Neutrophil elastase modulates cytokine expression: contribution to host defense against Pseudomonas aeruginosa-induced pneumonia.

There is accumulating evidence that following bacterial infection, the massive recruitment and activation of the phagocytes, neutrophils, is accompanied with the extracellular release of active neutrophil elastase (NE), a potent serine protease. Using NE-deficient mice in a clinically relevant model of Pseudomonas aeruginosa-induced pneumonia, we provide compelling in vivo evidence that the absence of NE was associated with decreased protein and transcript levels of the proinflammatory cytokines TNF-α, MIP-2, and IL-6 in the lungs, coinciding with increased mortality of mutant mice to infection. The implication of NE in the induction of cytokine expression involved at least in part Toll-like receptor 4 (TLR-4). These findings were further confirmed following exposure of cultured macrophages to purified NE. Together, our data suggest strongly for the first time that NE not only plays a direct antibacterial role as it has been previously reported, but released active enzyme can also modulate cytokine expression, which contributes to host protection against P. aeruginosa. In light of our findings, the long held view that considers NE as a prime suspect in P. aeruginosa-associated diseases will need to be carefully reassessed. Also, therapeutic strategies aiming at NE inhibition should take into account the physiologic roles of the enzyme.

Myeloperoxidase-dependent inactivation of surfactant protein D in vitro and in vivo.

Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H(2)O(2)-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity.

Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa.

According to the widely accepted view, neutrophil elastase (NE), a neutrophil-specific serine protease, is a major contributor to Pseudomonas aeruginosa infection-associated host tissue inflammation and damage, which in severe cases can lead to death. Herein, we provide for the first time compelling evidence that the host rather employs NE to protect itself against P. aeruginosa infection. Using a clinically relevant model of pneumonia, targeted deficiency in NE increased the susceptibility of mice to P. aeruginosa. We found that NE was required for maximal intracellular killing of P. aeruginosa by neutrophils. In investigating the mechanism of NE-mediated killing of P. aeruginosa, we found that NE degraded the major outer membrane protein F, a protein with important functions, including porin activity, maintenance of structural integrity, and sensing of host immune system activation. Consistent with this, the use of an isogenic mutant deficient in outer membrane protein F negated the role of NE in host defense against P. aeruginosa infection.

Differential modulation of nuclear texture, histone acetylation, and MDR1 gene expression in human drug-sensitive and -resistant OV1 cell lines.

Image cytometric study of pathological specimens or cell lines has suggested that epigenetic mechanisms are likely to play a major role in determining chromatin patterns evaluable through nuclear texture analysis. We previously reported that nuclear textural changes observed in the OV1-VCR etoposide-resistant ovarian carcinoma cell line were associated with an increased acetylated histone H4 level. In this study we analyzed the effects of treatments with the HDAC inhibitor trichostatin A (TSA) or with nickel subsulfide on histone H4 acetylation, nuclear texture, and MDR1 gene expression in drug-sensitive IGROV1 and drug-resistant OV1-VCR cell lines. In IGROV1 cells, TSA induced an increase in acetylated H4 level associated with a chromatin textural decondensation and an increase in MDR1 gene expression. In OV1-VCR cells, a similar increase in H4 acetylation was observed, but nuclear texture or MDR1 gene expression remained unchanged. ChIP analysis revealed that MDR1 gene expression remained stable in TSA-treated OV1-VCR cells despite a localized increase in H4 acetylation at the promoter level. Analysis of the methylation status of MDR1 promoter showed an increase in DNA methylation at 3 specific sites in OV1-VCR cells, that could participate to TSA low responsiveness in these cells. Treatment with nickel subsulfide induced a decrease in H4 acetylation without any effect on nuclear texture characteristics in both cell lines. In OV1-VCR cells, nickel subsulfide induced a significant down-regulation of the MDR1 gene expression. These results indicate that modulation of histone H4 acetylation level can be associated with up- or down-regulation of the MDR1 gene in OV1 cells. However, this modulation does not always result in chromatin pattern alterations and these data emphasize the complexity of chromatin texture regulation in tumor cells.