Bruton’s Tyrosine Kinase Mediates FcγRIIa/Toll-Like Receptor–4 Receptor Crosstalk in Human Neutrophils

Abstract

Previous observations by our laboratory indicate that the presence of anti-IL-8 autoantibody:IL-8 immune complexes in lung fluids from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) comprises an important prognostic indicator in the development and ultimate outcome of ALI/ARDS. We also showed that these complexes display proinflammatory activity toward neutrophils through the engagement of FcgRIIa receptors. Because sepsis is one of the most common risk factors for ALI/ARDS, the initial goal of our present study involved investigating the effects of LPS on the expression of FcgRIIa receptors in neutrophils. Our results indicate that LPS triggers an increase in the expression of FcgRIIa on the neutrophil surface, which leads to shortening of the molecular distance between FcgRIIa and Toll-like receptor-4 (TLR4). When such neutrophils are stimulated with anti-IL-8:IL-8 complexes, the TLR4 cascade becomes activated via the engagement of FcgRIIa. The underlying molecular mechanism has been subsequently examined and involves Bruton's tyrosine kinase (Btk). In conclusion, our study reveals the existence of Btk-dependent molecular cooperation between FcgRIIa and TLR4 signaling cascades in LPS-"primed" human neutrophils. Furthermore, we used fluorescence lifetime imaging to study the interactions between TLR4 and FcgRIIa in human alveolar neutrophils from patients with ALI/ARDS. The results from these experiments confirm the existence of the molecular cooperation between TLR4 and FcgRIIa.Keywords: neutrophil; FcуRIIa; TLR4; Btk Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterized by diffuse, acute lung injury with a significant increase in both the total number of neutrophils and the proportion of neutrophils within alveolar spaces (1). Although neutrophils constitute only 0.8-3% of nonstructural alveolar cells in normal subjects, they account for 70-80% of the cells in bronchoalveolar (BAL) fluid from patients with ARDS (2, 3). Previous observations by our laboratory indicate that the presence of anti-IL-8 autoantibody:IL-8 immune complexes (IL-8 associated with anti-IL-8 autoantibodies) in lung fluid from patients with ALI/ARDS comprises an important prognostic indicator of the development and ultimate outcome of ALI/ARDS (4-6). In addition, our studies were the first to show that purified anti-IL-8 autoantibody:IL-8 immune complexes display proinflammatory activity toward neutrophils and endothelial cells through the engagement of FcgRIIa (7-9). Further, when we examined lung tissue from patients with lung injury for anti-IL-8 autoantibody: IL-8 immune complexes, we found these complexes in the lungs of patients with ARDS, in association with FcgRIIa (10). Our previous findings also indicate that the expression of FcgRIIa is substantially increased in the lungs of patients with ARDS (9, 10).Sepsis is a major risk factor for the development of ALI/ARDS, and LPS is a likely cause of progression to ALI/ARDS (1). Tolllike receptor-4 (TLR4), a receptor for LPS, ...

Silencing Bruton's tyrosine kinase in alveolar neutrophils protects mice from LPS/immune complex-induced acute lung injury

“…This was in agreement with our previous findings that showed that anti-KC:KC ICs contribute in a significant way to severe lung inflammation in LPS-treated mice (28). Furthermore, Btk was detected in close proximity to the cell membrane in lung neutrophils from the two-hit model of ALI (LPS/IC-induced ALI), which indicated the activation of this kinase (25,48,57).…”

“…Furthermore, our recent study has shown that LPS triggers an increase in expression of Fc␥RIIa on the neutrophil surface, and this leads to augmentation of neutrophil responses to stimulation with anti-IL-8:IL-8 ICs (25). Importantly, we have noted the existence of the molecular cooperation between Fc␥RIIa and TLR4 receptors in alveolar neutrophils from patients with ALI/ARDS (25). To mimic closely the succession of inflammatory events in lungs of patients with ALI/ARDS, we developed a two-hit model of lung injury in which we treat mice first with LPS, then after 8 h with anti-KC:KC ICs (LPS/IC).…”

“…In human neutrophils Btk mediates signaling via TLR4 receptor and G proteincoupled receptor (16,57). Moreover, our recent studies have shown that engagement of Fc␥RIIa receptors by ICs can also trigger Btk activation in these cells (25).…”

“…The inactive form of Btk resides in the cytoplasm. Once activated, Btk typically migrates to the cell membrane (25,48). Btk has been primarily studied in B lymphocytes where the engagement of the B cell receptor leads to its phosphorylation (7,22).…”

“…Our previous studies presented a novel concept in the field of human ALI/ARDS, i.e., that Btk (Bruton's tyrosine kinase)-associated pathways may play an important role in the pathophysiology of ALI/ARDS by influencing local inflammation (25). Btk, a Tec kinase, belongs to a family of nonreceptor intracellular tyrosine kinases (38).…”