Abstract
According to the 1997 National Health Interview Study by the National Center for Health Statistics and the Center for Disease Control and Prevention, it was estimated that 25.7 million people had been diagnosed with asthma. This number increased to 26.3 million in 1998. While the etiology of asthma is complex and may involve a number of triggering factors such as external allergens, irritants, cold, stress and exercise, the manifestation is universal. Asthmatic patients experience labored breathing due to spasm of the bronchioles.Atopic asthma is a chronic inflammatory disorder of the airways where upon exposure to allergens, the body mounts an immune response. This response involves the release of mediators including but not limited to histamine, cytokines, leukotrienes, prostaglandins, bradykinin and tryptase.|Histamine is one of the important mediators of allergic disease, asthma and inflammation. The development of some allergic reactions, infections and tumors are associated with excessive histamine production. T lymphocytes, especially T helper lymphocytes, also play an important role in the pathogenesis of atopic asthma. Helper T cells can be divided into two subsets: T helper type 1 cells (Th1) and T helper type 2 cells (Th2). Both subsets play distinctive roles in the development, initiation and regulation of the immune response. Histamine regulates the development of an allergic state by increasing the number of T helper type 2 lymphocytes and decreasing the number of T helper type 1 lymphocytes. In doing so, histamine increases the secretion of Th2 cytokines such as IL-4 (interleukin-4), IL-5, IL-10 and IL-13 and inhibits the production of Th1 cytokines IL-2 and IFNy (interferon y) and monokine 1L-12.|IL-13 (interleukin-13), produced primarily by T helper cells, is an important autacoid mediator that has been implicated in the development of allergic disease. IL-13 plays a central role in allergic inflammatory eosinophilic reaction in allergic asthma through IgE-mediated isotype switching. Furthermore, it has been shown that selective neutralization of IL-13 in murine models leads to amelioration of the asthma phenotype, including airway hyperresponsiveness, eosinophil recruitment and mucus over-production. Because of the implication of IL-13 in the pathogenesis of atopic asthma, this study was designed to investigate the mechanisms of regulation of IL-13 by histamine in Th2 cells. D10.G4.1 cells, a murine Th2 cell line, were treated with histamine (10'8-10~*M) and then activated with PMA (phorbol 12 myristate 13- acetate) plus ionomycin or aCD3. aCD3 was used to activate Th2 cells because it binds to surface glycoprotein CD3 and ultimately stimulates T cell activation and the release of cytokines. Phorbol ester PMA and calcium ionophore ionomycin were employed in downstream T cell activation by stimulating the protein kinase C pathway that also stimulates T cell activation and the release of cytokines. Levels of IL-13 production were then measured by ELISA (enzyme-linked immunosorbent assay) and semiquantitative RT-PCR (reverse transcription-polymerase chain reaction). Cells were pretreated with histamine receptor antagonists pyrilamine, ranitidine, cimetidine and thioperamide to determine the involvement of histamine receptors. Cells were also pretreated with PKA (protein kinase A) inhibitors H-8 (N-[2-(methylaminoethyl)]-5-isoquinoline-sulfonamide) and Rp-cAMPS (Rp-diastereomer of adenosine cyclic 3’5’-phosphorothionate) and Jak-STAT (janus kinase - signal transducers and activators of transcription) inhibitor tyrphostin AG490 prior to the addition of histamine. H-8 is an inhibitor of the catalytic subunit of PKA while Rp-cAMPS is an inhibitor of the regulatory subunit of PKA. Tyrphostin is an inhibitor of Jak2, Jak3, STAT1, STAT3 and STAT5. Finally, it has also been shown that IL-12 is capable of repressing STAT6 DNA binding, which led to downregulation of Th2 cytokines IL-4 and IL-5. Based on these findings, we also pretreated cells with monokine IL-12 to determine the effects of IL-12 on histamine-mediated IL-13 production. We found that histamine dose- dependently enhanced IL-13 secretion and mRNA levels in Th2 cells via H1 (histamine type 1) and H2 (histamine type 2) receptors. Pretreatment of cells with H-8, Rp-cAMPS and tyrphostin prevented histamine-induced secretion and transcription of IL-13. Likewise, pretreatment of Th2 cells with IL-12 also reversed histamine’s effects on IL-13 secretion from stimulatory to inhibitory. These observations suggest a role for PKA and the Jak-STAT pathway in histamine-mediated elevation of IL-13 secretion and transcription.