Fundamental studies of regularion for endotoxin shock by CD14

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1.Separation of rabbit and rat CD14 cDNA : Rabbit and rat CD14 (rabCD14 and ratCD14) cDNAs were separated and sequenced. 2.Production of recombinant CD14 (rCD14) : Full length and N-terninal side encoding amino acid position from 1 to 71 of mouse CD14 (mCD14) and the latter of rabCD14 and ratCD14 were expressed in E.coil. 3.Preparation of anti-CD14 antibodies : Polyclonal (pAb) and monoclonal (mAb) anti-mCD14 antibodies were prepared. Each mAb detected different epitopes. mAb designated rmC5-3 detected most carboxyterminal epitope of mCD14.4.Functional features of mAbs : rmC5-3 enhanced LPS-induced TNF-alpha production in vitro. mAbs designated rmA4-2, rmF4-3 and rmA12-10, all of which recognized epitopes of middle area of mCD14, slightly inhibited LPS-induced TNF-alpha production in vitro. Because of the low inhibitory effect of mAbs. we are continuing to produce mAbs using the N-terminal protein described above. 5.Effect of pAb and mAbs on LPS-induced TNF-alpha production in vivo : A … More dministration of F (ab') 2 of pAb significantly reduced LPS-induced TNF-alpha production in vivo. However, effect of mAbs were unclear. 6.Analysis of soluble mCD14 (smCD14) : Serum levels of smCD14. TNF-alpha and IL-1beta were analyed by Western blotting using mAbs and ELISA befoce and after administration of LPS.7.Features of expression of mCD14 and cytokines including TNF-alpha, IL-1beta and IL-6 in Kupffer cells (KC) and peritoneal macrophages (Mphi) after LPS stimulation in vivo : mRNA and protein levels of mCD14 and cytokines including TNF-alpha, IL-1beta and IL-6 in Kupffer cells (KC) and peritoneal macrophages (Mphi) after LPS stimulation in vivo TNF-alpha, IL-1beta and IL-6 in Kupffer cells (KC) and peritoneal macrophages (Mphi) after LPS stimulation in vivo were analyzed. Signal for cytokine production could be triggered by small numbers of mCD14.8.Close localization of mCD14 and F_<CY>RII/III and possible involvement of F_<CY>RII/III in LPS triggering. 9.Establishment of mCD14 transgenic mice : Transgenic mice expressing membrane or soluble forms of mCD14 mice designated M14M and M14S respectively were established. M14S mice showed significantly higher levels of smCD14 than M14M and control mice. Levels of TNF-alpha and IL-1beta in M14S mice were significantly lowere than those of M14M and control mice. However, lethality in M14S mice after a high dose of LPS administration was indistinguishable from that in M14M and control mice. 10.Production of mCD14 knock out mice : Production of mCD14 knock out mice is now underway. Less