BACKGROUND AND OBJECTIVE: Eosinophil is a major inflammatory cell in allergic diseases and parasitic infestations. Various cytokines such as GM-CSF, IL-3 and IL-5 are known to activate eosinophils and prolong their survival. Among them, IL-5 is the most potent stimulator of eosinophil survival. Recently, it was reported that increased expression of Bcl-2 is related to prolonged survival of IL-5 stimulated eosinophil. Theophylline is a useful drug in bronchial asthma, due not only to bronchial dilation but also to its anti-inflammatory effects. It has been suggested that anti inflammatory action of theophylline derives from the reduction of inflammatory cells in the airways which is mechated by stimulat on of apoptosis of inflammatory cells. In this study, we investigated, by measuring Bcl-2 expression of IL-5 stimulated eosinophil, the effect of theophylline on apoptosis as one of the anti-inflammatory action. MATERIAL AND METHOD: Peripheral eosinophils were isolated from atopic patients by using Perco- 11 discontinuous gradient and purified by negative selection technique using MACS. Eosinophil viability and apoptosis were measured by FACscan. Expression of Bcl-2 protein in eosinophils was detected by Western blot and ELISA. RESULTS: IL-5 increased the percentage of viable eosinophils and reduced the apoptosis of eosinophils in a dose dependent manner. The increased survival of IL-5 stimulated eosinophils was reduced by theophylline via activation of apoptosis. Bcl-2 was increased when eosinophils were cultured with IL-5 only, but when theophylline was cocultured, reduced Bcl-2 was seen with Western blot and ELISA. CONCLUSION: IL-5 increases the survival of eosinophil through the enhanced expression of Bcl- 2. Theophylline has counter action against IL-5 via inhibition of Bcl-2 induced by IL-5. Inhibiting the prolongation of eosinophil survival caused by IL-5 might be one possible mechanism of antiinflammatory effects of theophylline.
Apoptosis ; Asthma ; Blotting, Western ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Eosinophils* ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Interleukin-3 ; Interleukin-5 ; Theophylline*

BACKGROUND: Interleukin-5 (IL-5), IL-3, and GM-CSF are known to prolong the survival of eosinophils, and IL-5 has the most potent effect on eosinophil survivaL It is also known that divergent signals induce apoptosis in different cells. But, There have been few reports on about the intracellular signals that trigger the effectors of apoptosis. Cyclic AMP (cAMP) can modulate apoptosis in many cells. But, the role of intracellular cAMP in the IL-5 induced eosinophil survival is still not completely understood. OBJECTIVES: This study was aimed to elucidate the role of intracellular cAMP in IL-5 induced eosinophil survival. MATERIAL AND METHOD: Eosinophils were isolated from peripheral blood of atopic patients. Eosinophil viability was measured by means of propidium iodine (PI) method and the number of viable cells was counted by FAC scan (Becton Dickinson, USA). Cells were cultured with or without IL-5, and also with various cAMP-elevating agents (dibutyryl cAMP, 8-bromo-cAMP, N6- benzoyl cAMP). The concentrations of cAMP were measured by cAMP enzyme immunoassay system(BiotrakTM, Amersham). Finally cAMP dependent protein kinase A inhibitor (H8) was added to eosinophils to examine the effect of decreased intracellular cAMP activity on the viability of eosinophils stimulated with IL-5. RESULTS: The percentage of viable eosinophils was reduced rapidly from 92.1+/-1.8% to 8.23+/- 3.41% without IL-5 (p<0.05; n=ll, 4-day incubation). Upon addition of IL-5, it was increased to 33.02+7.8% (p<0.05; n=ll). In the absence of IL-5, the addition of cAMP-elevating agent increased eosinophil viability in a dose-dependent manner. Upon addition of H8 (24 uM), the eosinophil viability increased by IL-5 (52.5+/-6.4%) was significantly reduced to 27.2+/-5.4% (p<0.05;n=7). Compared with tissue culture media (TCM) only, IL-5 produced persistent elevation of intracellular cAMP of eosinophils in a time and dose dependent manner.
8-Bromo Cyclic Adenosine Monophosphate ; Apoptosis* ; Culture Media ; Cyclic AMP ; Cyclic AMP-Dependent Protein Kinases ; Eosinophils* ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Immunoenzyme Techniques ; Interleukin-3 ; Interleukin-5 ; Iodine ; Propidium