Dispersive locomotion of human neutrophils in response to a steroid-induced factor from monocytes.

A monocyte-derived factor that stimulates the locomotion of human neutrophils on an albumin-coated glass surface has been prepared from the culture supernatant of dexamethasone-treated human monocytes and called STMS (steroid-treated monocyte supernatant). A modified cell tracking program has been developed and the parameters of locomotion determined by the analysis of Gail and Boone for cells moving in a persistent random walk. Cells moving in uniform concentrations of STMS, interleukin-8 (IL-8) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) chosen to give a sub-maximal speed of locomotion show persistent, random and constrained random diffusion, respectively, with augmented diffusion coefficients of 0.8 +/- 0.1, 0.14 +/- 0.02 and 0.12 +/- 0.03 microns 2 per second for STMS, IL-8 and fMLP, respectively. The augmented diffusion coefficient and the underlying persistence are therefore sensitive quantitative assay parameters for STMS activity and the qualitative characteristics of locomotion allow STMS activity to be distinguished from that of all other factors tested. The contribution of lowered adhesion to locomotion was examined in a novel tilt-assay, which demonstrated that cells in the presence of STMS, but not other factors, moved down slope with significantly increased speed while maintaining contact with the substratum. The results were interpreted in terms of the bipolar form of STMS-treated cells, contrasting with multipolar forms in response to other agents. This together with low adhesiveness plus an inherent tendency of a single locomotor focus to continue motion in its original direction has been used to explain the difference between response to STMS and other factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Addition-order dependent modulation of the sensitivity of rabbit erythrocyte membrane to bee venom phospholipase A2 by oleic acid, lysophosphatidyl choline and albumin.

The addition of exogenous oleic acid to erythrocyte membranes induces a characteristic membrane crenation and sensitises the cells to the lytic action of phospholipase A2 enzymes. Both effects are extremely sensitive to inhibition by endogenous lysophosphatidyl choline (LPC), but the strength of inhibition depends of the order in which the reagents are added to the cells. These responses are further enhanced when the reagents are extracted from the cell membranes by treatment with albumin. Thus the inhibitory action of LPC added before oleic acid increases when the reagents have been extracted but that of LPC added after oleic acid decreases after extraction. The results are discussed in terms of the stimulation of PLA2 activity by enhanced membrane curvature.

Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms.

The mode of inhibition of the phospholipase A2 (PLA2) enzyme from the Chinese cobra (Naja naja atra) by Zn2+ is qualitatively different from inhibition by Ba2+. Inhibition by Ba2+ shows the kinetic characteristics of a conventional competitive inhibitor acting to displace Ca2+ from a single essential site, but Zn2+ has the paradoxical property of being more inhibitory at high than at low Ca2+ concentration. Kinetic analysis of the Ca(2+)-dependence of enzymic activity shows a bimodal response, indicating the presence of two Ca(2+)-binding sites with affinities of 2.7 microM and 125 microM respectively, and we propose that these can be identified with the two Ca(2+)-binding sites revealed by crystallographic analysis [White, Scott, Otwinowski, Gleb and Sigler (1990) Science 250, 1560-1563]. The results are consistent with the model that the enzyme is activated by two Ca2+ ions, one that is essential and can be displaced by Ba2+, and one that modulates the activity by a further 5-10-fold and which can be displaced by Zn2+. An alternative model is also presented in which the modulating Zn(2+)-binding site is a phenomenon of the lipid/water interface.

Effect of lysophosphatidic acid on motility, polarisation and metabolic burst of human neutrophils.

The effect of lysophosphatidic acid (LPA) on human neutrophil activation was examined by a combination of automated tracking assays, cell shape measurements and assays of the metabolic burst by means of 7-dimethylamino-naphthalene-1,2-dicarbonic acid hydrazide (DNDH)-dependent chemiluminescence. LPA powerfully stimulated polarisation and motility. Polarisation became detectable at 2 microM LPA and virtually 100% of cells were polarised at 20 microM LPA. Cell motility increased with the degree of polarisation, and was diminished at high LPA concentration, but this decrease was reversed by albumin. LPA also inhibited the metabolic burst response to both n-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorobol 12-myristate 13-acetate (PMA). Inhibition of the PMA-induced metabolic burst by LPA was not affected by pertussis toxin, showing that the effect was not mediated by the pertussis toxin-sensitive heterotrimeric G protein, and that inhibition of the PMA-stimulated metabolic burst by LPA could result from a direct action of LPA on the small cytosolic GTP-binding proteins. These results indicate that lysophosphatidic acid production by thrombin-activated platelets could play a significant role in the regulation of the inflammatory response.

A factor released by monocytes in the presence of dexamethasone stimulates neutrophil locomotion.

1. Steroid-treated monocyte supernatants cause a dramatic increase in the speed of locomotion of human neutrophils and a significant decrease in their adhesion to protein-coated glass. In contrast, control monocyte supernatants have a smaller effect on the speed of locomotion, but cause a large increase in their adhesiveness. 2. This supernatant activity was produced equally well in the presence or absence of serum after 24 h culture at 37 degrees C with 10(-6) M dexamethasone. 3. The effect of the steroid-treated monocyte supernatants on the speed of locomotion of human peripheral blood neutrophils was not altered by rabbit polyclonal antisera against lipocortins 1-6. 4. Rabbit anti-interleukin-8 antibody which blocked the effect of IL-8 on the speed of locomotion of neutrophils did not antagonize the locomotion stimulating action of steroid-treated monocyte supernatants. 5. The exocellular release of this factor(s) by human mononuclear leucocytes suggests that it may be an in vivo mediator of the anti-inflammatory effect of glucocorticoids.

Rapid activation of the non-toxic basic isoform of phospholipase A2 from Naja mossambica mossambica (spitting cobra) by long-chain fatty acylation.

Purified phospholipase A2 from Naja naja (common Indian cobra) venom, a basic non-toxic isoform and a toxic isoform from Naja mossambica mossambica venom were treated with an equimolar amount of oleoyl imidazolide, a known activator of bee venom phospholipase A2. The ability of the first two enzymes to induce erythrocyte lysis was strongly activated while hydrolysis of dioctanoyl phosphatidyl choline was only weakly activated. The toxic enzyme showed little change in either assay. The susceptible enzyme from Naja mossambica mossambica venom reacted very much more rapidly with oleoyl imidazolide than did the bee venom enzyme and oleic acid was shown to be a relatively weak antagonist. Activation had very little effect on the metal ion dependence of these enzymes. Although activation was a progressive response having the characteristics of a chemical reaction which could not be mimicked by free fatty acids, the adduct did not appear to be stable under acidic conditions. This evidence suggested that primary amino groups were not the target for acylation.

High resolution of honey bee (Apis mellifera) venom peptides by propionic acid/urea polyacrylamide gel electrophoresis after ethanol precipitation.

A new and simple gel electrophoretic method is described which enables the protein and polypeptide components of bee venom to be resolved on a single gel. The electrophoretic method allows octapeptides to be resolved and species as small as decapeptides can be detected at high sensitivity using the Coomassie blue staining method without prior fixation. This has been achieved by replacing acetic acid by propionic acid in acid/urea polyacrylamide gels and by controlling the amount of TEMED catalyst for the polymerisation of high concentration gels in order to obtain a low effective pore size. We demonstrated the value of ethanol precipitation as a rapid and efficient desalting the fractionation technique and propose that it could be used in combination with gel filtration to purify many of the peptides to homogeneity.