Structural and physicochemical requirements of endotoxins for the activation of arachidonic acid metabolism in mouse peritoneal macrophages in vitro

Abstract

Lipopolysaccharides of different wild-type and mutant gram-negative bacteria, as well as synthetic and bacterial free lipid A, were studied for their ability to activate arachidonic acid metabolism in mouse peritoneal macrophages in vitro. It was found that lipopolysaccharides of deep-rough mutants of Saln~onellu minnesotu and Escherirhia coli (Re to Rc chemotypes) stimulated macrophages to release significant amounts of leukotriene C4 (LTC,) and prostaglandin E2 (PGE,). Lipopolysaccharides of wild-type strains (S. abortus equi, S.friedenau) only induced PGE2 and not LTC, formation. Unexpectedly, free bacterial and synthetic E. coli lipid A were only weak inducers of LTC, and PGE2 production. Deacylated Re-mutant lipopolysaccharide preparations were inactive. However, co-incubation of macrophages with both deacylated lipopolysaccharide and lipid A lead to the release of significant amounts of LTC, and PGE2, similar to those obtained with Re-mutant lipopolysaccharide. The significance of the lipid A portion of lipopolysaccharide for the induction of LTC4 was indicated by demonstrating that peritoneal macrophages of endotoxin-low-responder mice or of mice rendered tolerant to endotoxin did not respond with the release of arachidonic acid metabolites on stimulation with Re-mutant lipopolysaccharide and that polymyxin B prevented the Re-lipopolysaccharide-induced LTC, and PGE2 release. Physical measurements showed that the phase-transition temperatures of both free lipid A and S-form lipopolysaccharide were above 37 "C while those of R-mutant lipopolysaccharides were significantly lower (30 -35 "C). Thus, with the materials investigated, an inverse relationship between the phase-transition temperature and the capacity to elicit LTC4 production was revealed.In a previous paper we demonstrated that, under specified conditions, lipopolysaccharides of a Salmonella minnesota Re mutant (strain R595) are capable of inducing, in cultured mouse peritoneal macrophages, leukotriene C4 (LTC,) formation and release in a dose-and time-dependent manner [l]. Chemically, this lipopolysaccharide consists of lipid A carrying an a(2,4)-linked 3-deoxy-~-manno-octu~osonic acid (dOclA) disaccharide [2-41. It has recently been proven that the lipid A component represents the endotoxic principle of lipopolysaccharides and that it elicits many of the biological effects seen with endotoxins [5]. On the other hand, a number of studies indicate that also the polysaccharide portion possesses intrinsic biological activity or that it may modulate lipid A activity [6, 71. I t was, therefore, of interest to investigate the significance of the lipid A and the polysaccharide region in the lipopolysaccharide-induced formation of LTC4 in phagocytes.The present paper describes experiments involving lipopolysaccharides from wild-type bacteria (S-form lipopolysaccharides), rough mutants (R-form lipopolysaccharides) and free bacterial, as well as synthetic lipid A (preparation 506). Our results suggest that the physicochemical state of Abbreviations. P,/NaC...

Macrophages cultured in vitro release leukotriene B4 and neutrophil attractant/activation protein (interleukin 8) sequentially in response to stimulation with lipopolysaccharide and zymosan.

“…The above data differ from those of Brown and colleagues (34) who determined that normal human AM release only cyclooxygenase products after challenge with endotoxin. Luderitz and co-workers (35) have established that different LPS vary in their capacity to effect leukotriene release from mouse peritoneal macrophages. Thus, the difference between the findings of Brown and co-workers and those of our study possibly may be explained in part, by the use of a different serotype of LPS and/or by other undefined conditions.…”

Investigation into the fluidity of lipopolysaccharide and free lipid A membrane systems by Fourier‐transform infrared spectroscopy and differential scanning calorimetry

“…We have described in an earlier paper a correlation between the /3 ++ ci phase-transition temperatures of wild-type and various rough mutant lipopolysaccharide and of free lipid A from S. minnesota with their ability to induce the release of leukotriene C4 from mouse peritoneal macrophages [25]. We showed that this ability is highest for lipopolysaccharide Re (lowest t,) and lowest for free lipid A (highest t,) and decreases with increasing completeness of the sugar moiety (tc increasing with respect to Re lipopolysaccharide).…”

Chemical structure of the carbohydrate backbone of Vibrio parahaemolyticus serotype 012 lipopolysaccharide

“…In view of the known significance of the polysaccharide portion for serological specificity [ l l ] and of the inner core region for the modulation of lipid A toxicity [12,131, the activation of complement [14], and the binding of 28-kDa serum factor [15] it seemed rewarding to analyze its primary chemical structure.…”