Immunological characterization of A2B adenosine receptors in human mast cells

There is increasing evidence that adenosine modulates mast cell function. Such an interaction may be particularly important in asthma. Inhaled adenosine provokes bronchoconstriction in asthmatic patients through processes associated with mast cell activation. The adenosine receptor type that mediates these actions has not been identified. For this purpose, we generated antibodies against a 22-mer peptide corresponding to C-terminus of the human A(2B) adenosine receptor. This antibody recognized adenosine A(2B) receptors on Western blots as a band with an apparent molecular mass of 31 kDa, and a smear of immunoreactivity in the region from 60 to 130 kDa. Enzymatic deglycosylation of adenosine A(2B) receptor, or prevention of natural glycosylation with tunicamycin resulted in accumulation of protein with an apparent molecular mass of 29 kDa. The specificity of this antibody was confirmed in experiments with HA-tagged A(2B) receptor fusion protein. Anti-HA and anti-A(2B) antibodies, used for immunoprecipitation and Western blotting of the fusion protein, labeled the same glycoprotein in membranes of transfected, but not mock-transfected cells. These antibodies were also found useful in immunocytochemistry, as evidenced by specific staining of cells expressing A(2B), but not other subtypes of adenosine receptors. Human lung mast cells obtained from bronchoalveolar lavage fluid of asthmatic patients were identified by specific monoclonal anti-tryptase antibody. Double immunostaining demonstrated expression of A2B receptors in tryptase-positive cells. Our results, therefore, demonstrate the presence of A2B receptors in human lung mast cells and support the hypothesis that A2B receptors are involved in activation of mast cells in asthma. (C) 2003 Wiley-Liss, Inc.