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Tumor necrosis factor-alpha and FMLP receptors are functionally linked during FMLP-stimulated activation of adherent
K J Balazovich1, S J Suchard, D G Remick
1Department of Pediatrics, University of Michigan School of Medicine, Ann Arbor, MIC, USA.
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.
Insights
Tumor necrosis factor-alpha (TNF alpha) is crucial for the activation of human neutrophils adherent to fibrinogen. Blocking TNF alpha or its receptors significantly inhibits the release of hydrogen peroxide (H2O2) and lactoferrin, key inflammatory mediators.
Area of Science:
- Immunology
- Cell Biology
- Inflammation Research
Background:
- Human peripheral blood neutrophils (PMN) play a key role in the innate immune response.
- Neutrophil activation involves the release of specific granule components and reactive oxygen species.
- Tumor necrosis factor-alpha (TNF alpha) is a pro-inflammatory cytokine with complex roles in neutrophil function.
Purpose of the Study:
- To investigate the role of TNF alpha in the activation of adherent human neutrophils.
- To determine if TNF alpha mediates the release of hydrogen peroxide (H2O2) and lactoferrin from FMLP-stimulated PMN.
- To elucidate the involvement of TNF alpha receptors in this process.
Main Methods:
- Adherent human PMN were stimulated with FMLP (formyl-methionyl-leucyl-phenylalanine).
- TNF alpha release was measured using ELISA.
- The effects of anti-TNF alpha antibodies and receptor-blocking antibodies on H2O2 and lactoferrin release were assessed.
- The role of protein synthesis was examined using actinomycin D and cycloheximide.
Main Results:
- FMLP-stimulated adherent PMN released TNF alpha, H2O2, and lactoferrin in parallel kinetics.
- Neutralizing anti-TNF alpha antibodies significantly inhibited FMLP-induced H2O2 and lactoferrin release.
- Antibodies targeting both TNF alpha receptors (p55 and p75) were required to fully block FMLP-mediated activation.
- Inhibition of protein synthesis partially reduced mediator release, which was overcome by exogenous TNF alpha.
Conclusions:
- TNF alpha production and subsequent receptor ligation are central events in the FMLP-mediated activation of fibrinogen-adherent human neutrophils.
- Both p55 and p75 TNF alpha receptors are involved in this activation pathway.
- These findings highlight a critical role for TNF alpha in neutrophil inflammatory responses.