This application examines the ability of human basophils to synthesize IL-4 and IL-13 utilizing a variety of agonists and contrasts conditions that modulate (augment or inhibit) production of each. The initial aim, and the one examined in greatest detail, examines the effects upon each cytokine by agonists that stimulate c-AMP, steroids, cyclosporin, FK506, and tyrosine kinase inhibitors. IL-13 protein and mRNA production will be examined, the former utilizing ultrapure basophil preparations and stimuli such as IL-3, ionomycin, and PMA. Flow cytometry will be used to determine intracellular cytokine expression. Primers acting through PKC such as IL-1b and TNFa will be tested as well as non-IgE dependent agonists such as C5a or FMLP, and IL-3-like growth factors such as GMCSF and IL-5. Perturbation of CD40L or CD32 (IgG receptor) will be tested for effects on IL-4/IL-13 secretion. The cells of atopic versus non-atopic subjects will be compared and correlation of levels of production of each cytokine with atopic symptoms sought. The kinetics of mRNA production versus protein accumulation will be compared, since preliminary data indicate that the synthesis of IL-4 precedes that of IL-13 but rates of up or down regulation at the mRNA level are unknown. Subpopulations of basophils secreting each cytokine will be sought or simultaneous or sequential production of IL-4 and/or IL-13 by single cells demonstrated. They will utilize flow cytometry with three color fluorescence, and monensin to inhibit protein secretion. Experiments in which IL-4 or IL-13 are added prior to stimulation will test whether each one can regulate the other. Drugs that inhibit apoptosis in B-cells by preventing NFkB activation will be tested for effects on IL-4 or IL-13 production as well as selected tyrosine kinase inhibitors.
Aim 2 attempts to differentiate PKC isozymes in the regulation of IL-4 and IL-13 since phorbol esters examined thus far inhibit IL-4 synthesis, but seem to stimulate production of IL-13. The effect of depletion of PKC by prolonged PMA stimulation on IL-3 and IgE-stimulated IL-13 secretion will be examined. Calcium dependent versus calcium independent PKC inhibitors will be employed seeking differential effects. RT-PCR will be used for qualitative and semi-quantitative assessment of IL-4 and IL-13 mRNA levels. Finally, a recombinant human HRF will be tested for its ability to stimulate IL-13 secretion in IgE(+) versus IgE(-) cell donors. Direct stimulation and priming of other secretogogues by HRF will be examined. A variety of cytokine primers will be tested with HRF as agonist and IL-4 versus IL-13 secretion compared. The time of priming preincubation will be varied and any inhibitors of secretion sought.
| Schroeder, John T; Chichester, Kristin L; Bieneman, Anja P (2009) Human basophils secrete IL-3: evidence of autocrine priming for phenotypic and functional responses in allergic disease. J Immunol 182:2432-8 |
