Our studies of cytokine and chemokine-dependent acute lung injury in rodents have implicated the role of the complement system and the anaphylatoxin, C5a, which enhances production of pro-inflammatory mediators. The proposed studies will evaluate acute inflammatory lung injury induced in vivo by airway deposition of IgG immune complexes or bacterial lipopolysaccharide (LPS). We will determine the roles of the two C5a receptors, C5aR and C5L2. C5aR is a pro-inflammatory receptor while C5L2 is postulated to be a C5a """"""""default"""""""" or scavenger receptor in which signaling events fail to occur. In the two models of lung injury, we will measure quantitative changes in C5aR and C5L2 in both the vasculature and in the airways. C5aR and C5L2 receptors in lung will be selectively blocked by the use of antibodies and by the use of siRNA technology. Conversely, C5aR and C5L2 levels will be enhanced in lung by the use of adenoviral vector technology. We will determine in the two models of lung injury if enhanced levels of C5aR in lung intensifies lung injury, while enhanced C5L2 expression depresses lung injury, and vice-versa in the case of siRNA approach. The parameters of lung injury will be 125I-albumin leak, buildup of lung myeloperoxidase (MPO), and content of cytokines and chemokines in bronchoalveolar lavage (BAL) fluids. We will conduct parallel in vitro studies using blood neutrophils and alveolar macrophages which will be stimulated by LPS, C5a or the combination. Other mediators will be employed to see if we can achieve mediator specific changes in C5aR and C5L2 on macrophages. Macrophages will be transfected with adenovirus vectors to enhance receptor content of C5aR or C5L2. Cells will then be exposed to LPS or C5a, or the combination, and the production of TNFalpha, IL-1beta and specific CXC and CC chemokines will be measured. The proposed studies may have implications for our understanding of inflammatory lung diseases of humans. Project I will have close ties with Project II (Kunkel) and Project IV (Lukacs) as described in the application.
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