Role of cell surface contact in the kinetics of superoxide production by granulocytes

Abstract

The complement-derived anaphylatoxin C5a and a putative analogue of bacterial chemotactic factor (N-formyl-methionyl-leucyl-phenylalanyl [fMLP]), as well as bacterial lipid A, all stimulate human granulocyte (PMN) adhesiveness and superoxide (O2-) production in a concentration-dependent manner. Since attachment of particulate matter to the PMN membrane is an early event in the triggering of respiratory burst of these cells, we further examined how adherence might modulate the release of O2- induced by soluble mediators of inflammation. We found that both the quantity and kinetics of O2- production depend on prior attachment of the cells to a surface. In stirred suspensions of PMN, fMLP induces only a short burst (2.5 min) of O2- release associated with reversible PMN aggregation. The magnitude, but not the time course, of both these responses depend on the fMLP concentration. Unlike the short respiratory response of cells in suspension, PMN allowed to settle onto stationary petri dishes, then overlaid with fMLP, rapidly spread and attach to the surface where they remain and release O2- throughout the 60-min test period. Prolonged O2- release also follows fMLP stimulation in suspensions of PMN pretreated with cytochalasin B, in which case aggregation becomes irreversible during the 20-min burst. If fMLP is slowly infused into a suspension of cells at 37°C or if PMN are challenged at 0°C, and then warmed to 37°C, O2- release greatly decreases or becomes undetectable. Suspended PMN do not respond to a second challenge by the same stimulus regardless of the rate or temperature at which the first stimulus was added, a phenomenon formerly described as desensitization. However, if PMN challenged with fMLP in suspension undergo the short respiratory response and then are later placed in petri dishes, they adhere and resume production of O2- without further stimulation. Chemotactic factor-induced adherence and O2- release of PMN on a surface is entirely independent of either the mode of activation or prior O2- release during preincubation in suspension. Human C5a also promotes PMN adherence and prolonged O2- release in petri dishes. Furthermore, lipid A increases O2- release and adherence of settled PMN, but fails to elicit either response from suspended PMN. These results indicate that cell surface contact plays an essential role in triggering the respiratory burst of PMN activated by soluble stimuli. This long-lasting O2- release by chemotactic factor-stimulated MPN may play a significant role in inflammatory reactions when PMN become adherent in vivo.