TGF-β1 Regulates Adhesion of Mucosal Mast Cell Homologues to Laminin-1 Through Expression of Integrin α7

Steven Wright, A. Rosbottom, Cheryl L Scudamore, H von der Mark, Elisabeth Thornton, H R P Miller

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-β1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-β1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 ± 3.6% (mean ± SEM) in the absence of TGF-β1 to 58.7 ± 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-β1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcεRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin α7 by MMC homologues grown in the presence of TGF-β1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the α7-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1. Cell trafficking into and through tissues depends upon complex interactions, including adhesion to extracellular matrix (ECM)4 proteins (1); integrins are the major molecules that mediate this process (2). Mast cells are involved in the pathophysiology of immediate-type hypersensitivity reactions, wound healing, and chronic inflammatory diseases (3, 4). The ability to move through tissues is mandatory for their physiological function, and integrins are likely to be involved in mast cell migration. The roles of several integrins and other adhesion molecules in mast cell adhesion to ECM proteins has been studied using murine IL-3-dependent, bone marrow-derived cultured mast cells (CMC). These cells adhere to laminin-1 and -2 (5, 6, 7, 8), fibronectin (9), and vitronectin (10), and integrins α6β1 (5, 8), α5β1 (11), and αvβ3 (10), respectively, have been shown to play a role in this adhesion. Additionally, Abs to the non-integrin 67-kDa laminin binding protein (LBP) block adhesion of IL-3-dependent CMC to laminin-1 (6), indicating a role for this molecule. In vivo, mast cells are phenotypically classified into two major subsets, connective tissue and mucosal (12, 13), and mucosal mast cells are important in the immune response to gut-dwelling nematodes. Nematode infection of the gut results in recruitment of the precursors of mucosal mast cells (MMC) (14) and their migration intraepithelially, where expression of the MMC-specific chymase, mouse mast cell protease-1 (mMCP-1), indicates the mature mucosal phenotype (15). As IL-3-dependent CMC are thought to represent an immature mast cell phenotype (16) and lack mMCP-1 expression (17), they may be a poor model for study of the molecular interactions involved in this process, especially since it is widely recognized that the cytokine stem cell factor (SCF) is a key growth and differentiation factor for mast cells in vivo and in vitro (18). We have recently developed an in vitro model of mast cell differentiation in which addition of the cytokine TGF-β1 to bone marrow cells supplemented with IL-3, IL-9, and SCF regulates differentiation of bone marrow cells into a close homologue of the mucosal phenotype, as shown by abundant expression of mMCP-1 (19). Adhesion molecule expression in mast cells may be modulated during differentiation. The integrin α4 is expressed by IL-3-dependent CMC, is down-regulated in older cultures (11, 20), and is not present in mature tissue-derived connective tissue mast cells (20), but is required for the intestinal recruitment of MMC (14). In our in vitro model and in IL-3-dependent CMC (21), TGF-β1 up-regulates the expression of integrin αE (22), which may facilitate retention of MMC intraepithelially by binding E-cadherin (23). Therefore, as the key cytokine mediating differentiation of the mucosal phenotype, TGF-β1 may also change the spectrum of integrins expressed to reduce adhesion to ECM proteins and increase adhesion to the basement membrane (BM) protein laminin, thus facilitating intraepithelial migration. In support of this, one study showed that supplementing IL-3-dependent CMC with TGF-β1 for 48 h increased adhesion to laminin-1 (7); the mechanism was thought to be increased adhesion receptor expression. To test the proposed hypothesis that the adhesion properties of CMC are regulated by TGF-β1, we have compared adhesion of mast cells cultured with and without TGF-β1 to the ECM proteins fibronectin and vitronectin and the BM protein laminin-1. We have also examined the expression of several integrins using RT-PCR and flow cytometry. In addition to those integrins previously implicated in mast cell adhesion, we have investigated the role of the laminin-binding integrin α7 in adhesion of TGF-β1-dependent CMC. This integrin was originally thought to be skeletal muscle specific (24), but expression of the α7B isoform has since been found in other tissues, including intestinal epithelium, where it correlates with intestinal cell differentiation (25). In this study we show the expression of α7B integrin in cultured MMC homologues, which is regulated by TGF-β1. We also demonstrate, by use of the blocking Ab 6A11, a novel role for α7 in promoting adhesion of mucosal mast cell homologues to laminin-1.
Original languageEnglish
Pages (from-to)5689-5695
JournalThe Journal of Immunology
Publication statusPublished - 15 Nov 2002


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