| Systemic inflammation response syndrome (SIRS) and its complications are the main cause of mortality in intensive care units (ICU) patients. However, the mechanism of SIRS is still not well understood. It is generally recognized as an uncontrolled inflammation caused by severe insults and shock etc. The main effector cells such as monocytes and macrophages are primed and then activated; produce cytokines such as TNF-α, IL-1, IL-6, enzymes, superoxide anion and nitric oxide that may cause tissue injury. Bacterial endotoxin (LPS) plays a key role in the development of SIRS. LPS can also induce, hypo-responsiveness (tolerance) of macrophages. This phenomenon has been demonstrated in man and experimental animals, and provides an active protection from tissue injury but may decrease host defense abilities.There were contrary reports about the responsiveness of macrophages in SIRS. Both priming and tolerance related to the severities of the disease. The doses or time of LPS pretreatment on priming and tolerance induction of macrophage is similar. So we hypothesize that LPS can induce both tolerant and priming state simultaneously in a macrophage (referred to as "co-existence"). It has been reported that LPS pretreatment induces decreased TNF- and increased IL-1 production to second LPS challenge, but the studies were performed with murine peritoneal macrophages and cannot exclude the possibility of hyper- and hypo-responsiveness being performed by different macrophage subgroups separately. Macrophage cell lines (RAW264.7, J774A.1 and P388D1), which can stand for single cell level, was used to built a cell model in that LPS pretreatment may induce hyper- and hypo-responsiveness simultaneously in a macrophage by measuring many effectors. Cell lines were precultured with or without 10ng/ml LPS for 1-24h, then challenged with containing LPS, MDP, Zymosan (Zyms), PAF, FMLP or PMA for 24h. TNF-a, IL-1, IL-6, NO, PLA2 and O2 in supernatantswere measured. The results showed that LPS pretreatment inhibited TNF-a and NO production, but increased IL-1, PLA2 and O2 release from LPS challenge. LPS pretreatment also alters macrophage responsiveness to the other stimuli, some effectors increased, the others decreased. CONCLUTIONS: LPS can induce hyper- and hypo-responsiveness simultaneously in one macrophage. Determination of the macrophage responsive state depends on different stimuli and effectors, which were measured.One of the mechanisms of LPS induced "co-existing" state may be caused by the dissociate changes of PKC and [Ca2+]i, that is, LPS pretreatment increases resting [Ca2+]i but decrease PKC activities which results LPS priming and tolerant effects separately. Resting [Ca2+]i of RAW264.7 after LPS pretreatment was measured. The results showed that [Ca2+]i elevation is dose- and time- dependent on LPS in a certain extent, and LPS priming effect on O2 release is also dose- and time- dependent on LPS. Pretreatment of the cell with calcium inophore A23187 can mimic the LPS priming effects in O2 production. Pretreatment of the cell with Ca2+ chelator BAPTA and EGTA can block this priming effect. The results indicated that LPS priming effect on O2 production is depend on the elevation of resting [Ca2-H|i.As a PKC activator, PMA can either activates PKC by Ih exposure or depletion PKC by prolonged (18h) exposure. Our results showed that pretreatment of PMA (18h) can mimic LPS pretreatment on decreased TNF-a production to LPS challenge; PKC activation with PMA for Ih between LPS pretreatment and LPS challenge can restore the TNF-a production partially. The results of western blot for PKC isoforms and activities showed LPS pretreatment for 18h decreased PKC-a expression in three cell lines, and PKC activities are down-regulated when challenged with LPS again. As PKC defective cell line, P388D1 produce less TNF-a and NO, which are related to PKC than other two cell lines, but the production of PLA2 and O2 is similar to them when treating with LPS. All the results suggested that the down-regulated PKC is related t...
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