Anti–IL-8 autoantibody:IL-8 immune complexes are present in 55–70% of normal human plasmas (1
). We have reported that lung fluids of patients with ALI/ARDS contain high concentrations of these complexes, and our previous studies indicate that anti–IL-8 autoantibody:IL-8 immune complexes could contribute to pathogenesis of ALI/ARDS (1
). Importantly, our group discovered that anti–IL-8 autoantibody:IL-8 immune complexes are deposited in the lungs of patients with ARDS via FcγRIIa (6
). Moreover, anti–IL-8 autoantibody:IL-8 immune complexes purified from normal human plasma or from edema fluids from patients with ALI, and finally formed between recombinant IL-8 and monoclonal anti–IL-8 antibody, trigger chemotaxis of human blood neutrophils, induce neutrophil activation, and modulate survival of these cells (4
). Significantly, activity of these complexes is mediated by IgG receptors—specifically, FcγRIIa (4
There is limited information regarding the functional effects of soluble immune complexes on endothelial cells and, importantly, anti–IL-8:IL-8 complexes have never been studied before. Preliminary studies from our laboratory revealed that anti–IL-8:IL-8 complexes have the ability to promote an inflammatory phenotype of endothelial cells (9
). Moreover, because the level of FcγRIIa is increased in lungs of patients with ARDS, and proinflammatory activity of anti–IL-8:IL-8 complexes is controlled by this receptor (6
), the goal of the current study was to determine whether HUVECs express FcγRIIa, and to test its role in the process of activation of endothelial cells triggered by anti–IL-8 autoantibody:IL-8 immune complexes. We found that anti–IL-8:IL-8 complexes interact with FcγRIIa on HUVECs and induce activation of signaling proteins (Syk, ERK, Akt, and JNK) in the FcγRIIa cascade. Importantly, the role of FcγRIIa in mediating activity of the complexes was confirmed by blocking of this receptor with specific antibodies that inhibited activation (phosphorylation) of analyzed signaling proteins.
NF-κB has been implicated in the development and progression of ALI, and increased levels of NF-κB were detected in neutrophils that accumulated in the lungs and airways of patients with ALI (28
). We found that binding of anti–IL-8 autoantibody:IL-8 immune complexes to FcγRIIa on HUVECs results in activation of NF-κB subunit, p65. Phosphorylation of p65 was inhibited by blocking of FcγRIIa. To the best of our knowledge, soluble immune complexes, such as anti–IL-8 autoantibody:IL-8 immune complexes, have not been extensively studied in relation to evoking of downstream signals in the FcγRIIa cascade in endothelial cells. There are few reports addressing the role of NF-κB in the FcγRIIa signaling cascade (33
). Furthermore, Alonso and colleagues (36
) demonstrated activation of NF-κB in response to stimulation of FcγRs on human monocyte/macrophage cell line (THP-1) by insoluble aggregates of human IgG; however, the authors did not distinguish between the subclasses of FcγRs.
In the current study, we observed that binding of anti–IL-8 autoantibody:IL-8 immune complexes to HUVECs causes the increase in surface level of adhesion molecule ICAM-1. This effect was also mediated by FcγRIIa. Up-regulation of ICAM-1 affected the recruitment of neutrophils to HUVECs that was 2.4- to 2.9-fold higher than spontaneous adhesion of these cells to nonstimulated HUVECs. Furthermore, increased expression of ICAM-1 was detected on endothelial cells in lungs of mice with lung injury caused by deposition of anti-KC:KC immune complexes (murine KC [CXCL1/KC] is a functional equivalent of human IL-8) (8
). Furthermore, even more importantly, expression of ICAM-1 remained unchanged in mice deficient in IgG receptors capable of interacting with anti-KC:KC immune complexes.
In our model of anti-chemokine autoantibody:chemokine (anti-KC:KC) immune complex–induced lung injury, IgG receptors (FcγRs) are essential for lung tissue deposition of anti-KC:KC complexes and, by association, for maintaining the increased expression of ICAM-1 (Ref. 7
and this study). Accordingly, ICAM-1 levels are normal in mice deficient in FcγRs, in which we did not detect deposition of anti-KC:KC complexes in lungs (Ref. 7
and this study). There have been several studies describing the presence of ICAM-1 on endothelial cells in IgG immune complex–mediated lung injury in rats (37
). The level of ICAM-1 expression is regulated by complement in these animals (39
). The animal model used in these studies differs substantially from our model. It is based on the local formation of heterologous immune complexes, which then trigger the alveolar inflammatory response (reverse passive Arthus reaction). A foreign antigen is given intravenously, whereas an antibody against this antigen is administered intratracheally. We developed a model in which mice are immunized with murine antigen (KC) for several weeks. After autoantibodies develop, the antigen (KC) is administered intratracheally, and autologous immune complexes (anti-KC:KC complexes) form in the lung. This model mimics very well the situation observed in patients with ARDS who have anti–IL-8 autoantibody complexes in their lungs (1
In conclusion, we found that anti–IL-8:IL-8 complexes are capable of activating endothelial (HUVEC) cells and inducing an increase in expression of ICAM-1 in these cells. Both of these activities are mediated by FcγRIIa. Analogous anti-chemokine autoantibody:chemokine immune complexes are essential for triggering enhanced expression of ICAM-1 via murine FcγRs. Anti–IL-8:IL-8 immune complexes are also found in lungs of patients with ARDS, and are associated with lung endothelial cells by interacting with endothelial cell FcγRIIa. Moreover, these complexes are deposited on endothelial cells that express increased levels of ICAM-1. Tsokos (40
) also observed enhanced expression of ICAM-1 on pulmonary endothelium in sepsis-induced ALI employing immunohistochemical analysis of human lung tissues. It is likely that anti–IL-8 autoantibody:IL-8 immune complexes, by engaging FcγRIIa present on lung endothelial cells, could contribute to pathophysiological changes observed in these cells during the course of ARDS.
Our findings are important in relation to inflammatory responses associated with ALI/ARDS because up-regulated expression of adhesion molecules on the surface of activated endothelial cells facilitates neutrophil adherence leading to accumulation of these cells in lungs (12
). It is known that influx of neutrophils may initiate or amplify lung injury contributing to edema formation and causing fibrosis (10
). By activating of endothelial cells, anti–IL-8 autoantibody:IL-8 immune complexes may facilitate neutrophil migration in patients with ALI/ARDS. These results implicate a novel mechanism by which anti–IL-8 autoantibody:IL-8 immune complexes may contribute to the pathogenesis of lung inflammation in ALI/ARDS.