Mechanical loading promotes mast cell degranulation via RGD-integrin dependent pathways
Introduction
Classical mast cell activation occurs through the aggregation and cross-linking of the high affinity FcεR1 receptor by antigen-bound IgE antibodies, leading to degranulation and release of inflammatory mediators (Bischoff, 2007, Galli et al., 2008). Other studies have found mast cells to be activated by environmental (e.g., bacterial toxins, chemical agents), biochemical (e.g., complement component 5, endothelin-1) and physical (osmotic pressure, shear stress) stimuli (Cochrane and Douglas, 1973; Murray et al., 2004, Yang et al., 2007). Studies have also illustrated changes in mast cell accumulation in response to alterations in the mechanical environment in vivo. For example, increased mast cell density has been observed within hypertrophic dermal scars of mice subjected to mechanical stress (Aarabi et al., 2007) and within the soleus muscles of rats subjected to hindlimb unloading (Dumont et al., 2007). Furthermore, many cardiovascular diseases (e.g. volume overload, hypertension, and myocardial infarctions) exhibit altered mechanical loading and impaired contractile function (Gupta and Grande-Allen, 2006; Abraham et al., 2007), followed by an increase in mast cell activation and density. To date, the cellular and molecular mechanisms mediating mast cell response to physical stimuli have received little attention.
The mechanical loads applied to extracellular matrices (ECM) are essential in maintaining tissue homeostasis (Riser et al., 1992, Wang et al., 1993, Gupta and Grande-Allen, 2006); however deviations in matrix mechanics can activate pathological signaling pathways and/or induce cellular stress responses (O'Donnell et al., 1988; Duranti et al., 1995, Gupta and Grande-Allen, 2006). Numerous pathways have been identified through which mechanical forces are transduced into biological signals (Hamill and Martinac, 2001). Cell surface adhesion molecules, including integrins, serve critical roles in transmitting mechanical forces from the ECM to cells (Wang et al., 1993, Wilson et al., 1995, Ingber, 2006). Integrins are heterodimeric transmembrane glycoproteins composed of α and β subunits (Arnaout et al., 2005), and members of the β1 and β3 subfamilies bind ECM proteins containing an arginine-glycine-aspartic acid (RGD) sequence (e.g. fibronectin, laminin and fibrin). These receptor–ligand interactions mediate outside-in signaling (e.g. mechanotransduction), thus allowing cells to perceive changes in the deformation and stiffness of the ECM (Gao et al., 2007, Kock et al., 2009). Although mast cells express members of the RGD-integrin family, including α5β1, αvβ3 and αIIbβ3 (Ra et al., 1994, Yasuda et al., 1995), and most of the in vivo studies previously mentioned correlate changes in mast cell number with alterations in mechanical load, the role of integrins in the response of mast cells to mechanical loading have not been investigated.
The present studies utilized a novel 3-dimensional model system to evaluate the effects of uniaxial cyclic mechanical load on mast cell degranulation to elucidate the role of integrin–ECM interactions in modulating these effects. By varying the peak strains applied to the constructs, load-dependent cell degranulation was observed. The mechanism mediating this load-dependent degranulation was investigated using a competitive inhibitor of RGD-integrins. Our results suggest a novel mechanism by which mechanical loading induces mast cell degranulation via RGD-integrins.
Section snippets
Materials and methods
All chemicals were purchased from Sigma (Sigma Chemical Co., St. Louis, MO) except where noted.
Mechanical loading induces degranulation of RBL-2H3 cells
RBL-2H3 cells undergo degranulation and release of pro-inflammatory mediators when stimulated. To investigate whether mechanical loading can induce degranulation, RBL-2H3 cells were seeded in 3D fibrin scaffolds, subjected to 1.2 Hz cyclic loading at 5% or 10% peak strain and 0% (static control) for 4, 12 and 24 h (Fig. 1), and assayed for degranulation via β-hexosaminidase release (Fig. 2). RBL-2H3-fibrin constructs deformed to peak tensile strains of 5% or 10% demonstrated significant (p<0.01)
Discussion
Several studies have illustrated that mechanical stresses, such as compression or tension, can elicit cellular responses (Carver et al., 1991, Smith et al., 1997, MacKenna et al., 1998, Bishop and Lindahl, 1999, Li et al., 2003, Barbee et al., 1994). The goal of this study was to determine if mechanical loading stimulates activation and degranulation of mast cells. Our results demonstrate that RBL-2H3 cells degranulate when subjected to 5% or 10% strain, and although there were no detectable
Conflict of interest
None of the authors have a conflict of interest.
Acknowledgments
The authors would like to thank Cheryl Cook and Charity Fix for assisting with cell culture maintenance and Mike Gore and Joseph Farrow for the contributions to the design and fabrication of the loading device. These studies were supported by funds from the National Institute of Health Grant nos. HL083441 (W.C) and HL097214 (E.C.G.). C.G.W. was supported by a fellowship administered through NIH Grant no. P20 RR-016461 from the National Center for Research Resources and V.F. was supported
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Current address: University of Michigan School of Dentistry, Center for Craniofacial Regeneration, Ann Arbor, MI 48109, USA.