Role of Mitogen-activated protein kinases in regulating human lung mast cell responses

Linda Kay, Suzanne Havard, Peter Peachell

University of Sheffield, Sheffield, UK

Asthma is a complex disease in which the mast cell plays a central role. Mitogen-activated protein kinase (MAPK) signalling cascades may play an important role in the release of mediators from mast cells. The potential involvement of the extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) on the IgE-dependent release of histamine, cysteinyl-leukotrienes (cys-LT) and prostaglandin D2 (PGD2) from human lung mast cells was evaluated by studying selective inhibitors. The expression and phosphorylation of these MAPKs was also investigated.

Mast cells were isolated from macroscopically normal human lung tissue by a modification of the method described by Ali and Pearce (1985). On occasion mast cells were further purified (85-100 % purity) by immunomagnetic bead separation. Cells were incubated with or without a MAPK inhibitor (10 μM) for 1 h before challenge with anti-IgE (1:300) for 25 min. Mediators released into the supernatants were determined by an automated fluorometric technique (histamine) or commercially available ELISA kits (cys-LT and PGD2). Expression of MAPKs in purified mast cells was determined by western blotting. Phosphorylation profiles of MAPKs, following challenge of mast cells with anti-IgE for various times (0, 3, 9 and 21 min), were also investigated. The membranes were probed using antibodies to the phosphorylated and non-phosphorylated forms of ERK, p38 and JNK. Band intensities were analysed using densitometery (Kodak Digital Science 1D software) and intensities normalised relative to actin or mononuclear cells (MNC). Values cited are means±S.E.M.

As inhibitors of IgE-dependent histamine release from mast cells, the efficacy of SP600125 (JNK-selective), SB202190 (p38-selective) and PD98059 (ERK-selective) varied. The order of efficacy was SP600125>SB202190>PD98059 (n=7-10). However, all three MAPK inhibitors almost completely blocked the IgE-dependent release of both cys-LT and PGD2 (n=4). Immunoblotting studies indicated that the expression of MAPKs, in purified mast cells (n=6-7), was 1.4±0.3, 0.8±0.2, 1.6±0.1 (ERK, p38 and JNK, respectively) fold different compared to MNC on a per cell basis. Further studies of purified mast cells (n≥4) revealed a dramatic increase (3-8 fold) in IgE-dependent phosphorylation of ERK shortly after activation followed by a gradual decline over time. However, IgE-dependent phosphorylation of either JNK or p38 was modest at best.

These findings suggest that ERK, p38 and JNK may all influence the IgE-dependent generation of mediator release from mast cells but that the relative contributions of each may vary.

Ali et al., (1985). AgentsActions 16: 138-140