Syk deficiency in human non-releaser lung mast cells

Abstract

Human lung mast cells, like peripheral blood basophils, can have a nonreleaser phenotype characterized by a failure to respond to FceRI-IgE-mediated stimulation (1). Previous studies on human basophils demonstrated that approximately 10–15% of donors fail to release histamine in response to FceRI crosslinking (2–5) and that depressed protein levels of the tyrosine kinase Syk correlated with this nonreleaser status (6;7). Here we present evidence for diminished Syk protein in a second preparation of lung mast cells with a nonreleasing phenotype. Mast cells were isolated from discarded surgical lung obtained within 24 h of surgery as approved by the VCU IRB. Mast cells were dispersed by mincing the lung and digesting the pieces with collagenase and hyaluronidase, enriched by Percoll density-dependent sedimentation, and purified to >95% purity by positive selection using mouse IgG anti-Kit mAb and bead-conjugated anti-mouse Ab. Because initial activation experiments with anti-FceRI mAb did not cause degranulation (not shown), these putative non-releaser mast cells were examined in greater detail. Skin derived mast cells, obtained as described (8) were examined in parallel with the lung mast cells as a positive control. Skin and lung mast cells were challenged with various concentrations of anti-FceRI mAb and examined for degranulation (β-hexosaminidase, Figure 1A) and cytokine (GMCSF, Figure 1B) release. As expected, skin mast cells degranulated in response to each dose of anti-FceRI mAb stimulation. In contrast, the lung mast cells failed to degranulate in response to any concentration of anti-FceRI mAb. While incubation with IL-3 has been shown to revert nonreleasing basophils to releasing basophils (5) with concomitant recovery in Syk levels, (9) incubation of the lung mast cells with this cytokine (50 ng/ml for 4 days) did not convert them to a releasing phenotype (not shown). The FceRI-mediated release of GM-CSF was also impaired. However, the abilities of the lung mast cells to degranulate and release GM-CSF in response the calcium ionophore, {type:entrez-nucleotide,attrs:{text:A23187,term_id:833253,term_text:A23187}}A23187, a non-FceRI-dependent stimulus, were intact. Thus, these lung mast cells had a defect that was selectively manifested in their FceRI-initiated signal transduction pathway. Figure 1 Specific impairment of FceRI-dependent degranulation in lung mast cells. Lung mast cells or releasing skin mast cells were challenged with different concentrations of anti-FceRI mAb, calcium ionophore ({type:entrez-nucleotide,attrs:{text:A23187,term_id:833253,term_text:A23187}} ... This non-releaser phenotype was not due to absent FceRI. After 21 days in culture, as seen in Figure 2A, these mast cells expressed high amounts of FceRI and Kit by flow cytometry (Figure 2-top panel) and tryptase by immunocytochemistry (Figure 2-bottom panel). No obvious morphological abnormalities were apparent. Figure 2 Characterization of non-releaser human lung mast cells. Top Panel. Expression if FceRI and Kit on non-releasing human lung mast cells. Lung mast cells were incubated at 4°C with mouse IgG mAbs against FceRI-α and Kit (5 ... We next tested these lung mast cells for protein levels of Syk, Lyn, and Fyn; molecules previously associated with signaling after FceRI aggregation. As seen in Figure 3, the anti-FceRI-stimulated non-releasing lung mast cells do not express detectable levels of Syk protein by Western blotting after 14 to 30 days of culture, in contrast to a releaser lung mast cell preparation. However, Lyn and Fyn expression were readily detected in non-releaser lung mast cells Longer exposure times did not reveal Syk protein expression. We conclude that the lung mast cells in these experiments were defective in their IgE-mediated signaling, most likely related to a deficiency in Syk protein, analogous to the IgE non-releaser phenotype described for basophils. (6;7) Figure 3 Non-releaser lung mast cells lack Syk protein. Nonreleasing lung mast cells (~200,000 cell-equivalents/lane) after the indicated days in culture were collected and then lysed and subjected to Western blotting as described (6;9). Actin was used as a protein ...