Role of Complement C5 in Experimental Blunt Chest Trauma-Induced Septic Acute Lung Injury (ALI)

Abstract

Background: Severe blunt chest trauma is associated with high mortality. Sepsis represents a serious risk factor for mortality in acute respiratory distress syndrome (ARDS). In septic patients with ARDS complement activation products were found to be elevated in the plasma. In single models like LPS or trauma complement has been studied to some degree, however in clinically highly relevant double hit models such as the one used here little data is available. Here, we hypothesized that absence of C5 is correlated with a decreased inflammatory response in trauma induced septic acute lung injury.

Methods: 12 hrs after DH in mice the local and systemic cytokines and chemokines were quantified by multiplex bead array or ELISA, activated caspase-3 by western blot. Data were analyzed using one-way ANOVA followed by post-hoc Sidak's multiple comparison test (significance, p≤ 0.05).

Results: In lung tissue interleukin (IL)-6, monocyte chemo attractant protein-1 (MCP-1) and granulocyte-colony stimulating factor (G-CSF) was elevated in both C5-/- mice and wildtype littermates (wt), whereas caspase-3 was reduced in lungs after DH in C5-/- mice. Systemically, reduced keratinocyte-derived chemokine (KC) levels were observed after DH in C5-/- compared to wt mice. Locally, lung myeloperoxidase (MPO), protein, IL-6, MCP-1 and G-CSF in brochoalveolar lavage fluid (BALF) were elevated after DH in C5-/- compared to wt.

Conclusions: In the complex but clinically relevant DH model the local and systemic inflammatory immune response features both, C5-dependent and C5-independent characteristics. Activation of caspase-3 in lung tissue after DH was C5-dependent whereas local inflammation in lung tissue was C5-independent.

Fig 1. Cytokines and chemokines in lung tissue after sham procedure or double hit (DH) in wild type (wt) mice (black bars) or in absence of C5 (C5^-/-) mice (grey bars). Systemic effects of the absence of complement C5 in double hit (DH).

A. Increased IL-6 levels in lung tissue of wt and in C5^-/- mice after DH compared to sham. B. Increased MCP-1 levels in lung tissue of wt and in C5^-/- after DH compared to sham. C. Increased G-CSF levels in lung tissue of wt and in C5^-/- mice after DH compared to sham. D. Increased KC levels in lung tissue of wt mice. E. Increased KC plasma concentration in wt mice after DH; decreased KC plasma concentrations after DH in C5^-/- compared wt mice. F. Increased MCP-1 plasma concentrations in wt mice after DH, decrease of MCP-1 after DH in absence of C5. For each bar, n = 7 separate mice. * p<0.05, n.s. = not significant.

Fig 2. Activated caspase-3 in lung tissue in C5^+/+, wt (black bar) or C5^-/- (grey bar) mice after sham procedure or double hit (DH).

A. Representative western blot of activated caspase-3 in two wt and two C5^-/- mice after DH and corresponding western blot of anti-β-actin. B. Densitometry of three independent western blots for activated caspase-3. For each bar, n = 7 separate mice. * p<0.05.

Fig 3. Bronchoalveolar lavage fluid (BALF) cytokine, chemokine and protein concentrations in mice after sham procedure or double hit (DH) in C5^+/+ (wt) mice (black bars) and C5^-/- mice (grey bars) and MPO activity in lung tissue.

Increased IL-6 BALF concentration in C5^-/- mice after DH compared to sham and compared to DH in wt mice. B. Increased MCP-1 levels after DH in absence of C5 compared to sham and compared to DH in wt mice. C. Increased G-CSF BALF concentration after DH in presence and absence of C5. Further increase of G-CSF concentration after DH in absence of C5 compared to wt. D. Increased KC levels in wt mice after DH. E. Increased protein concentrations in bronchoalveolar lavage fluids (BALF) after DH in both wt (black bars) and in absence of C5 (grey bars). Further increase in BALF protein concentrations in C5^-/- after DH compared to DH in wt mice. F. MPO activity in lung tissue is increased after DH compared to sham in both wt and in absence of C5 and further increased after DH in absence of C5. For each bar, n = 7 separate mice. * p<0.05, n.s. = not significant.