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The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.

The JAK2 tyrosine kinase is known to associate with the receptors for growth hormone (GH) and erythropoietin (EPO) and with the interleukin-6 receptor signal transducing protein, gp130. Here we demonstrate that chimeric cytokine receptors which contain the cytoplasmic domain of the receptors for GH and EPO or for gp130 can form complexes with JAK2 when transiently co-expressed in HeLa cells. Mutational analyses of chimeras for the the GH and EPO receptors and gp130 demonstrated that box 1, a motif critical for cytokine receptor signal transduction, was required for the association of JAK2. Although JAK2 was capable of associating with all three of the chimeras, JAK1 co-precipitated only with the gp130 chimera. Association of JAK1 and JAK2 with cytokine receptor proteins, therefore, requires the highly conserved box 1 domain, but other sequences within the receptor proteins may influence the specificity of JAK binding. Mutational analysis of JAK2 revealed that multiple or complex protein sequences within JAK2 are required for association with cytokine receptors.

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