HEAT-SHOCK IN HUMAN NEUTROPHILS - SUPEROXIDE GENERATION IS INHIBITED BY A MECHANISM DISTINCT FROM HEAT-DENATURATION OF NADPH OXIDASE AND ISPROTECTED BY HEAT-SHOCK PROTEINS IN THERMOTOLERANT CELLS

Independently of the stimulating agent used, generation of O2- by huma n neutrophils is transiently inhibited when the cells have been expose d to elevated temperatures. This phenomenon is concomitant with the sy nthesis of heat shock proteins (HSPs). We have investigated a possible relationship between HSPs and modulation of NADPH oxidase activity in human neutrophils exposed to heat. HSPs were not involved in the inhi bition of O2- generation since 1) in enucleated cytoplasts, which are unable to synthesize proteins, the generation of O2- was inhibited aft er exposure to 43-degrees-C, 2) using actinomycin D (Act D) in intact cells, it was possible selectively to inhibit the synthesis of HSPs wi thout modifying the inhibition of NADPH oxidase activity that followed HS. Furthermore, the recovery of NADPH oxidase activity was not under the control of HSPs because the enzyme recovered as well in Act D-tre ated neutrophils. The NADPH oxidase activity was reconstituted in a ce ll-free assay by combining the cytosol with the plasma membrane-enrich ed fraction in the presence of arachidonic acid (AA) and NADPH. Subcel lular fractions obtained from control or heated neutrophils exhibited similar oxidase activities suggesting that heat exposure did not induc e denaturation of the oxidase components but rather altered the mechan isms of translocation and/or assembly of these components with the pla sma membrane. This hypothesis was supported by the inhibition of the g ranule release in heated cells, a process which also requires transloc ation and association fusion with the plasma membrane. On the other ha nd, preexposure of neutrophils to HS prevented the inhibition of O2-ge neration during a second challenging HS. This acquired thermotolerance was abolished when the synthesis of HSPs was inhibited during the fir st HS with Act D, indicating a direct relationship between protection of O2-generation and synthesis of HSP. Here we demonstrate that synthe sis of HSPs and inhibition or recovery of NADPH oxidase activity are c oncomitant but unrelated phenomena. In contrast, accumulation of HSPs in thermotolerant neutrophils appeared to play an important role in th e prevention of NADPH oxidase inhibition. These results provide furthe r insights into the behavior of human neutrophils and NADPH oxidase up on heat injury. (C) 1993 Wiley-Liss, Inc.