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Recent work suggests that various neutrophil agonists affect the rate of apoptosis in these cells. On the basis of these observations, we hypothesized that signals triggered in neutrophils via their adhesion receptors might also modify their life span. This hypothesis has been tested using human neutrophils adherent to tissue culture plastic, either untreated or coated with extracellular matrix (ECM) proteins or with monolayers of human umbilical vein endothelial cells. To detect and quantitate apoptotic changes in adherent cells, we developed a microtiter plate assay using a cell-permeable DNA-binding fluorescent dye, Hoechst 33342. Use of this assay demonstrated that 1) the number of apoptotic cells among neutrophils adherent to plastic after 6-20 h of incubation was significantly lower than that among neutrophils adherent to the ECM proteins fibronectin or laminin; 2) adhesion to interleukin-1-activated endothelial cells delayed apoptosis, whereas adhesion to nonactivated endothelium accelerated neutrophil death; and 3) monoclonal antibodies directed against intercellular adhesion molecule 1 or against the common beta 2-chain of the leukocyte integrins abolished the protective effect of interleukin-1-activated endothelial cells on apoptosis of adherent neutrophils. These results suggest that the life span of adherent neutrophils. depends on the activating signals triggered by the surface of adhesion.

A simple, rapid, and quantitative method for estimation of the degree of spreading of fluorescently labeled cells adherent to an artificial or biological surface has been designed, based on the principle that the medium between adherent cells and the surface of adhesion absorbs fluorescent light emitted by the adherent cells. Using the Beer-Lambert equation for extinction coefficient of monochromatic light, we have calculated the factor by which fluorescence signal actually emitted from cells adherent to the bottom of the microtiter well would differ from the fluorescence of these cells detected by a bottom-scanning plate reader. The physical nature of cell-cell ligand-receptor bridges depends on many parameters, such as the type of the receptor(s), the intensity and direction of a stress force, blockade of the receptors with monoclonal antibodies (MAb), and transfer of adhesion mediation to different ligand-receptor pairs. When employed in various adhesion models, this new technique has demonstrated quantitative changes of cellular spreading of human neutrophils adherent to different extracellular matrix protein-coated surfaces and to endothelial cells after neutrophil/endothelial cell activation or in the presence of MAb directed against neutrophil integrins. These measurements of spreading of adherent cells appear more sensitive than visual observation of cellular morphology on biological surfaces or cells.

Extravasation of leukocytes at the sites of ischemia-reperfusion is thought to exacerbate the tissue injury. It has been proposed that leukocyte accumulation is a secondary effect of the ischemic damage, mediated by inflammatory cytokines. We have recently demonstrated that physiologically low levels of oxygen tension alone can have a direct effect on the adhesive characteristics of mesenchymal cells for lymphocytes. We now report that decrease of oxygen tension in the environment induces the adhesion of neutrophils to human endothelial cells in culture. Adhesion of human neutrophils to human umbilical vein, bovine aortic, and mouse microvascular endothelial cell monolayers, which had been incubated at pO2 of 50 torr for 3 hours, increased 2.5-fold, 2-, and 1.5-fold, respectively. The effects of decreased oxygen concentration on adhesion were not mediated by a soluble factor elaborated by the hypoxic cells. Low oxygen tension upregulates a saturable, endothelial cell-associated adhesion mechanism, capable of withstanding centrifugation forces greater than 160g. Hypoxia-induced adhesion was inhibited by LFA-1-specific (CD11a/CD18 integrin) antibodies, but not by antibodies directed against the ICAM-1 ligand for the LFA-1 receptor. These studies demonstrate that decreases in oxygen tension alone increase the adhesive properties of endothelial cells for leukocytes. In addition, they provide evidence for the existence of a new ligand for the LFA-1 molecule on endothelial cells which can be affected by hypoxic environments.

Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.

The mechanism by which unstimulated human neutrophils initiate a respiratory burst on adherence to a surface has been examined. When neutrophils adhere to a plastic surface, they immediately generate a sustained burst of superoxide (O2-). However, this respiratory burst is not initiated by adherence alone, since neutrophils attached to fibronectin fail to mount a response. Adhesion to plastic is calcium (Ca2+) independent, but O2- production requires Ca2(+)-containing buffer in the initiation phase, that is, during adhesion and the early phase of O2- production. The Ca2(+)-dependent step was shown to involve protein kinase C (PK-C) in that the O2- production, but not adherence, was blocked with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and PK-C was found to translocate from the cytosol to the membrane on adhesion. Furthermore, it may be inferred that this translocation results in the generation of a Ca2+ independent form of PK-C, PK-M, since leupeptin, which inhibits the generation of PK-M, also blocked O2- production. This finding was corroborated by showing that after 5 minutes in a Ca2(+)-containing buffer, enough time to initiate O2- production and PK-C translocation, Ca2+ is no longer required for sustained O2- release. These results, in aggregate, demonstrate that neutrophils are activated by adhesion to plastic to generate O2-, a PK-C-dependent process that appears to involve a Ca2(+)-independent form of the kinase, PK-M. Why adherent neutrophils generate a respiratory burst on plastic and not fibronectin surfaces probably reflects activation of distinct receptors, whose nature must still be defined. Another issue to address is the priming effect of adhesion, since cells adherent to plastic- or fibronectin-coated surfaces have an enhanced O2- response to formylmethionyl-leucine-phenylalanine (FMLP) compared with neutrophils stimulated in suspension. This may relate to increased Ca2+ mobilization, an important mediator of priming for FMLP responses. Thus, adhesion as a priming event does not necessarily initiate cell effector function, and the further elucidation of the plastic and fibronectin models suggests a means of characterizing the crucial event that control neutrophil activation.