Effects of integrin clustering on human lung mast cells and basophils.

The interaction of cells with the extracellular matrix can alter cell responses and is regulated by integrins on the cell surface. We used monoclonal antibodies to the VLA-4 integrins CD29 and CD49d followed by an F(ab')2 fragment of rabbit anti-mouse immunoglobulin G1 to crosslink integrins on the surface of human lung mast cells and basophils. Crosslinking either CD29 or CD49d caused a significant histamine release (HR) from the basophils of most asthmatic donors (10 of 14 for CD49d and 7 of 10 for CD29) (HR = 21 +/- 5%, n = 10, P < 0.005 for CD29 and HR = 19 +/- 4%, n = 14, P < 0.01 for CD49d) yet failed to initiate HR from the basophils of non-atopic and atopic donors (HR was 1 +/- 0.5% for CD29 and 1 +/- 0.5% for CD49d, n = 10, P = NS). Crosslinking either CD29 or CD49d also failed to initiate histamine release from human lung mast cells (HR was 1 +/- 1% for CD29 and 2 +/- 1% for CD49d). The basophils of asthmatic donors responded to 100 and 30 micrograms/ml tissue fibronectin (HR = 12 +/- 2% and 10 +/- 3% for 100 and 30 micrograms/ml fibronectin, respectively, n = 18, P < 0.05), whereas basophils of nonasthmatic patients again failed to degranulate (HR was 0 +/- 0.4% and 1 +/- 0.6%, respectively, n = 11, P = NS). In contrast to the basophil, crosslinking of either CD29 or CD49d failed to initiate histamine release in human lung mast cells (HR = 1 +/- 1% for CD29 and 2 +/- 1%, n = 15). Human lung mast cells were also unresponsive to tissue fibronectin (100 and 30 micrograms/ml) (HR = 1 +/- 1%, n = 5). The tyrosine kinase inhibitor, genistein, significantly reduced CD29- and CD49d-induced HR (inhibition = 83 +/- 7% for CD29 and 77 +/- 6% for CD49d, n > or = 5, P < 0.05). A second tyrosine kinase inhibitor, piceatannol, also significantly reduced both CD29- and CD49d-induced HR (inhibition was 62 +/- 19% for CD29 and 56 +/- 14% for CD49d, n = 7, P < or = 0.05). Integrin crosslinking also affected the response to a second, immunoglobulin E (IgE)-dependent stimulus. Both CD29 and CD49d clustering significantly inhibited anti-IgE-induced histamine release from the human basophil. Inhibition was 30 +/- 5%, n = 18, P < or = 0.001 for CD29 versus 40 +/- 6% for CD49d. In summary, we have shown that crosslinking the beta 1 integrins using either monoclonal antibodies or tissue fibronectin can initiate mediator release from the basophils of asthmatic patients by a mechanism which appears to be tyrosine kinase-mediated. In addition, clustering of integrins modulates the response to a second IgE-dependent signal.

Regulation of integrin-dependent release in human lung mast cells and basophils.

The interaction of cells with surfaces or components of the extracellular matrix alters cell responses and is regulated by integrins on the cell surface. We have used monoclonal antibodies to CD29 and CD49d followed by an F(ab)2 fragment of rabbit anti-mouse IgG1 to cross-link the integrins on the surface of human lung mast cells and basophils. We found that cross-linking either CD29 or CD49d failed to initiate mediator release from the basophils of non-atopic and atopic donors [histamine release (HR) = 1 +/- 0.5% for CD29 and 1 +/- 0.5% for CD49d, n = 10, NS]. In contrast we found that clustering CD29 caused significant HR from the basophils of asthmatic donors (HR = 21 +/- 5%, n = 10, p < 0.005). Clustering of CD49d also caused significant degranulation in the same donors (HR = 9 +/- 3%, n = 10, p < 0.11). Incubating the basophils of these asthmatic donors with a synthetic RGD peptide significantly reduced CD29- and CD49d-induced histamine release. CS-1 peptide was also found to inhibit CD29-induced histamine release but had no significant effect on CD49d-induced histamine release. The tyrosine kinase inhibitors, genistein and piceatannol, completely ablated CD29- and CD49d-induced degranulation. In summary, we have shown that cross-linking integrins can initiate mediator release from the basophils of asthmatic patients and that this appears to involve recognition of RGD and activation of tyrosine kinase.

A sensitive colorimetric assay for the release of tryptase from human lung mast cells in vitro.

Studies of human lung mast cells have usually focused on histamine release, although the enzymes stored in the granules may also contribute to the pathophysiology of the allergic response. We have used a simple colorimetric assay for tryptase to follow the release of proteolytic enzymes from human lung mast cells in vitro. Either human lung mast cell supernatants or authentic mast cell tryptase were mixed with benzoyl-DL-arginine-p-nitroaniline and incubated for up to 72 h at 37 degrees C. The appearance of nitroaniline was then measured at 410 nm in an ELISA plate reader. Cells were sonicated in H2O to measure total tryptase and histamine. Human lung mast cells contained the equivalent of 11.2 +/- 0.7 pg tryptase per cell and 3.2 +/- 0.3 pg of histamine. The amount of tryptase measured colorimetrically correlated with the level of tryptase measured by radioimmunoassay (Pharmacia), r = 0.92, P < 0.01. The inhibition profile of the proteolytic enzyme measured by the cleavage of BAPNA, was found to be identical to that of authentic lung mast cell tryptase. Over 90% of the maximum tryptase release was complete within 15 min whilst histamine release occurred within 5 min. In cells stimulated with 10 micrograms/ml anti-IgE we found a strong correlation between the release of tryptase and histamine, r = 0.95, P < 0.005. Finally, investigations with various pharmacological agents have supported our initial hypothesis that tryptase would mimic histamine release and provide an alternative marker for mast cell activation. In summary, we have utilised a simple enzymic assay as an indicator of human lung mast cell degranulation. In washed lung mast cells this assay appears be specific for granule tryptase and release of this activity into the supernatants of challenged cells correlates well with the presence of histamine. This assay offers several advantages over current methods of measuring mediator release from human lung mast cells in vitro and should provide an inexpensive and sensitive technique for following mast cell degranulation.

Role of tyrosine kinases in IgE-mediated signal transduction in human lung mast cells and basophils.

Recent evidence suggests that tyrosine kinases play an important role in signal transduction mechanisms utilized by a range of different agonists in many cell types. We have investigated the effects of four different inhibitors of tyrosine kinases on IgE-dependent histamine release from human lung mast cells and basophils. Genistein inhibited the anti-IgE-induced histamine release from human basophils (at 10 microM genistein, inhibition = 55 +/- 5%, n = 17, P < 0.005) with an IC50 of 8 microM, but was much less effective in the human lung mast cell (at 10 microM, inhibition = 18 +/- 6%, n = 11, P < 0.05). Two inactive analogs of genistein, genistin and diadzein, failed to affect anti-IgE-induced histamine release significantly in either mast cells or basophils. A second inhibitor of tyrosine kinases, tyrphostin 25, inhibited IgE-dependent release from basophils (at 10 microM, inhibition = 25 +/- 7%, n = 6, P < 0.05) though it was less effective than genistein and failed to affect IgE-induced histamine release from human lung mast cells (at 10 microM, inhibition = 22 +/- 16%, n = 5, P = NS). In contrast, methyl 2,5 dihydroxycinnamate (MDC) failed to inhibit anti-IgE-dependent histamine release in human basophils (at 10 microM, inhibition = 3 +/- 3%, n = 5, P = NS) but proved to be an effective inhibitor of anti-IgE-induced degranulation in human lung mast cells (at 10 microM, inhibition = 53 +/- 16%, n = 5, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)