Lipopolysaccharide binding protein and CD14 interaction induces tumor necrosis factor-α generation and neutrophil sequestration in lungs after intratracheal endotoxin

Abstract

It has been proposed that lipopolysaccharide (LPS) bound to the 60-kD LPS binding protein (LBP) forms an LPS/LBP complex that, in turn, binds to the CD14 receptor on monocytes/macrophages and stimulates the release of cytokines. We examined the role of LBP and CD14 in tumor necrosis factor-α (TNF-α) production and neutrophil (polymorphonuclear leukocyte [PMN]) sequestration in lungs induced by intratracheal instillation of LPS using rabbit lungs perfused at constant flow with lactated Ringer-albumin solution. LPS alone (Salmonella Minnesota, wild type; 20 ng) or in the presence of LBP (500 ng) was injected intratracheally. In some experiments, human PMNs (5×107) were added to the perfusate after a 2-hour period of perfusion. Samples of lung perfusate were collected every 30 minutes for 180 minutes when bronchoalveolar lavage was also performed. TNF-α concentrations in the perfusate and bronchoalveolar lavage fluid were determined by use of a bioassay with L-929 fibroblasts, and PMN accumulation in lungs was determined by myeloperoxidase assay of lung homogenates. LPS alone did not significantly increase TNF-α production or lung PMN accumulation, whereas the LPS/LBP complex increased TNF-α concentration in perfusate twofold and PMN accumulation twofold compared with the effect of LPS alone. Intratracheal instillation of anti-CD14 monoclonal antibody MY4 (40 μg) with the LPS/LBP complex prevented TNF-α release and PMN sequestration, whereas an isotype-matched control monoclonal antibody was ineffective. Therefore, LBP in the airspace enhances the LPS effect on TNF-α production via a CD14-dependent pathway, and as a result, CD14 activation can contribute to lung PMN sequestration. Airspace accumulation of LBP secondary to increased vascular and airway epithelial injury may play a critical role in development of acute lung injury by promoting TNF-α production via a CD14-dependent mechanism.