These results suggest that in severe asthma, epithelial damage has the potential to contribute to neutrophilic inflammation through enhanced production of IL-8 via EGFR- dependent mechanisms.

SUMMARYFollowing skin sensitization a proportion of epidermal Langerhans cells (LC) are stimulated to leave the skin and to migrate, via afferent lymphatics, to draining lymph nodes where they accumulate as immunostimulatory dendritic cells (DC). It has been demonstrated previously that tumour necrosis factor-a (TNF-a), an inducible product of epidermal keratinocytes, and interleukin (IL)-1b, produced exclusively by LC in murine epidermis, provide important signals for the initiation of this response. Recently, it has been demonstrated that IL-18, a cytokine produced by both LC and keratinocytes within the epidermis, may also participate in immune responses induced following skin sensitization. In the present investigations, the ability of IL-18 to contribute to the regulation of LC migration and the accumulation of DC in draining lymph nodes has been examined. It was found that, like IL-1b, IL-18 administered intradermally to mice resulted in a signi®cant reduction in epidermal major histocompatibility complex (MHC) class II + LC densities and a marked increase in lymph node DC numbers. Using neutralizing anti-TNF-a and blocking anti-type I IL-1 receptor (IL-1RI) antibodies, it was shown also that the induction by IL-18 of both LC mobilization and DC accumulation in regional lymph nodes was dependent upon availability of TNF-a and the integrity of IL-1RI signalling. Furthermore, using IL-1b converting enzyme (caspase-1) knockout mice, IL-18-induced LC migration was found to have a mandatory requirement for active IL-1b. Importantly, not only was IL-18 able to contribute to the regulation of LC migration, it was found to be essential for the manifestation of these processes in response to topical sensitization with the contact allergen oxazolone.

A disease-related, corticosteroid-insensitive increase in the expression of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase in asthmatic bronchial epithelium has been shown previously by the current authors. To determine whether this is associated with enhanced intracellular signalling, the aim of this study was to evaluate epithelial tyrosine phosphorylation.Bronchial biopsies were analysed for the presence of phosphotyrosine by immunohistochemistry. Bronchial epithelial cells were exposed to EGF, hydrogen peroxide or tumour necrosis factor-a in vitro for measurement of tyrosine phosphorylated signalling intermediates and interleukin (IL)-8 release.Phosphotyrosine was increased significantly in the epithelium of severe asthmatics when compared with controls or mild asthmatics; however, in mild asthma, phosphotyrosine levels were significantly decreased when compared with controls. There was no significant difference between phosphotyrosine levels before or after 8 weeks of treatment with budesonide. Stimulation of bronchial epithelial cells resulted in tyrosine phosphorylation of several proteins, including EGFR, Shc and p42/p44 mitogen-activated protein kinase. In the presence of salbutamol, a transient partial suppression of EGFR phosphorylation occurred, whereas dexamethasone was without effect. Neither salbutamol nor dexamethasone inhibited EGFstimulated IL-8 release.These data indicate that regulation of protein tyrosine kinase activity is abnormal in severe asthma. The epidermal growth factor receptor and/or other tyrosine kinase pathways may contribute to persistent, corticosteroid-unresponsive inflammation in severe asthma.

SUMMARYActivated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-a mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-a protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNFa mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-a release but inhibited IL-4 release; notably, the ratio of TNF-a : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Aginduced IL-4 and IL-6 mRNA more readily than TNF-a mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre-and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.

The murine local lymph node assay (LLNA) is a method for the identification of skin sensitizing chemicals in which activity is measured as a function of proliferative responses induced in draining lymph nodes following topical exposure of mice to the test material. More recently, the LLNA has also been used for the determination of relative skin sensitizing potency based upon the mathematical derivation of an EC3 value, this being the estimated concentration of test chemical necessary to provoke a 3-fold increase in lymph-node cell-proliferative activity compared with concurrent vehicle-treated controls. Here we describe the use of the LLNA to determine the influence of vehicle on the skin-sensitizing potency of methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), the active ingredient of preservatives such as Kathon CG. To this end, LLNA responses to MCI/ MI were measured using the vehicles 4:1 acetone:olive oil (AOO), methyl ethyl ketone, dimethylsulfoxide, dimethylformamide, propylene glycol (PG) and acetone. It was found that the vehicle in which MCI/MI was applied had a substantial impact on activity, with derived EC3 values varying from 0.0049% with AOO to 0.048% with PG. With the other vehicles, EC3 values ranged from 0.0068 to 0.0076%. The skin sensitizing potency of MCI/MI as judged from LLNA responses is consistent with what is known of the requirements for sensitization in humans. It is proposed that the LLNA not only provides a method for determination of relative skin sensitizing potency, but is also appropriate for assessing the influence of vehicle matrix on sensitizing activity.