Induction of neutrophil respiratory burst by tumour necrosis factor-alpha; priming effect of solid-phase fibronectin and intervention of CD 11b-CD18 integrins

Abstract

SUMMARY Human neutrophils, added to fibronectin (FN)‐coated polystyrene wells and exposed to tumour necrosis factor‐alpba (TNF‐α, were found to exhibit a prolonged production of superoxide anion (Q2) after a lag period of approx 30 min. The O2 production, but not the cell adherence to FN, was completely inhibited by two MoAbs against CD18 and by a MoAb against CD11b, suggesting the involvement of CD11b‐CD18 integrins in the neutrophil oxidative response. When neutrophils were induced to adhere to FN by incubation for 30 min on FN‐coated surfaces and then washed to remove non‐adherent cells, FN‐anchored cells exhibited a rapid onset of O2 production in response to TNF‐α. This suggests that FN primes neutrophils for the TNF‐α‐mediated respiratory burst. The O2− production by adherent neutrophils could be inhibited by anti‐CD11b and anti‐CD18 MoAbs only when the MoAbs were present both during the induction of adherence and during the subsequent exposure of FN‐bound cells to TNF‐α. The incapacity of MoAbs, added to neutrophils during the induction of adherence, to modify the characteristics of the subsequent neutrophil response to TNF‐α suggests that the FN‐mediated cell priming is independent of the interaction of CD11b‐CD18 integrins with the FN substrate. The results are consistent with the intervention of three classes of cell receptors in the TNF‐α‐induced oxidative burst of neutrophils plated on FN: (i) neutrophil FN‐binding sites, distinct from CD11b‐CD18 and responsible for the cell priming; (ii) CD11b‐CD18 integrins, absolutely required for permitting the cell triggering; and (iii) TNF‐α receptors, responsible for switching on a rapid cell response in primed cells. The requirement of multiple classes of receptors for the full expression of the cell function can be envisaged as a natural precautionary measure to control the neutrophil responsiveness to TNF‐α and, in turn, the TNF‐α‐dependent neutrophil‐mediated oxidative injury at sites of inflammation.

Cyclic AMP-elevating agents down-regulate the oxidative burst induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in adherent neutrophils

“…Nevertheless, experiments planned to test this possibility showed that the lag period preceding the onset of O^ production could be abolished by I h incubation of neutrophils on FN bef~ore the addition of GM-CSF. Consequently, as previously shown for tumour necrosis factor (TNF) [10]. the results suggest ihat neutrophil interaction with FN represents a time-consuming cell-priming event by which neulrophils arc converted to a GM-CSF-rcsponsive functional state.…”

“…'Neutrophils Heparinized (heparin lOUml) venous blood was obtained from healthy male volunteers. Neutrophils were isolated by dextran sedimentation and subsequent centritugation on a Ficoll-Hypaque density gradient, as previously described [10]. Contaminating erythrocytes were removed by hypotonic lysis [l(l|.…”

“…neutrophils were washed three times with the incubation medium and resuspended at appropriate concentrations. Final cell suspensions contained ^ 98% neutrophils and >98''/o viable cells, as evaluated by the ethidium bromidefluorescein diacetate test [10].…”

“…Adherence as.say Neutrophil adherence to FN was measured under conditions identical to those used for Oi production assay. Tests were carried out in triplicate, as previously described [10]. After incubation, non-adliercnt cells were removed by washing the plate twice with ()15M NaCI.…”

Priming and de‐priming of neutrophil responses in vitro and in vivo

“…Engagement of integrins during transmigration stimulates “outside‐in” signalling, which together with exposure to endothelial‐bound mediators such as PAF ensures that neutrophils are primed as they egress (Ref; Figure ). The engagement of such receptors can alter neutrophil responsiveness dramatically; for example cells adherent to fibronectin release substantial ROS on exposure to TNF (normally regarded as a priming agent rather than an activating stimulus) and display sequential PI3K and δ‐PKC activation, with δ‐PKC‐dependent phosphorylation of p47 phox . Of note, these effects are agonist specific, as treatment of adherent cells with fMLF resulted in δ‐PKC‐independent ROS generation .…”