Complement effectors, C5a and C3a, in cystic fibrosis lung fluid correlate with disease severity.

In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, inflammation and tissue destruction. The complement system is a major mediator of inflammation for many diseases with the effectors C5a and C3a often playing important roles. We have previously shown in a small pilot study that CF sputum soluble fraction concentrations of C5a and C3a were associated with clinical measures of CF disease. Here we report a much larger study of 34 CF subjects providing 169 testable sputum samples allowing longitudinal evaluation comparing C5a and C3a with clinical markers. Levels of the strongly pro-inflammatory C5a correlated negatively with FEV1% predicted (P < 0.001), whereas the often anti-inflammatory C3a correlated positively with FEV1% predicted (P = 0.01). C5a concentrations correlated negatively with BMI percentile (P = 0.017), positively with worsening of an acute pulmonary exacerbation score (P = 0.007) and positively with P. aeruginosa growth in sputum (P = 0.002). C5a levels also correlated positively with concentrations of other sputum markers associated with worse CF lung disease including neutrophil elastase (P < 0.001), myeloperoxidase activity (P = 0.006) and DNA concentration (P < 0.001). In contrast to C5a, C3a levels correlated negatively with worse acute pulmonary exacerbation score and correlated negatively with sputum concentrations of neutrophil elastase, myeloperoxidase activity and DNA concentration. In summary, these data suggest that in CF sputum, increased C5a is associated with increased inflammation and poorer clinical measures, whereas increased C3a appears to be associated with less inflammation and improved clinical measures

C5a and C3a correlations with sputum concentration of Neutrophil Elastase (NE), myeloperoxidase (MPO) and DNA.

<p>C5a and C3a correlations with sputum concentration of Neutrophil Elastase (NE), myeloperoxidase (MPO) and DNA.</p

Longitudinal C5a level (red) and FEV1% predicted (black) for the six subjects with the most sputum samples.

<p>(A-C) For 3 subjects elevations in C5a concentration (purple arrows) appear to precede declines in FEV1% predicted. (D–F) For three subjects elevations in C5a concentration (blue arrows) appear to be coincident with declines in FEV1% predicted.</p

Changes in C5a and C3a over time or increasing age.

<p>(A) Changes in C5a (ng/ml) concentration over time and effect of systemic corticosteroid use. First sample for each subject is day zero. (B) C3a (ng/ml) compared with subjects age at specimen collection. Best fit line is shown for each graph.</p

Correlation of C5a and C3a with FEV1% predicted and BMI percentile.

<p>(A) FEV1% predicted compared with C5a (ng/ml). (B) FEV1% predicted compared with C3a (ng/ml). (C) FEV1% predicted compared with age. (D) C5a concentration correlation with BMI percentile in ≤ 20 year olds. Best fit line is shown for each graph.</p

Longitudinal C3a level (blue) and FEV1% predicted (black) for the six subjects with the most sputum samples.

<p>(A-F) For 6 subjects changes in C3a do not appear to demonstrate a pattern with respect to acute changes in FEV1% predicted.</p

Demographics at study entry.

<p>Demographics at study entry.</p

C5a and C3a correlations with Pulmonary Exacerbation Score (PES), bronchiectasis, <i>S</i>. <i>aureus</i>, <i>P</i>. <i>aeruginosa</i>, and Cystic Fibrosis Related Diabetes (CFRD).

<p>C5a and C3a correlations with Pulmonary Exacerbation Score (PES), bronchiectasis, <i>S</i>. <i>aureus</i>, <i>P</i>. <i>aeruginosa</i>, and Cystic Fibrosis Related Diabetes (CFRD).</p

Incompatible erythrocyte transfusion with lipopolysaccharide induces acute lung injury in a novel rat model.

Acute transfusion reactions can manifest in many forms including acute hemolytic transfusion reaction, allergic reaction and transfusion-related acute lung injury. We previously developed an acute hemolytic transfusion reaction rat model mediated by transfusion of incompatible human erythrocytes against which rats have preexisting antibodies resulting in classical complement pathway mediated intravascular hemolysis. In this study, the acute hemolytic transfusion reaction model was adapted to yield an acute lung injury phenotype. Adolescent male Wistar rats were primed in the presence or absence of lipopolysaccharide followed by transfusion of incompatible erythrocytes. Blood was collected at various time points during the course of the experiment to determine complement C5a levels and free DNA in isolated plasma. At 4 hours, blood and lung tissue were recovered and assayed for complete blood count and histological acute lung injury, respectively. Compared to sham animals or animals receiving increasing amounts of incompatible erythrocytes (equivalent to a 15-45% transfusion) in the absence of lipopolysaccharide, lungs of animals receiving lipopolysaccharide and a 30% erythrocyte transfusion showed dramatic alveolar wall thickening due to neutrophil infiltration. C5a levels were significantly elevated in these animals indicating that complement activation contributes to lung damage. Additionally, these animals demonstrated a significant increase of free DNA in the blood over time suggestive of neutrophil extracellular trap formation previously associated with transfusion-related acute lung injury in humans and mice. This novel 'two-hit' model utilizing incompatible erythrocyte transfusion in the presence of lipopolysaccharide yields a robust acute lung injury phenotype