|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥2,100,000 (Direct Cost : ¥2,100,000)
This study aimed at elucidating role of IL-1beta converting enzyme (ICE) and intracellular pH in processing and release of IL-1alpha.
(1) We first examined various types of cells regarding production of IL-1alpha and beta. When human macrophage cell line, THP-1, was stimulated with LPS and nigericin, it produced and secreted a mature form of IL-1beta, but not IL-1alpha. The same was true for murine macrophage cell line, J774.l, although it produced IL-1alpha and beta mRNA in response to LPS.Human bladder carcinoma, HTB9 5637, constitutively produces IL-1alpha and beta precursor, and it turned out that this cell line contains ICE mRNA as assayed by RT-PCR.However, ICE activity was not detected in the cell lysate using in vitro synthesized preIL-lbeta as a substrate. We therefore decided to use human monocytes for elucidating role of ICE in processing and release of IL-lalpha. Human monocytes were first stimulated with LPS, followed by activation of ICE with nigericin treatment. These cells were then stimulated with A23187 to induce processing and release of IL-1alpha ICE inhibitor peptide, however, didn't inhibit processing and release of IL-1alpha, suggesting that ICE does not regulate processing and release of IL-1alpha directly.
(2) When intracellular pH was measured by BCECF, A23187 didn't change the pH in HTB9 5637 cells. We then determined the change of intracellular pH in these cells upon treatment with ammonium chloride and sodium acetate. Both reagents decreased intracellular pH till 5 min, but the pH returned back to the normal level after 30 min. When processing and release of IL-1alpha was examined under this condition, processing was accelerated at 5 min but not other time points. On the other hand, a mature form of IL-1alpha was secreted similarly. Although decrease of intracellular pH was transient, this finding does not support the possibility that intracellular acidification is required for secretion of a mature form of IL-1alpha.