[COPD: An early disease]. / La BPCO : une maladie qui commence précocement.

This general review deals with the mechanisms which underlie the genetic factors in COPD. Many cellular and biochemical mechanisms occur in bronchial inflammation. We present the experimental models of COPD, insisting on the importance of oxydative stress, and on recent knowledge about the lung microbiome. Starting from this pathophysiology basis, we show how various genetic targets are able to interfere with the disease model. Thanks to these genetic targets, new markers in exhaled breath condensates and new drug targets are rising.

Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-ß (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation.

Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-ß (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3(-/-) mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3(-/-) progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-XL rescued survival; however, Bcl-XL-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3(-/-) cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-XL permitted differentiation of G6PC3(-/-) cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation and survival thus underlies neutropenia in G6PC3(-/-) deficiency, both originating from a reduced ability to utilize glucose. Hoxb8-dependent cells are a model to study differentiation and survival of the neutrophil lineage.

Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis.

The myeloperoxidase system of neutrophils uses hydrogen peroxide and chloride to generate hypochlorous acid, a potent bactericidal oxidant in vitro. In a mouse model of polymicrobial sepsis, we observed that mice deficient in myeloperoxidase were more likely than wild-type mice to die from infection. Mass spectrometric analysis of peritoneal inflammatory fluid from septic wild-type mice detected elevated concentrations of 3-chlorotyrosine, a characteristic end product of the myeloperoxidase system. Levels of 3-chlorotyrosine did not rise in the septic myeloperoxidase-deficient mice. Thus, myeloperoxidase seems to protect against sepsis in vivo by producing halogenating species. Surprisingly, levels of 3-bromotyrosine also were elevated in peritoneal fluid from septic wild-type mice and were markedly reduced in peritoneal fluid from septic myeloperoxidase-deficient mice. Furthermore, physiologic concentrations of bromide modulated the bactericidal effects of myeloperoxidase in vitro. It seems, therefore, that myeloperoxidase can use bromide as well as chloride to produce oxidants in vivo, even though the extracellular concentration of bromide is at least 1,000-fold lower than that of chloride. Thus, myeloperoxidase plays an important role in host defense against bacterial pathogens, and bromide might be a previously unsuspected component of this system.

Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase.

In determining the mechanism of neutrophil elastase (NE)-mediated killing of Escherichia coli, we found that NE degraded outer membrane protein A (OmpA), localized on the surface of Gram-negative bacteria. NE killed wild-type, but not OmpA-deficient, E. coli. Also, whereas NE-deficient mice had impaired survival in response to E. coli sepsis, as compared to wild-type mice, the presence or absence of NE had no influence on survival in response to sepsis that had been induced with OmpA-deficient E. coli. These findings define a mechanism of nonoxidative bacterial killing by NE and point to OmpA as a bacterial target in host defense.

Matrix metalloproteinases cleave tissue factor pathway inhibitor. Effects on coagulation.

The capacity of inflammatory cell-derived matrix metalloproteinases (MMPs) to cleave tissue factor pathway inhibitor (TFPI) and alter its activity was investigated. MMP-7 (matrilysin) rapidly cleaved TFPI to a major 35-kDa product. In contrast, MMP-1 (collagenase-1), MMP-9 (gelatinase B), and MMP-12 (macrophage elastase) cleaved TFPI into several fragments including the 35-kDa band. However, rates of cleavage were most rapid for MMP-7 and MMP-9. NH(2)-terminal amino acid sequencing revealed that MMP-12 cleaved TFPI at Lys(20)-Leu(21)(close to Kunitz I domain and producing a 35-kDa band), Arg(83)-Ile(84) (between Kunitz I and II domains), and Ser(174)-Thr(175) (between Kunitz II and III domains). MMP-7 and MMP-9 cleaved TFPI at Lys(20)-Leu(21) with additional COOH-terminal processing. These MMPs did not cleave tissue factor (TF), factor VII, and factor Xa. Proteolytic cleavage by MMP-1, MMP-7, MMP-9, and MMP-12 resulted in considerable loss of TFPI activity. These observations indicate specific cleavage of TFPI by MMPs, which broadens their substrate profile. Co-localization of MMPs, TF, and TFPI in atherosclerotic tissues suggests that release of MMPs from inflammatory cell leukocytes may effect TF-mediated coagulation.

Normal neutrophil function in cathepsin G-deficient mice.

Cathepsin G is a neutral serine protease that is highly expressed at the promyelocyte stage of myeloid development. We have developed a homologous recombination strategy to create a loss-of-function mutation for murine cathepsin G. Bone marrow derived from mice homozygous for this mutation had no detectable cathepsin G protein or activity, indicating that no other protease in bone marrow cells has the same specificity. Hematopoiesis in cathepsin G-/- mice is normal, and the mice have no overt abnormalities in blood clotting. Neutrophils derived from cathepsin G-/- mice have normal morphology and azurophil granule composition; these neutrophils also display normal phagocytosis and superoxide production and have normal chemotactic responses to C5a, fMLP, and interleukin-8. Although cathepsin G has previously shown to have broad spectrum antibiotic properties, challenges of mice with Staphylococcus aureus, Klebsiella pneumoniae, or Escherichia coli yielded survivals that were not different from those of wild-type animals. In sum, cathepsin G-/- neutrophils have no obvious defects in function; either cathepsin G is not required for any of these normal neutrophil functions or related azurophil granule proteases with different specificities (ie, neutrophil elastase, proteinase 3, azurocidin, and/or others) can substitute for it in vivo.

Genomic organization and chromosomal localization of mouse proteinase 3 (Myeloblastin).

Proteinase 3 (PR3), is a matrix-degrading serine proteinase expressed in different hematopoietic cell lineages. The PR3 protein appears to regulate the myeloid differentiation and was found to be the autoantigen associated with Wegener granulomatosis. We have isolated and characterized the gene for mouse PR3 (mPR3) and determined its chromosomal location. The gene has been localized to Chromosome (Chr) 10. Comparison of mouse PR3 genomic structure with that of its human counterpart indicates that: 1) the mPR3 gene spans 7 kb organized in 5 exons and 4 introns, 2) the codons of His-Asp-Ser of the catalytic site are conserved and spread out over different exons, similar to the human gene, and 3) the gene product encodes a pre-proform of the protein. Knowledge of the structure and chromosomal location of the mPR3 gene may help better the understanding of the temporal and cell-specific expression of mouse PR3.

Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis.

Neutrophil elastase (NE) is a potent serine proteinase whose expression is limited to a narrow window during myeloid development. In neutrophils, NE is stored in azurophil granules along with other serine proteinases (cathepsin G, proteinase 3 and azurocidin) at concentrations exceeding 5 mM. As a result of its capacity to efficiently degrade extracellular matrix, NE has been implicated in a variety of destructive diseases. Indeed, while much interest has focused on the pathologic effects of this enzyme, little is known regarding its normal physiologic function(s). Because previous in vitro data have shown that NE exhibits antibacterial activity, we investigated the role of NE in host defense against bacteria. Generating strains of mice deficient in NE (NE-/-) by targeted mutagenesis, we show that NE-/- mice are more susceptible than their normal littermates to sepsis and death following intraperitoneal infection with Gram negative (Klebsiella pneumoniae and Escherichia coli) but not Gram positive (Staphylococcus aureus) bacteria. Our data indicate that neutrophils migrate normally to sites of infection in the absence of NE, but that NE is required for maximal intracellular killing of Gram negative bacteria by neutrophils.

Characterization of the mouse neutrophil elastase gene and localization to chromosome 10.

Neutrophil elastase (NE), a serine proteinase, is considered to play a role in normal tissue turnover and host defense, NE may also cause tissue damage in acute and chronic inflammatory diseases. We have isolated and characterized the gene for mouse NE and determined its chromosomal location. The mouse NE gene has been localized by interspecific backcross analysis to Chromosome (Chr) 10. The gene for mouse NE is composed of 5 exons and 4 introns, similar to the human NE. Mouse NE shares the highly conserved exon size and intron-exon borders with human NE. The coding exons of the mouse NE gene predict a translation product in a pre-pro form, similar to human NE. Knowledge of the genomic organization and chromosomal location of mouse NE may allow us to further define mechanisms responsible for cell and tissue-specific expression of mouse NE.

Human macrophage metalloelastase. Genomic organization, chromosomal location, gene linkage, and tissue-specific expression.

Human macrophage metalloelastase (HME) is a recent addition to the matrix metalloproteinase (MMP) family that was initially found to be expressed in alveolar macrophages of cigarette smokers. To understand more about HME expression, analysis of the structure and location of the gene was performed. The gene for HME is composed of 10 exons and 9 introns, similar to the stromelysins and collagenases, and HME shares the highly conserved exon size and intron-exon borders with other MMPs. The 13-kilobase (kb) HME gene has been localized by fluorescence in situ hybridization to chromosome 11q22.2-22.3, the same location of the interstitial collagenase and stromelysin genes. We determined that HME and stromelysin 1 genes are physically linked within 62 kb utilizing pulse-field gel electrophoresis. The promoter region of the HME gene contains several features common to other MMP genes including a TATA box 29 bp upstream to the transcription initiation site, an AP-1 motif, and a PEA3 element. HME mRNA is not detectable in normal adult tissues but is induced in rapidly remodeling tissues such as the term placenta. In situ hybridization and immunohistochemistry of placental tissue demonstrated HME mRNA and protein expression in macrophages and stromal cells. Cell-specific expression and response to inflammatory stimuli such as endotoxin is conferred within 2.8 kb of the HME 5'-flanking sequence as demonstrated by HME promoter-CAT expression constructs. Knowledge of the genomic organization and chromosomal location of HME may allow us to further define mechanisms responsible for cell- and tissue-specific expression of HME.

Nucleotide sequences of the genes coding for the TEM-like beta-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2).

Two blaTEM-like genes were characterized that encoded IRT beta-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with beta-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 beta-lactamase resembled the blaTEM-1B gene, and that for IRT-2 resembled blaTEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the blaIRT-1 gene and C to A transversion in the blaIRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to beta-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic pI (pI = 5.2) of these IRT enzymes compared to that of TEM-1 (pI = 5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this beta-lactamase to inhibition by p-chloromercuribenzoate.

Clinical isolates of Escherichia coli producing TRI beta-lactamases: novel TEM-enzymes conferring resistance to beta-lactamase inhibitors.

Two different strains of Escherichia coli exhibiting unusual patterns of resistance to beta-lactam antibiotics were isolated from patients at Cochin Hospital. Both isolates showed a low level of resistance to amoxycillin, ticarcillin and ureidopenicillins but were susceptible to cephalosporins, aztreonam and imipenem; beta-lactamase inhibitors potentiated the activities of the beta-lactams to only a limited extent. All resistance characteristics of the strains were transferable by conjugation to E. coli K12. Resistance was shown to be due to beta-lactamases of pI 5.20 and relative molecular masses of 24,000. The hydrolytic and inhibition profiles of these enzymes were similar to each other but differed from those of broad-spectrum beta-lactamases (TEM-1). The rates of hydrolysis (Vmax) of amoxycillin (c. 200%) were higher than that for TEM-1 (84%). Ticarcillin, ureidopenicillins and cephaloridine were hydrolyzed slowly. However, as for TEM-1, no hydrolysis was observed with cefoxitin, third generation cephalosporins, aztreonam and imipenem. The high Km values demonstrated the poor affinity of these enzymes for their substrates. Unlike TEM-1, they were poorly inhibited by beta-lactamase inhibitors. These two enzymes differed from each other as follows: (i) the concentrations of clavulanic acid required for 50% beta-lactamase inhibition were 31 mumol/L for one enzyme (E-SAL) and 9.4 mumol/L for the other (E-GUER); (ii) p-chloromercuribenzoate was a more active inhibitor of E-SAL then E-GUER. The titration curve method and DNA-DNA hybridization studies demonstrated that both enzymes were structurally related to TEM-1. The novel plasmid-encoded enzymes produced by the two isolates of E. coli appeared to be almost identical and to be derived from TEM-enzymes. On the basis of their presumed phylogeny and their biological properties, we propose that these beta-lactamases be given the generic name TRI (TEM Resistant to beta-lactamase Inhibitors).