Mutagenesis studies of interleukin-8. Identification of a second epitope involved in receptor binding.

Interleukin-8 (IL-8) is a dimeric, C-X-C chemokine, produced by a variety of cells and which elicits proinflammatory responses from the neutrophil. As a prelude to drug design, we have investigated the interactions between IL-8 and its receptor by preparing a number of single-site mutants of IL-8 and determining their activity in receptor-binding and functional assays. In order to define the binding surface as precisely as possible, we have used chemical shifts obtained from nuclear magnetic resonance spectroscopy to screen mutant proteins for structural changes which affect regions of the IL-8 surface remote from the site of mutation. In addition to a previously recognized sequence, Glu4-Leu5-Arg6 in the N-terminal peptide, we have identified a second epitope comprising a contiguous group of non-sequential, solvent-exposed, hydrophobic residues, Phe17, Phe2l, Ile22, and Leu43. These two receptor-binding regions are separated by over 20 A in the IL-8 structure and are important both for receptor binding and function. In addition, we have shown through the production of a covalently linked IL-8 dimer, that subunit dissociation is not necessary for biological activity.

Enhanced adhesion of polymorphonuclear leukocytes to anoxic cultured vascular endothelium.

This study showed that the adherence of human polymorphonuclear leukocytes (PMN) to monolayer cultures of human umbilical vein endothelial cells (HUVEC) was increased when the latter were rendered anoxic. This adhesion was greater after 4-5 h than after 8 h of anoxia, but even at 8 h was significantly above the level of adhesion to HUVEC maintained under normoxic conditions for the same period. The changes in adhesion were not dependent on the viability of HUVEC during anoxia. Anoxia-induced adhesion was prevented by addition of cycloheximide (5 micrograms/ml) to the cultures, suggesting that it depended on HUVEC protein synthesis. Enhanced adhesion was also prevented by addition of a monoclonal antibody directed against the cytokine, interleukin-1 alpha (IL-1 alpha). These findings are consistent with a role of endogenous IL-1 alpha as a mediator of the anoxia-induced adhesion of PMN to HUVEC.

Prostaglandin E1: a review.

Work on the structure of prostaglandin E1 (PGE1), isolated from natural sources, was completed 25 years ago (1). Shortly after, methods for the chemical synthesis of PG with their natural configuration were developed in the laboratories of the UpJohn Company (2) and of E. J. Corey (3) and, by the late sixties, PGE1 became widely available. The information since accumulated about its biological and clinical effects is more substantial than for any other PG. This review will draw together some of this information, focusing on recent studies of its mechanisms of action.

The effect of dihomogammalinolenic acid on platelet aggregation and prostaglandin release, erythrocyte membrane fatty acids and serum lipids: evidence for defects in PGE1 synthesis and delta 5-desaturase activity in insulin-dependent diabetics.

The effect of dihomogammalinolenic acid (DHLA) administration on platelet aggregation and prostaglandin production, erythrocyte fatty acid composition and serum lipids was compared in healthy subjects and insulin-dependent diabetics (IDDs). In healthy subjects, DHLA caused a significant inhibition of ADP-induced platelet aggregation and an increase in platelet PGE1 release; IDDs did not show these changes. There were no differences, however, in platelet thromboxane A2 (TXA2) or PGE2 release between healthy subjects and IDDs before and after DHLA. Following DHLA, the arachidonic acid content of erythrocytes increased in healthy subjects; this increase was not observed in IDDs. DHLA induced a significant fall in serum non-esterified fatty acid concentrations in both groups without altering either cholesterol or triglyceride concentrations. These data show for the first time that IDD platelets may have a specific defect of PGE1 synthesis quite distinct from the delta 5- and delta 6-desaturase defects known to be associated with experimental diabetes; this defect may contribute to platelet hyper-aggregability in diabetes; and DHLA has a potent antilipolytic effect in vivo; and erythrocytes from IDDs may have a delta 6-desaturase defect.

Dihomo-gamma-linolenic acid reverses hypertension induced in rats by diets rich in saturated fat.

This study has shown that hypertension induced in rats by a diet rich in saturated fat (16% coconut oil, 4% palmitic acid by weight) is reversed by the addition of the essential fatty acid, dihomo-gamma-linolenic acid (DHLA), at 5.0% but not at 0.5% of dietary energy. This potent effect of DHLA has been attributed to modulation of prostaglandin biosynthesis.

The metabolism of dihomo-gamma-linolenic acid in man.

Orally administered dihomo-gamma-linolenic acid (DHLA) is well absorbed in man; it appears in blood after ca. 4 hr first as triglyceride ester and later as phospholipid. After sustained-dosing, DHLA penetrated membrane pools and all phospholipid components but, depending on the dosage, reached a metabolic equilibrium in 4-16 days. Intact platelets do not accumulate arachidonate following DHLA administration, and species differences occur in the capacity of animals to metabolize DHLA to arachidonic acid (AA). The rat appears to be unusual in having a very active hepatic delta5-desaturase enzyme system. Potentially antithrombotic changes in platelet function which followed the administration of DHLA to man were accompanied by a significant increase in the capacity of platelets to synthesize PGE1. Concomitant increases in PGE2 synthesis do not apparently result from an increased production of AA and suggest that DHLA, or a DHLA metabolite, interferes with the metabolism of AA. Effects on thromboxane and prostacyclin synthesis are being studied.