The role of heat shock proteins (HSP) during the inflammatory response has been controversial. The effect of heat shock (HS) on the synthesis of the monokines tumor necrosis factor (TNF) and interleukin 1 (IL-1) by endotoxin-stimulated thioglycollate-elicited peritoneal macrophages was investigated. HS was deemed to have affected macrophages if a 70 kD HSP appeared on SDS gels; identity of this protein as the highly conserved HSP70 was then confirmed by immunoprecipitation. Maximal increases in HSP70 were apparent 2-5 h after HS at 45 degrees C for 12 min. Synthesis of HSP70 was no longer detected 24 h post HS. A reciprocal relationship between HSP70 and TNF was apparent in kinetic studies. TNF was not detected in culture supernatants if macrophages were endotoxin-stimulated 2 to 6 h after HS; however, the same stimulation 24 h later induced significant TNF secretion. RNA analysis of HS and non-HS macrophage cultures demonstrated a 60-fold reduction in TNF message in the HS macrophages 1 h after endotoxin stimulation. TNF mRNA levels remained depressed at 6 h while the HSP70 message had increased 30-fold. The ability of HS macrophages to ingest antibody-coated erythrocytes was not significantly affected following heat treatment. Macrophage response to HS can be said to inhibit transcription of inducible monokines while retaining other macrophage functions.
Thioglycollate-elicited murine peritoneal macrophages produce significant quantities of TNF 2 to 4 h after induction with bacterial endotoxin, LPS. However, macrophages exposed to a second LPS stimulus are refractory and the amount of TNF detected in these supernatants is reduced 10- to 50-fold. The acquisition of the refractory state in vitro or in vivo requires the continued presence of LPS for a minimum of 6 to 8 h, is optimal by 20 h, and is reversible. Refractory macrophages incubated for an additional 48 h in the absence of LPS produce significant quantities of TNF after reexposure to endotoxin. Although LPS refractory macrophages do not release TNF in response to a secondary endotoxin challenge, riboprobe ribonuclease protection assays demonstrated amplification of TNF message, suggesting that post-transcriptional events are involved in the regulation of TNF production in endotoxin refractory macrophages. Immunoprecipitation studies revealed the accumulation of the 26-kDa TNF precursor in lysates of refractory macrophages, thus demonstrating a post-translational regulatory process. Although LPS refractory macrophages do not release TNF in response to a second LPS stimulus, ingestion of zymosan by these cells results in the release of significant quantities of TNF. Furthermore, LPS-refractory macrophages do not demonstrate a reduction in other effector functions including Fc-mediated erythrophagocytosis. Therefore, the LPS refractory state is a metabolically dependent post-translational regulatory process, which requires continuous LPS exposure, is specific in which macrophage effector functions are inhibited, and is reversible with further incubation or by non-LPS-related stimuli.
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