Tumor Necrosis Factor-α and FMLP Receptors Are Functionally Linked During FMLP-Stimulated Activation of Adherent Human Neutrophils

Abstract

Human peripheral blood neutrophils (PMN) plated onto fibrinogen and activated with FMLP release H2O2 and lactoferrin, a specific granule component, with parallel kinetics. Although tumor necrosis factor-alpha (TNF alpha) only primes PMN in suspension, it is a potent agonist of adherent PMN. Activation of adherent PMN by FMLP (10(-7) mol/L) stimulated detectable release of TNF alpha within 45 minutes of stimulation, with maximal release (45.5 pg/10(6) cells) detected by 90 minutes. TNF alpha release paralleled the release of both lactoferrin and H2O2. To determine if TNF alpha plays a role in H2O2 and lactoferrin release, we investigated the effect of anti-TNF alpha antibodies on FMLP-stimulated activation of adherent PMN. A neutralizing rabbit anti-TNF alpha antibody inhibited both H2O2 and lactoferrin release stimulated by FMLP, whereas rabbit lgG, anti-HLA-A,B,C, anti-CD 14, and anti-interleukin-8 antibodies were without effect. The simultaneous addition of TNF alpha (1,000 U/mL) with anti-TNF alpha antibody reversed the inhibition seen with anti-TNF alpha alone. Furthermore, treatment of PMN with either actinomycin D or cylcoheximide resulted in partial (33%) inhibition of H2O2 and lactoferrin release, suggesting that protein synthesis is required for FMLP-mediated activation of adherent PMN. The addition of TNF alpha to either cycloheximide or of actinomycin D-treated PMN overcame the inhibition, indicating that the effect was specific for TNF alpha. The addition of antibodies against either the 55-or 75-kD TNF alpha receptors (referred to as p55 and p75, respectively) resulted in partial (32%) inhibition of FMLP-mediated activation of H2O2 and lactoferrin release, whereas a combination of both antibodies reduced their release to control levels. These data indicate that both p55 and p75 are involved in FMLP activation of adherent PMN. Taken together, these findings indicate that the production of TNF alpha and ligation of TNF alpha receptors are central to FMLP activation of PMN adherent to fibrinogen.