Search

BACKGROUND: Mast cells are involved in many distinct pathological conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by qPCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins are small GTPases, receptors and transporters. One such cell surface protein is CD98 heavy chain (CD98hc) encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Our study indicates that glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, we found that CD98hc plays an important role in mast cell function.

Background Mast cells are significantly involved in IgE-mediated allergic reactions; however, their roles in health and disease are incompletely understood. Objective We aimed to define the proteome contained in mast cell releasates on activation to better understand the factors secreted by mast cells that are relevant to the contribution of mast cells in diseases. Methods Bone marrow–derived cultured mast cells (BMCMCs) and peritoneal cell–derived mast cells were used as "surrogates" for mucosal and connective tissue mast cells, respectively, and their releasate proteomes were analyzed by mass spectrometry. Results Our studies showed that BMCMCs and peritoneal cell–derived mast cells produced substantially different releasates following IgE-mediated activation. Moreover, we observed that the transglutaminase coagulation factor XIIIA (FXIIIA) was one of the most abundant proteins contained in the BMCMC releasates. Mast cell–deficient mice exhibited increased FXIIIA plasma and activity levels as well as reduced bleeding times, indicating that mast cells are more efficient in their ability to downregulate FXIIIA than in contributing to its amounts and functions in homeostatic conditions. We found that human chymase and mouse mast cell protease-4 (the mouse homologue of human chymase) had the ability to reduce FXIIIA levels and function via proteolytic degradation. Moreover, we found that chymase deficiency led to increased FXIIIA amounts and activity, as well as reduced bleeding times in homeostatic conditions and during sepsis. Conclusions Our study indicates that the mast cell protease content can shape its releasate proteome. Moreover, we found that chymase plays an important role in the regulation of FXIIIA via proteolytic degradation.

Background Group B Streptococcus (GBS) or Streptococcus agalactiae are β-hemolytic gram-positive bacteria that colonize the lower genital tracts of women and are frequently associated with infections during pregnancy. Innate immune defenses are critical for controlling GBS dissemination and systemic infection. Mast cells are resident sentinel cells that come into contact with pathogens early during colonization and infection. Objective We aimed to investigate the contribution of chymase to systemic GBS infection and rates of preterm birth. Methods Pharmacologic and genetic approaches using mice deficient in mast cell protease (MCPT) 4, the mouse functional homologue of human chymase, were used. Results Our studies show that mast cells release a protease with chymotrypsin-like cleavage specificity in response to GBS. Additionally, increased GBS systemic infection and preterm births were observed in MCPT4-deficient mice versus MCPT4-sufficient mice. Furthermore, we observed that proteolytic cleavage of the host extracellular matrix protein fibronectin by peritoneal cell–derived mast cell lysates diminished GBS adherence. Consistent with this observation, the increase in GBS dissemination and preterm births observed in MCPT4-deficient mice was abolished when GBS was deficient in expression of the fibronectin-binding protein SfbA. Conclusions Taken together, our results suggest that the protective effect of MCPT4 against GBS dissemination and preterm labor can be attributed in part to MCPT4-mediated proteolysis of fibronectin. Our studies reveal a novel role of mast cells in defense against bacterial infections.

Background Eotaxin/CCL11 plays an important role in asthma. It acts through the chemokine receptor CCR3 expressed on hematopoietic and nonhematopoietic cells in the lung. Objective To determine whether eotaxin/CCL11 modulates lung and bronchial fibroblast properties and thereby might contribute to airway remodeling. Methods CCR3 expression was characterized on a lung fibroblast line (MRC-5; flow cytometry, fluorescent microscopy, RT-PCR, and Northern blotting), on primary bronchial fibroblasts (flow cytometry), and on fibroblasts in human lung tissue (confocal laser microscopy). The effects of eotaxin/CCL11 on lung fibroblast migration (Boyden chamber), proliferation (tritiated thymidine incorporation), α-smooth muscle actin expression (ELISA), 3-dimensional collagen gel contraction (floating gel), pro-α1(I) collagen mRNA (Northern blotting), total collagen synthesis (tritiated proline incorporation), matrix metalloproteinase activity (gelatin zymography), and TGF-β 1 release (ELISA) were evaluated. The contribution of eotaxin/CCL11/CCR3 binding on lung fibroblasts was also investigated by neutralizing experiments. Results CCR3 is constitutively expressed in cultured lung and primary bronchial fibroblasts and colocalizes with specific surface markers for human fibroblasts in lung tissue. Eotaxin/CCL11 selectively modulates fibroblast activities by increasing their proliferation, matrix metalloproteinase 2 activity, and collagen synthesis but not their differentiation into myofibroblasts, contractility in collagen gel, or TGF-β 1 release. Eotaxin/CCL11 enhances migration of lung fibroblasts in response to nonspecific chemoattractants, and this effect is completely inhibited by anti-CCR3–neutralizing antibodies. Conclusion These data demonstrate that eotaxin/CCL11 has a direct and selective profibrogenic effect on lung and bronchial fibroblasts, providing a novel mechanism whereby eotaxin/CCL11 can participate in airway remodeling in asthma.