Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations

Divalent cations are thought essential for motile function of leukocytes in general, and for the function of critical adhesion molecules in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the effects of 10 mM EDTA on locomotion of human blood polymorphonuclear leukocytes (PMN). In very thin slide preparations, EDTA did not impair either random locomotion or chemotaxis; motile behavior appeared to benefit from the close approximation of slide and coverslip (“chimneying”). In preparations twice as thick, PMN in EDTA first exhibited active deformability with little or no displacement, then rounded up and became motionless. However, on creation of a chemotactic gradient, the same cells were able to orient and make their way to the target, often, however, losing momentarily their purchase on the substrate. In either of these preparations without EDTA, specific antibodies to β2 integrins did not prevent random locomotion or chemotaxis, even when we added antibodies to β1 and αvβ3 integrins and to integrin-associated protein, and none of these antibodies added anything to the effects of EDTA. In the more turbulent environment of even more media, effects of anti-β2 integrins became evident: PMN still could locomote but adhered to substrate largely by their uropods and by uropod-associated filaments. We relate these findings to the reported independence from integrins of PMN in certain experimental and disease states. Moreover, we suggest that PMN locomotion in close quarters is not only integrin-independent, but independent of external divalent cations as well.

The crucial role of polymorphonuclear leukocytes in resistance to Salmonella dublin infections in genetically susceptible and resistant mice

Macrophages are considered to be the mediators of resistance to extra-intestinal Salmonella infections. Nevertheless, the initial cellular response to Salmonella infections consists primarily of polymorphonuclear leukocytes (PMN). To determine whether PMN serve an important function for the infected host, we made mice neutropenic with the rat mAb to RB6–8C5 and infected them i.v. with ≈103 Salmonella dublin or an isogenic derivative that lacks the virulence plasmid (LD842). We infected BALB/c mice, which have a point mutation in the macrophage-expressed gene Nramp1 that makes them susceptible to Salmonella, and BALB/c.D2 congenic mice, which have the wild-type Nramp1 gene that makes them resistant to Salmonella. Both mouse strains were resistant to LD842, and neutropenia made only the BALB/c strain susceptible to this infection. Neutropenic congenic mice, however, were susceptible only to wild-type S. dublin (plasmid+). These results show a complex interplay between plasmid-virulence genes in Salmonella, host macrophages, and PMN. Mice with normal macrophages need PMN to defend against nontyphoid Salmonella that carry a virulence plasmid but not against Salmonella without virulence plasmids. Mice with a mutant Nramp1 gene need PMN to defend against all Salmonella, even those that lack virulence plasmids. These results, plus the evidence that PMN kill Salmonella efficiently in vitro, suggest that Salmonella have adapted to grow inside macrophages where they are relatively sheltered from PMN. The adaptations that allow Salmonella to survive in macrophages do not protect them from PMN.

Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions.

Aspirin [acetylsalicylic acid (ASA)], along with its analgesic-antipyretic uses, is now also being considered for cardiovascular protection and treatments in cancer and human immunodeficiency virus infection. Although many of ASA's pharmacological actions are related to its ability to inhibit prostaglandin and thromboxane biosynthesis, some of its beneficial therapeutic effects are not completely understood. Here, ASA triggered transcellular biosynthesis of a previously unrecognized class of eicosanoids during coincubations of human umbilical vein endothelial cells (HUVEC) and neutrophils [polymorphonuclear leukocytes (PMN)]. These eicosanoids were generated with ASA but not by indomethacin, salicylate, or dexamethasone. Formation was enhanced by cytokines (interleukin 1 beta) that induced the appearance of prostaglandin G/H synthase 2 (PGHS-2) but not 15-lipoxygenase, which initiates their biosynthesis from arachidonic acid in HUVEC. Costimulation of HUVEC/PMN by either thrombin plus the chemotactic peptide fMet-Leu-Phe or phorbol 12-myristate 13-acetate or ionophore A23187 leads to the production of these eicosanoids from endogenous sources. Four of these eicosanoids were also produced when PMN were exposed to 15R-HETE [(15R)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid] and an agonist. Physical methods showed that the class consists of four tetraene-containing products from arachidonic acid that proved to be 15R-epimers of lipoxins. Two of these compounds (III and IV) were potent inhibitors of leukotriene B4-mediated PMN adhesion to HUVEC, with compound IV [(5S,6R,15R)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoi c acid; 15-epilipoxin A4] active in the nanomolar range. These results demonstrate that ASA evokes a unique class of eicosanoids formed by acetylated PGHS-2 and 5-lipoxygenase interactions, which may contribute to the therapeutic impact of this drug. Moreover, they provide an example of a drug's ability to pirate endogenous biosynthetic mechanisms to trigger new mediators.