Tonic inhibition of chemotaxis in human plasma

Abstract

We found exaggerated chemotaxis in plasma treated with EDTA and thought that the EDTA might itself be inhibiting a tonic inhibitor(s) of chemotaxis. Our plasma fractionations suggested that evidence should be sought for a lipid moiety carrying this activity, and on spectrometry (LC-MS-MS together with GC-MS analyses), the biologically active but not the inactive fraction contained oleic and arachidonic acids. Because fatty acids are largely protein bound, we flooded plasma preparations with delipidated albumin, reasoning that it would bind enough fatty acids, including inhibitory ones, to counter their tonic inhibition. Indeed, we observed dramatic increases in chemotaxis. Hence, adding delipidated albumin to plasma has a similar effect to that of adding EDTA--amplification of the chemotactic response. Oleic acid in physiologic concentrations diminishes the magnifying effects of both EDTA and of delipidated albumin, and in fact diminishes the chemotactic response even without the presence of the amplifiers of chemotaxis. In contrast, arachidonic acid amplifies further the effect of EDTA but not of delipidated albumin, and this augmentation appears to be caused by an EDTA-dependent enrichment of the chemotactic gradient with leukotriene B4 (LTB4). We conclude that oleic acid, the blood levels of which vary among individuals, is at least one tonic inhibitor of chemotaxis in plasma.

Fig. 1.

Identification of oleic and arachidonic acid in active fractions. (A) GC/MS selected ion monitoring for m/z 87 = R-CH₂-CH₂-COO-Me, the common ion for PUFA, of active and inactive fractions. Samples were treated with BSTFA to produce Me₃Si derivatives before injection. (B) LC/MS/MS selected ion chromatogram of m/z 302 = M-H shows the presence of arachidonic acid in the active fraction, which co-elutes with authenticated standards.

Fig. 2.

Reversal by oleic acid of the outsized chemotaxis induced by EDTA. Both preparations contain EDTA. (A–C) EDTA control cells, having received only the solvent for oleic acid (EtOH); (D–F) cells that received oleic acid. (A and D) Fields just after central erythrocytes have been destroyed by ruby laser microirradiation, creating a chemotactic gradient. (B and E) 15 min later: with EDTA alone (B), neutrophils continue to arrive; with EDTA + oleic acid (E), maximum aggregation has occurred and cells have begun to disperse (as normal cells do without EDTA). (C and F) After an additional 21.5 min, with EDTA alone (C), neutrophils are still arriving; with EDTA + oleic acid (F), neutrophil locomotion is normal but direction is random. EDTA, 5 mM; oleic acid, 125 μM; EtOH, 0.2%.

Fig. 3.

Amplification by arachidonic acid of the outsized chemotaxis induced by EDTA. All preparations contain EDTA. (A–C) EDTA control cells, having received only the solvent for the fatty acids (EtOH); D has received oleic acid and E arachidonic acid. (A–C) EDTA control at laser flash (A), 7.6 min later (B), and after a total of 27.6 min (C) when the cell aggregate is at its maximum. (D) EDTA + oleic acid, maximum aggregation, 15.7 min after the laser flash. (E) EDTA + arachidonic acid, maximum aggregation, 32.6 min after the laser flash. EDTA, 10 mM; oleic acid, 250 μM; arachidonic acid, 400 μM; EtOH, 0.2%.