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Am. J. Respir. Cell Mol. Biol., Vol 10, No. 6, Jun 1994, 651-657.

Leukotriene B4 stimulates human polymorphonuclear leukocytes to synthesize and release interleukin-8 in vitro

RW McCain, EP Holden, TR Blackwell and JW Christman
Department of Veterans Affairs, Nashville, Tennessee.

Interleukin-8 (IL-8) is a potent chemotactic protein for polymorphonuclear leukocytes (PMN). Here we examine whether PMN synthesize and release IL-8 in response to stimulation by leukotriene B4 (LTB4). PMN isolated from normal heparinized peripheral human blood were incubated in RPMI culture medium at 37 degrees C in 5% CO2, with and without LTB4. The culture supernatants were tested for IL-8 bioactivity through chemotactic activity measurements with and without neutralizing anti-IL-8 serum. Immunoreactive IL-8 was quantified by ELISA, and de novo IL-8 synthesis was evaluated by metabolic labeling with [35S]cysteine followed by immunoprecipitation. LTB4 stimulated PMN to produce IL-8 in a dose- and time-dependent manner. The IL-8 concentrations reached maximal levels after 16 h of incubation with LTB4. Significant increases in IL-8 production occurred with LTB4 doses of 10 to 1,000 nM/ml. Immunoprecipitation of labeled IL-8 documented new synthesis of IL-8 by LTB4-treated PMN. Northern blot analysis of total RNA from PMN using a 30 mer oligonucleotide for IL-8 demonstrated increased mRNA expression in LTB4-stimulated PMN compared with untreated PMN. These data show that peripheral blood PMN can be stimulated by LTB4 to synthesize and secrete biologically active IL-8. PMN and other cells capable of producing LTB4 may induce IL-8 protein production by inflammatory PMN and thereby amplify or perpetuate the acute inflammatory response by recruiting additional PMN into an inflammatory site.


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