Abstract
Eosinophils are one of the major effector cells in asthma, and controlling the number and survival of eosinophils might attenuate the severity of asthma. This could be achieved by inducing eosinophil apoptosis. Apoptosis allows the removal of cells without inducing an inflammatory response. Our knowledge of the factors involved in regulating eosinophil apoptosis remains limited. CD30 molecule has been associated with T cell negative selection and in T cell receptor-mediated apoptosis. In this thesis dissertation I examined the expression and role of CD30 in apoptosis of human blood eosinophils. Percentage of apoptotic eosinophils was determined by Annexin V/PI labeling and CD30 expression was examined by flow cytometry. Spontaneous apoptosis was induced by serum deprivation, and survival was conferred by incubating cells with 10% FBS and 15 ng/ml IL-5. CD30 surface expression was upregulated in eosinophils incubated for 24 hours as compared to freshly isolated eosinophils, and both CD30 expression and eosinophil apoptosis increased in a time-dependent manner. I also measured CD30 mRNA expression by RT-PCR, and determined that CD30 transcripts increased in eosinophils undergoing apoptosis only under serum deprivation conditions. The agonistic CD30 antibodies, Ber-H8 and HeFi-1, significantly enhanced eosinophil apoptosis. FBS and IL-5 failed to inhibit or suppress the CD30 agonistic-induced apoptosis. CD30 stimulation induced a JNK pathway, and blocking JNK activity with SP600125 completely abrogated the CD30-induced apoptosis in human blood eosinophils in the presence of IL-5 and FBS. SP600125 only partially inhibited eosinophil spontaneous apoptosis. Furthermore, the CD30-induced apoptosis was inhibited by DPI,a nitric oxide synthase inhibitor, suggesting an essential role or iNOS expression and NO production in the mediation of apoptosis signals by CD30. These data combined supported a role for CD30 activation in the induction eosinophil apoptosis.|I also explored the expression of different Protein kinase C (PKC) isoforms in eosinophils induced to undergo apoptosis by serum withdrawal, and investigated the role of PKC-8 in regulating eosinophil survival and apoptosis. PKC expression was measured under survival and apoptotic conditions, as well as in freshly isolated eosinophils. I observed that protein expression of almost all PKC isozymes was down-regulated in eosinophils undergoing apoptosis as compared to fresh cells or cells incubated with FBS and IL-5. However, PKC-8 expression displayed less significant change, coupled with the appearance of an approximately 40 KDa fragment in the apoptotic serum-deficient cells. Interestingly, rottlerin, which at the concentration of 10|iM acts as a selective inhibitor of PKC-Ö, induced significant increase in eosinophil apoptosis in cells incubated with only 1% FBS, and not in cells incubated with 10% FBS and 15 ng/ml IL-5. These findings suggested a pivotal role for PKC-8 in modulating eosinophil survival.|This research will further advance our understanding of eosinophil apoptosis, and therefore might contribute to the development of better therapeutic modalities in the treatment and/or cure of allergic inflammation in bronchial asthma.