Original article CD40 and OX40 ligand are differentially regulated on asthmatic airway smooth muscle Asthma is a chronic disease of the airways associated with reversible airway obstruction, airway smooth muscle (ASM) cell hyperplasia, airway hyper-responsiveness and airway inflammation (1). Airway smooth muscle cells release cytokines and chemokines as well as express- ing cell-surface molecules that can bind to and modulate cells of the immune system. Specifically, ASM cells can bind activated T cells, which results in increased cellular proliferation (2). The recent discovery of the presence of two cell-surface molecules CD40 and OX40 Ligand (OX40L) on human ASM has highlighted the immunomodulatory role of this cell (3, 4). We have noted several important differences in the properties of ASM cells derived from asthmatic and nonasthmatic patients and amongst these is the observa- tion that the expression of CD40 and OX40L on asthmatic ASM is modulated by cytokines, which are relevant to asthma, viz. tumour necrosis factor (TNF) a and interleukin 1b (IL1b) (3). These co-stimulatory cell-surface molecules bind to CD154 and OX40, respec- tively, on activated CD4+ T cells. CD154 is expressed on CD4+ T cells immediately after their activation and provides a crucial Ôsecond signalÕ for T-cell activation (5). This is followed by the binding of OX40L to OX40 on CD4+ T cells, which activates a strong signalling pathway that augments cytokine secretion and cellular proliferation (6). Importantly, this interaction maintains long-term survival of CD4+ T cells (7). The expression of the cytokines TNF-a and interferon-c (IFN-c) is increased in asthmatic airways (8, 9). TNF-a facilitates inflammatory cell migration and increases the expression of intercellular adhesion molecule-1, CD40 and Background: CD40 and OX40 Ligand (OX40L) are cell-surface molecules ex- pressed on airway smooth muscle (ASM) that can enhance inflammatory cell activation and survival. The aim of this study was to examine the effect of tumour necrosis factor-alpha (TNF-a) and interferon-gamma (IFN-c) on ASM CD40 and OX40L expression. Methods: CD40 and OX40L expression on human ASM cells from asthmatic and nonasthmatic donors following stimulation with TNF-a and/or IFN-c was measured using cell-surface enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Involvement of signalling pathway was investigated with pharmacological inhibitors. Soluble TNF receptor levels were quantified by ELISA. Results: Interferon-c and TNF-a synergistically increased CD40 expression to a greater extent on asthmatic than on nonasthmatic ASM. In contrast, IFN-c reduced TNF-a-induced OX40L expression to a similar extent in both cell types. TNF-a and IFN-c induced CD40 via nuclear factor-jB (NF-jB) and signal transducer and activator of transcription-3 in both cell types and modulated OX40L via NF-jB and c-Jun N terminal kinase in nonasthmatic cells. Similar effects on the induction of OX40L in asthmatic cells were seen with NF-jB, but these were not statistically significant. The reduced OX40L expression with TNF-a and IFN-c involved extracellular regulated kinase 1/2 activation. Conclusion: Asthmatic ASM may modulate airway inflammation locally by increasing CD40 and OX40L expression in response to cytokines. IFN-c may regulate ASM pro-inflammatory actions by differentially modulating ASM CD40 and OX40L expression. D. I. Krimmer 1 , M. Loseli 1 , J. M. Hughes 2 , B. G. G. Oliver 1,3 , L. M. Moir 1,3 , N. H. Hunt 4 , J. L. Black 1,3 , J. K. Burgess 1,3 1 Discipline of Pharmacology and 2 Faculty of Pharmacy, The University of Sydney; 3 Woolcock Institute of Medical Research; 4 Department of Pathology, Bosch Institute, The University of Sydney, Sydney, NSW, Australia Key words: adhesion molecules; human airway smooth muscle cells; interferon-c; OX40 ligand; tumour necrosis factor-a. Dr Janette Burgess Respiratory Research Group Discipline of Pharmacology D05, Bosch Building University of Sydney Sydney NSW 2006 Australia This work was supported by the Australian National Health and Medical Research Council. J. K. Burgess is supported by a National Health & Medical Research Council R. Douglas Wright Fellowship #402835. Accepted for publication 5 December 2008 Allergy 2009: 64: 1074–1082 Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Munksgaard DOI: 10.1111/j.1398-9995.2009.01959.x 1074