Publications

Sepsis is a common, life-threatening disease for which there is little treatment. The cysteine protease dipeptidyl peptidase I (DPPI) activates granule-associated serine proteases, several of which play important roles in host responses to bacterial infection. To examine DPPI's role in sepsis, we compared DPPI(-/-) and DPPI(+/+) mice using the cecal ligation and puncture (CLP) model of septic peritonitis, finding that DPPI(-/-) mice are far more likely to survive sepsis. Outcomes of CLP in mice lacking mast cell DPPI reveal that the absence of DPPI in mast cells, rather than in other cell types, is responsible for the survival advantage. Among several cytokines surveyed in peritoneal fluid and serum, IL-6 is highly and differentially expressed in DPPI(-/-) mice compared with DPPI(+/+) mice. Remarkably, deleting IL-6 expression in DPPI(-/-) mice eliminates the survival advantage. The increase in IL-6 in septic DPPI(-/-) mice, which appears to protect these mice from death, may be related to reduced DPPI-mediated activation of mast cell tryptase and other peptidases, which we show cleave IL-6 in vitro. These results indicate that mast cell DPPI harms the septic host and that DPPI is a novel potential therapeutic target for treatment of sepsis.

Murine Mycoplasma pulmonis infection induces chronic lung and airway inflammation accompanied by profound and persistent microvascular remodeling in tracheobronchial mucosa. Because matrix metalloproteinase (MMP)-2 and -9 are important for angiogenesis associated with placental and long bone development and skin cancer, we hypothesized that they contribute to microvascular remodeling in airways infected with M. pulmonis. To test this hypothesis, we compared microvascular changes in airways after M. pulmonis infection of wild-type FVB/N mice with those of MMP-9(-/-) and MMP-2(-/-)/MMP-9(-/-) double-null mice and mice treated with the broad-spectrum MMP inhibitor AG3340 (Prinomastat). Using zymography and immunohistochemistry, we find that MMP-2 and MMP-9 rise strikingly in lungs and airways of infected wild-type FVB/N and C57BL/6 mice, with no zymographic activity or immunoreactivity in MMP-2(-/-)/MMP-9(-/-) animals. However, microvascular remodeling as assessed by Lycopersicon esculentum lectin staining of whole-mounted tracheae is as severe in infected MMP-9(-/-), MMP-2(-/-)/MMP-9(-/-) and AG3340-treated mice as in wild-type mice. Furthermore, all groups of infected mice develop similar inflammatory infiltrates and exhibit similar overall disease severity as indicated by decrease in body weight and increase in lung weight. Uninfected wild-type tracheae show negligible MMP-2 immunoreactivity, with scant MMP-9 immunoreactivity in and around growing cartilage. By contrast, MMP-2 appears in epithelial cells of infected, wild-type tracheae, and MMP-9 localizes to a large population of infiltrating leukocytes. We conclude that despite major increases in expression, MMP-2 and MMP-9 are not essential for microvascular remodeling in M. pulmonis-induced chronic airway inflammation.

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.

Mast cells and macrophages infiltrate healing myocardial infarcts and may play an important role in regulating fibrous tissue deposition and extracellular matrix remodelling. This study examined the time-course of macrophage and mast cell accumulation in healing infarcts and studied the histological characteristics and protease expression profile of mast cells in a canine model of experimental infarction. Although macrophages were more numerous than mast cells in infarct granulation tissue, macrophage density decreased during maturation of the scar, whereas mast cell numbers remained persistently elevated. During the inflammatory phase of infarction, newly recruited leucocytes infiltrated the injured myocardium and appeared to be clustered in close proximity to degranulating cardiac mast cells. During the proliferative phase of healing, mast cells had decreased granular content and were localized close to infarct neovessels. In contrast, macrophages showed no selective localization. Mast cells in healing canine infarcts were alcian blue/safranin-positive cells that expressed both tryptase and chymase. In order to explain the pro-inflammatory and angiogenic actions of tryptase--the major secretory protein of mast cells--its effects on endothelial chemokine expression were examined. Chemokines are chemotactic cytokines that play an important role in leucocyte trafficking and angiogenesis and are highly induced in infarcts. Tryptase, a proteinase-activated receptor (PAR)-2 agonist, induced endothelial expression of the angiogenic chemokines CCL2/MCP-1 and CXCL8/IL-8, but not the angiostatic chemokine CXCL10/IP-10. Endothelial PAR-2 stimulation with the agonist peptide SLIGKV induced a similar chemokine expression profile. Mast cell tryptase may exert its angiogenic effects in part through selective stimulation of angiogenic chemokines.

Mastin is a tryptic peptidase secreted by canine mast cells. This work reveals that mastin is composed of catalytic domain singlets and disulfide-linked dimers. Monomers unite non-covalently to form tryptase-like tetramers, whereas dimers aggregate with monomers into larger clusters stabilized by hydrophobic contacts. Unlike tryptases, mastin resists inactivation by leech-derived tryptase inhibitor, indicating a smaller central cavity, as confirmed by structural models. Nonetheless, mastin is strongly gelatinolytic while not cleaving native collagen or casein, suggesting a preference for denatured proteins threaded into its central cavity. Phylogenetic analysis suggests that mammalian mastins shared more recent ancestors with soluble alpha/beta/delta tryptases than with membrane-anchored gamma-tryptases, and diverged more rapidly. We hypothesize that gelatinase activity and formation of inhibitor-resistant oligomers are ancestral characteristics shared by soluble tryptases and mastins, and that secreted mastin is a mini-proteasome-like complex that breaks down partially degraded proteins without causing bystander damage to intact, native proteins.

BACKGROUND

Rejection and obliterative bronchiolitis are barriers to sustained graft function in recipients of transplanted lungs. Early detection is hindered by inadequate tests and an incomplete understanding of the molecular events preceding or accompanying graft deterioration.

METHODS

Hypothesizing that genes involved in immune responses and tissue remodeling produce biomarkers of rejection, we measured the expression of 192 selected genes in 72 sets of biopsy specimens from human lung allografts. Gene transcripts were quantified using a 2-step, multiplex, real-time polymerase chain reaction approach in endobronchial and transbronchial biopsy specimens from transplant recipients without acute infections undergoing routine surveillance bronchoscopy.

RESULTS

Comparisons of histopathology in parallel biopsy specimens identified 6 genes correlating with rejection as manifested by lymphocytic bronchitis, a suspected harbinger of obliterative bronchiolitis. For example, beta2-defensin and collagenase transcripts in inflamed bronchi increased 37-fold and 163-fold, respectively. By contrast, these transcripts did not correlate with acute rejection in transbronchial specimens. Further, no correspondence was noted between histopathologic bronchitis and parenchymal rejection when endobronchial and transbronchial samples were obtained from the same patient.

CONCLUSIONS

Our highly sensitive method permits quantitation of many gene transcripts simultaneously in small, bronchoscopically acquired biopsy specimens of allografts. Transcript signatures obtained by this approach suggest that airway and alveolar responses to rejection differ and that endobronchial biopsy specimens assess lymphocytic bronchitis and chronic rejection but are not proxies for transbronchial biopsy specimens. Further, they reveal changes in airway expression of the specific genes involved in host defense and remodeling and suggest that the measurement of transcripts correlating with lymphocytic bronchitis may be diagnostic adjuncts to histopathology.

RATIONALE

As the smallest free-living bacteria and a frequent cause of respiratory infections, mycoplasmas are unique pathogens. Mice infected with Mycoplasma pulmonis can develop localized, life-long airway infection accompanied by persistent inflammation and remodeling.

OBJECTIVE

Because mast cells protect mice from acute septic peritonitis and gram-negative pneumonia, we hypothesized that they defend against mycoplasma infection. This study tests this hypothesis using mast cell-deficient mice.

METHODS

Responses to airway infection with M. pulmonis were compared in wild-type and mast cell-deficient Kit(W-sh)/Kit(W-sh) mice and sham-infected control mice.

MEASUREMENTS AND MAIN RESULTS

Endpoints include mortality, body and lymph node weight, mycoplasma antibody titer, and lung mycoplasma burden and histopathology at intervals after infection. The results reveal that infected Kit(W-sh)/Kit(W-sh) mice, compared with other groups, lose more weight and are more likely to die. Live mycoplasma burden is greater in Kit(W-sh)/Kit(W-sh) than in wild-type mice at early time points. Four days after infection, the difference is 162-fold. Titers of mycoplasma-specific IgM and IgA appear earlier and rise higher in Kit(W-sh)/Kit(W-sh) mice, but antibody responses to heat-killed mycoplasma are not different compared with wild-type mice. Infected Kit(W-sh)/Kit(W-sh) mice develop larger bronchial lymph nodes and progressive pneumonia and airway occlusion with neutrophil-rich exudates, accompanied by angiogenesis and lymphangiogenesis. In wild-type mice, pneumonia and exudates are less severe, quicker to resolve, and are not associated with increased angiogenesis.

CONCLUSIONS

These findings suggest that mast cells are important for innate immune containment of and recovery from respiratory mycoplasma infection.

Hepatocyte growth factor (HGF) is a plasminogen-like protein with an alpha chain linked to a trypsin-like beta chain without peptidase activity. The interaction of HGF with c-met, a receptor tyrosine kinase expressed by many cells, is important in cell growth, migration, and formation of endothelial and epithelial tubes. Stimulation of c-met requires two-chain, disulfide-linked HGF. Portions of an alpha chain containing an N-terminal segment and four kringle domains (NK4) antagonize HGF activity. Until now, no physiological pathway for generating NK4 was known. Here we show that chymases, which are chymotryptic peptidases secreted by mast cells, hydrolyze HGF, thereby abolishing scatter factor activity while generating an NK4-like antagonist of HGF scatter factor activity. Thus, chymase interferes with HGF directly by destroying active protein and indirectly by generating an antagonist. The site of hydrolysis, Leu480, lies in the alpha chain on the N-terminal side of the cysteine linking the alpha and beta chains. This site appears to be specific for HGF because chymase does not hydrolyze other plasminogen-like proteins, such as macrophage-stimulating protein and plasminogen itself. Mast cell/neutrophil cathepsin G and neutrophil elastase generate similar fragments of HGF by cleaving near the chymase site. Mast cell and neutrophil peptidases are secreted during tissue injury, infection, ischemia, and allergic inflammation, where they may oppose HGF effects on epithelial repair. Thus, HGF possesses an "inactivation segment" that serves as an Achilles' heel attacked by inflammatory proteases. This work reveals a potential physiological pathway for inactivation of HGF and generation of NK4-like antagonists.

Tryptases secreted by tissue mast cells and basophils can enter the bloodstream. In human subjects tryptases are encoded by several genes and alleles, including alpha, beta, gamma, and delta. Common variations include complete absence of alpha genes. Until recently, alpha tryptase was considered to be the major tryptase secreted at baseline and in mastocytosis. However, lack of alpha tryptase genes has little effect on circulating tryptase levels, which are now thought mainly to consist of inactive pro-beta tryptase secreted constitutively rather than stored in granules with mature tryptases. Pro-beta tryptase levels thus might reflect total body mast cell content. In contrast, mature beta tryptase can increase transiently in severe systemic anaphylaxis and confirm the diagnosis. However, it might fail to increase in food anaphylaxis or might increase nonspecifically in samples acquired after death. Thus pro- and mature beta tryptase measurements are useful but associated with false-negative and false-positive results, which need to be considered in drawing clinical conclusions in cases of suspected anaphylaxis.

Prostasin, a trypsinlike serine peptidase, is highly expressed in prostate, kidney, and lung epithelia, where it is bound to the cell surface, secreted, or both. Prostasin activates the epithelial sodium channel (ENaC) and suppresses invasion of prostate and breast cancer cells. The studies reported here establish mechanisms of membrane anchoring and secretion in kidney and lung epithelial cells and demonstrate a critical role for prostasin in regulating epithelial monolayer function. We report that endogenous mouse prostasin is glycosylphosphatidylinositol (GPI) anchored to the cell surface and is constitutively secreted from the apical surface of kidney cortical collecting duct cells. Using site-directed mutagenesis, detergent phase separation, and RNA interference approaches, we show that prostasin secretion depends on GPI anchor cleavage by endogenous GPI-specific phospholipase D1 (Gpld1). Secretion of prostasin by kidney and lung epithelial cells, in contrast to prostate epithelium, does not depend on COOH-terminal processing at conserved Arg(322). Using stably transfected M-1 cells expressing wild-type, catalytically inactive, or chimeric transmembrane (not GPI)-anchored prostasins we establish that prostasin regulates transepithelial resistance, current, and paracellular permeability by GPI anchor- and protease activity-dependent mechanisms. These studies demonstrate a novel role for prostasin in regulating epithelial monolayer resistance and permeability in kidney epithelial cells and, furthermore, show specifically that prostasin is a critical regulator of transepithelial ion transport in M-1 cells. These functions depend on the GPI anchor as well as the peptidase activity of prostasin. These studies suggest that cell-specific Gpld1- or peptidase-dependent pathways for prostasin secretion may control prostasin functions in a tissue-specific manner.