Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors.

Airway epithelial cells can respond to infection by activating several signaling pathways. We examined the induction of apoptosis in response to Pseudomonas aeruginosa PAO1 in normal cells and several cystic fibrosis (CF) and corrected cell lines. Epithelial cells in monolayers with tight junctions, confirmed by apical ZO-1 staining demonstrated by confocal microscopy, were entirely resistant to PAO1-induced apoptosis. In contrast, cell lines such as 9HTEo(-) cells that do not form tight junctions were susceptible, with 50% of the population apoptotic after 6 h of exposure to PAO1. CF transmembrane conductance regulator (CFTR) dysfunction caused by different mechanisms (trafficking mutations, overexpression of the regulatory domain or antisense constructs) did not alter rates of apoptosis, nor were differences apparent in terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling detection of apoptotic airway cells from PAO1 infected cftr -/- or control mice. Bacterial expression of specific adhesins, complete lipopolysaccharide, and a functional type III secretion system were all necessary to evoke apoptosis even in susceptible epithelial cells. Unlike other mucosal surfaces, the airway epithelium is highly resistant to apoptosis, and this response is activated only when the appropriate epithelial conditions are present as well as fully virulent P. aeruginosa capable of coordinately expressing both adhesins and cytotoxins.

GFRalpha1 is an essential receptor component for GDNF in the developing nervous system and kidney.

Glial cell line-derived neurotrophic factor (GDNF) is a distant member of the TGFbeta protein family that is essential for neuronal survival and renal morphogenesis. We show that mice who are deficient in the glycosyl-phosphatidyl inositol (GPI) -linked protein GFRalpha1 (GDNFRalpha) display deficits in the kidneys, the enteric nervous system, and spinal motor and sensory neurons that are strikingly similar to those of the GDNF- and Ret-deficient mice. GFRalpha1-deficient dopaminergic and nodose sensory ganglia neurons no longer respond to GDNF or to the structurally related protein neurturin (NTN) but can be rescued when exposed to GDNF or neurturin in the presence of soluble GFRalpha1. In contrast, GFRalpha1-deficient submandibular parasympathetic neurons retain normal response to these two factors. Taken together with the available genetic and biochemical data, these findings support the idea that GFRalpha1 and the transmembrane tyrosine kinase Ret are both necessary receptor components for GDNF in the developing kidney and nervous system, and that GDNF and neurturin can mediate some of their activities through a second receptor.

Involvement of Interleukin (IL) 8 receptor in negative regulation of myeloid progenitor cells in vivo: evidence from mice lacking the murine IL-8 receptor homologue.

Expansion of mature neutrophils has been observed in mice lacking the murine interleukin (IL) 8 receptor homolog [mIL-8Rh(-/-)], and human (hu) IL-8 suppresses proliferation of primitive myeloid cells in vitro and in vivo. To evaluate involvement and relevance of murine IL-8 receptor homolog (mIL-8Rh) in negative regulation of myelopoiesis, we studied mIL-8Rh(-/-) and (+/+) mice raised in a normal or germ-free environment. Immature myeloid progenitors from mIL-8Rh(+/+) mice bred under normal or germ-free conditions were significantly suppressed in vitro by recombinant huIL-8, macrophage inflammatory protein (MIP)-1 alpha, platelet factor (PF) 4, interferon inducible protein (IP) 10, monocyte chemotactic peptide (MCP) 1, and H-ferritin. In contrast, progenitors from mIL-8Rh(-/-) mice were insensitive to inhibition by IL-8, but not to these other chemokines and H-ferritin. Mouse MIP-2, a ligand for mIL-8Rh, suppressed progenitors from normal but not mIL-8Rh(-/-) mice. Under normal environmental conditions, enhanced numbers of myeloid progenitors were found in femur, spleen, and blood of mIL-8Rh(-/-) compared with mIL-8Rh(+/+) mice. Numbers of myeloid progenitors were greatly decreased in mIL-8Rh(-/-)and (+/+) mice in germ-free conditions, and were either not significantly enhanced in mIL-8Rh(-/-) mice compared with (+/+) mice or were only moderately so. Differences in progenitors/organ between a germ-free and normal environment were greater for the mIL-8Rh(-/-) mice. These results document selective insensitivity of myeloid progenitor cells from mIL-8Rh(-/-) mice to inhibition by huIL-8 and mouse MIP-2 and a large expansion of myeloid progenitors in these mice, the latter effect being environmentally inducible. This provides strong support for a negative myeloid regulatory role played by the mIL-8Rh in vivo, whose active ligand may be MIP-2.

Hematopoietic defects in mice lacking the sialomucin CD34.

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34-null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34-null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.

Chemokine binding and activities mediated by the mouse IL-8 receptor.

In humans and rabbits two similar IL-8R mediate the chemotaxis and activation of neutrophils induced by alpha chemokines. We present data to suggest that there is only one such IL-8R gene in mice. We then use mice with a targeted deletion of this gene to characterize alpha chemokine ligands that signal via the mouse IL-8R. These experiments show that mouse macrophage inflammatory protein 2 binds the receptor with high affinity (Kd = approximately 1.5 nM) and potently activates both an intracellular Ca2+ flux and a chemotactic response, events absent in neutrophils from receptor-deleted mice. Mouse KC is approximately 10-fold less potent. These results show that macrophage inflammatory protein 2 and KC potently activate mouse neutrophils via a unique IL-8R, and these proteins may function as the major proinflammatory alpha chemokines in mice.

Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog.

Interleukin-8 (IL-8) is a proinflammatory cytokine that specifically attracts and activates human neutrophils. A murine gene with a high degree of homology to the two known human IL-8 receptors was cloned and then deleted from the mouse genome by homologous recombination in embryonic stem (ES) cells. These mice, although outwardly healthy, had lymphadenopathy, resulting from an increase in B cells, and splenomegaly, resulting from an increase in metamyelocytes, band, and mature neutrophils. Thus, this receptor may participate in the expansion and development of neutrophils and B cells. This receptor was the major mediator of neutrophil migration to sites of inflammation and may provide a potential therapeutic target in inflammatory disease.

The Pseudomonas cepacia 249 chromosomal penicillinase is a member of the AmpC family of chromosomal beta-lactamases.

Pseudomonas cepacia 249 produces an inducible beta-lactamase with penicillinase activity. The nucleotide sequence of the penA gene, which encodes this beta-lactamase, was determined and found to include regions with a significant homology to the ampC-encoded beta-lactamases of members of the family Enterobacteriaceae and Pseudomonas aeruginosa. The predicted amino acid sequence of the PenA beta-lactamase contained 17 amino acids immediately preceding the putative active-site serine which were highly conserved among the enzymes of the AmpC family. Although the penA-coding sequence had a total GC content of 60%, the predicted codon usage was more characteristic of Escherichia coli ampC-encoded beta-lactamase, with 53% of the codons having G or C in the third position, in contrast to the values for the P. aeruginosa ampC (88.5%) or Pseudomonas cepacia (88 to 92%) metabolic genes. The inducible expression of penA can be regulated by the E. coli gene product AmpD. A putative P. cepacia AmpR homolog was associated with the positive regulation of both Enterobacter cloacae ampC and P. cepacia penA expression, as confirmed by gel retardation studies. The E. cloacae AmpR did not regulate penA expression. Thus, by homology studies, codon usage, and genetic analysis, the P. cepacia penA beta-lactamase appears to have been acquired from members of the family Enterobacteriaceae and belongs to the class C group of beta-lactamases.

Comparison of adherence of Pseudomonas aeruginosa to respiratory epithelial cells from cystic fibrosis patients and healthy subjects.

The adherence of Pseudomonas aeruginosa PAO1 to primary cultures of cystic fibrosis nasal polyp (CFNP), normal human nasal polyp (NHNP), and immortalized CF and normal cell lines was studied. PAO1 bound significantly more to primary CFNP cells than to NHNP cells as the mean adherence +/- standard deviation of 5 x 10(7) CFU of 35S-labeled bacteria per ml per well was 15.09 x 10(6) +/- 4.25 x 10(6) CFU/ml per well and 7.62 x 10(6) +/- 2.11 x 10(6) CFU/ml per well, respectively (Mann-Whitney U test, P less than 0.0001). There was no significant difference in PAO1 adherence to the immortalized CF and normal cell lines. The primary CFNP cells had more receptors (115 per cell) than did NHNP cells (34 per cell). P. aeruginosa binding to CFNP was blocked by GlcNAc, NeuAc, L-Fuc, and D-Gal, while binding to NHNP was blocked only by GlcNAc, suggesting that receptors on the two cell types were qualitatively different. Pseudomonas supernatants containing protease, phospholipase C, and neuraminidase activity increased adherence to CFNP and NHNP cells. The Pseudomonas exoproducts modified epithelial cell glycoconjugates, as characterized by binding of fluorescein isothiocyanate-labeled lectins and the release of sialic acid. There was minimal release of fibronectin by the bacterial supernatants. The affinity of P. aeruginosa for CF epithelial cells appeared to be due to an increased number of receptors and modification of the epithelial cell surface by P. aeruginosa exoproducts that exposed asialoganglioside binding sites.

Production of the Pseudomonas aeruginosa neuraminidase is increased under hyperosmolar conditions and is regulated by genes involved in alginate expression.

The pathogenesis of Pseudomonas aeruginosa infection in cystic fibrosis (CF) is a complex process attributed to specific characteristics of both the host and the infecting organism. In this study, the properties of the PAO1 neuraminidase were examined to determine its potential role in facilitating Pseudomonas colonization of the respiratory epithelium. The PAO1 neuraminidase was 1000-fold more active than the Clostridium perfringens enzyme in releasing sialic acid from respiratory epithelial cells. This effect correlated with increased adherence of PAO1 to epithelial cells after exposure to PAO1 neuraminidase and was consistent with in vitro studies demonstrating Pseudomonas adherence to asialoganglioside receptors. The regulation of the neuraminidase gene nanA was examined in Pseudomonas and as cloned and expressed in Escherichia coli. In hyperosmolar conditions neuraminidase expression was increased by 50% (P less than 0.0004), an effect which was OmpR dependent in E. coli. In Pseudomonas the osmotic regulation of neuraminidase production was dependent upon algR1 and algR2, genes involved in the transcriptional activation of algD, which is responsible for the mucoid phenotype of Pseudomonas and pathognomonic for chronic infection in CF. Under the hyperosmolar conditions postulated to exist in the CF lung, nanA is likely to be expressed to facilitate the initial adherence of Pseudomonas to the respiratory tract.

Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa.

Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.

Isolation and characterization of a penicillinase from Pseudomonas cepacia 249.

Pseudomonas cepacia has an inducible beta-lactamase which is responsible for its novel ability to catabolize beta-lactam compounds. The gene encoding this enzyme, penA, was cloned from a genomic library of P. cepacia 249 on the broad-host-range cosmid pLAFR. This separated the penA gene from the gene encoding a second beta-lactamase in P. cepacia 249. Expression of penA was inducible in an Escherichia coli host strain by low levels of penicillin. The 33,500-molecular-weight enzyme had penicillinase activity not inhibited by clavulanic acid or sulbactam and was highly active against piperacillin and azlocillin. In comparison with other inducible beta-lactamases produced by gram-negative organisms, the penA enzyme had many properties which were similar to those of the penicillinase produced by Alcaligenes faecalis. It was unlike the ampC-type cephalosporinase produced by Pseudomonas aeruginosa.