMP-elevating agents, dibutyryl cyclic AMP (*EC 50 135 mM), forskolin (*EC 50 530 nM) and cholera toxin (*EC 50 575 pg ml 71) abolished IL-1b-induced release of GM-CSF, RANTES and eotaxin, but not IL-8. 4 IL-1b (1 ng ml 71) attenuated early increases (up to 1 h) in cyclic AMP formation induced by salbutamol (1 mM), but not by forskolin (10 mM). The cyclo-oxygenase inhibitor, indomethacin (1 mM) prevented later increases (3 ± 12 h) in IL-1b-stimulated cyclic AMP content, but did not prevent the attenuation by salbutamol of IL-1b-induced cytokine release. 5 We conclude in human ASM cells that activation of b 2 -adrenoceptors and generation of cyclic AMP is negatively-linked to the release, elicited by IL-1b or TNFa, of eosinophil-activating cytokines such as GM-CSF, RANTES and eotaxin, but not IL-8.