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Background: In acutely injured lungs, massively recruited polymorphonuclear neutrophils (PMNs) secrete abnormally neutrophil elastase (NE). Active NE creates a localized proteolytic environment where various host molecules are degraded leading to impairment of tissue homeostasis. Among the hallmarks of neutrophil-rich pathologies is a disrupted epithelium characterized by the loss of cell-cell adhesion and integrity. Epithelial-cadherin (E-cad) represents one of the most important intercellular junction proteins. E-cad exhibits various functions including its role in maintenance of tissue integrity. While much interest has focused on the expression and role of E-cad in different physio- and physiopathological states, proteolytic degradation of this structural molecule and ensuing potential consequences on host lung tissue injury are not completely understood., Methods: NE capacity to cleave E-cad was determined in cell-free and lung epithelial cell culture systems. The impact of such cleavage on epithelial monolayer integrity was then investigated. Using mice deficient in NE in a clinically relevant experimental model of acute pneumonia, we examined whether degraded E-cad is associated with lung inflammation and injury and whether NE contributes to E-cad cleavage. Finally, we checked for the presence of both degraded E-cad and NE in bronchoalveolar lavage samples obtained from patients with exacerbated COPD, a clinical manifestation characterised by a neutrophilic inflammatory response., Results: We show that NE is capable of degrading E-cad in vitro and in cultured cells. NE-mediated degradation of E-cad was accompanied with loss of epithelial monolayer integrity. Our in vivo findings provide evidence that NE contributes to E-cad cleavage that is concomitant with lung inflammation and injury. Importantly, we observed that the presence of degraded E-cad coincided with the detection of NE in diseased human lungs., Conclusions: Active NE has the capacity to cleave E-cad and interfere with its cell-cell adhesion function. These data suggest a mechanism by which unchecked NE participates potentially to the pathogenesis of neutrophil-rich lung inflammatory and tissue-destructive diseases.

Following phagocytosis in vivo, acidification of extracellular pH (pH(o)) and intracellular metabolic acid generation contribute to cytosolic proton loading in neutrophils. Cytosolic pH (pH(i)) affects neutrophil function, although its regulation is incompletely understood. Its effect on mechanisms of neutrophil death is also uncertain. Thus, we investigated pH(i) regulation in Escherichia coli-exposed neutrophils, at various pathogen-to-phagocyte ratios (0:1-50:1), under conditions simulating the inflammatory milieu in vivo and correlated pH(i) changes with mechanisms of neutrophil death. Following phagocytosis, proton extrusion was dominated early by passive proton conductance channels, and later by Na(+)/H(+) exchange (NHE). H(+)-translocating adenosine triphosphatase (V-ATPase) pH(i) regulation was evident primarily at lower bacterial densities. At physiologic pH(o), lower pathogen-to-phagocyte ratios alkalinized pH(i) and inhibited apoptosis, whereas higher ratios acidified pH(i) (despite proton extrusive mechanisms) and promoted apoptosis. Necrosis was induced by high-density bacterial exposure at reduced pH(o). Following phagocytosis, targeted inhibition of NHEs, proton conductance channels, or V-ATPases (amiloride, ZnCl(2), or bafilomycin, respectively) moderately hyperacidified pH(i) and accelerated apoptosis. However, in combination they profoundly acidified pH(i) and induced necrosis. Proinflammatory mediators in vivo might affect both pH(i) regulation and cell death, so we tested the effects of bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF) and healthy subjects. Only CF BAL fluid alkalinized pH(i) and suppressed apoptosis at physiologic pH(o), but failed to prevent necrosis following phagocytosis at low pH(o). Thus, a precarious balance between cytosolic proton loading and extrusion after phagocytosis dictates the mode of neutrophil cell death. pH(i)/pH(o) might be therapeutically targeted to limit neutrophil necrosis and protect host tissues during necrotizing infections.

Although neutrophils are a critical component of the inflammatory process, their functional regulation is incompletely understood. Of note, although pCO2 varies physiologically and pathologically in the neutrophilic milieu, its affect on neutrophil biological processes is unresolved. We demonstrate here that neutrophils respond to hypo- and hypercarbia, (0.04% and 10%) by increasing and decreasing, respectively, intracellular oxidant production (basally and in response to opsonized Escherichia coli and phorbol esters). Further, hypo- and hypercarbia increase and decrease, respectively, the release of IL-8 from LPS-stimulated cells; both effects are attenuated by the carbonic anhydrase inhibitor, acetazolamide. Anion exchange did not restore pH(i) under hypocarbic conditions, however partial restoration of pH(i) under hypercarbic conditions was achieved by Na+/H+ exchange and vacuolar ATPases. Abrogation of pCO2-induced changes in pH(i) prevented hypocarbia-induced generation of reactive oxidant species. These observations suggest that CO2 modifies neutrophil activity significantly by altering pH(i).

Cystic fibrosis (CF) is a condition characterized by neutrophil-mediated lung damage and bacterial colonization. The physiological basis for reported functional alterations in CF neutrophils, including increased release of neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These processes are, however, regulated by intracellular pH (pH(i)). We demonstrate here that pH(i) regulation is altered in neutrophils from CF patients. Although resting pH(i) is similar, pH(i) after acid loading and activation (N-formyl-methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate) is more acidic in CF cells than in normal cells. Furthermore, patients with non-CF-related bronchiectasis handle acid loading and activation in a fashion similar to subjects with normal neutrophils, suggesting that chronic pulmonary inflammation alone does not explain the difference in pH(i). This is further supported by data showing that normal neutrophils exposed to the CF pulmonary milieu respond by increasing pH(i) as opposed to decreasing pH(i) as seen in activated CF neutrophils. These pH(i) differences in activated or acid-loaded CF neutrophils are abrogated by ZnCl(2) but not by amiloride and bafilomycin A(1), suggesting that passive proton conductance is abnormal in CF. In addition, DIDS, which inhibits HCO(3)(-)/Cl(-) exchange, causes alkalinization of control but not of CF neutrophils, suggesting that anion transport is also abnormal in CF neutrophils. In summary, we have shown that pH(i) regulation in CF neutrophils is intrinsically abnormal, potentially contributing to the pulmonary manifestations of the condition.

Background: Neutrophil elastase (NE) activity is increased in lung diseases such as α1-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease 2 (MMP-2) in vitro and in a mouse model. It is postulated that increased cathepsin B and MMP-2 in acute and chronic lung diseases result from high levels of extracellular NE and that expression of these proteases could be inhibited by A1AT augmentation therapy. Methods: Cathepsin and MMP activities were assessed in bronchoalveolar lavage (BAL) fluid from patients with A1AT deficiency, pneumonia and control subjects. Macrophages were exposed to BAL fluid rich in free NE from patients with pneumonia following pretreatment with A1AT. MMP-2, cathepsin B, secretory leucoprotease inhibitor (SLPI) and lactoferrin levels were determined in BAL fluid from A1AT-deficient patients before and after aerosolisation of A1AT. Results: BAL fluid from both patients with pneumonia and those with A1AT deficiency containing free NE had increased cathepsin B and MMP-2 activities compared with BAL fluid from healthy volunteers. The addition of A1AT to BAL fluid from patients with pneumonia greatly reduced NE-induced cathepsin B and MMP-2 expression in macrophages in vitro. A1AT augmentation therapy to A1AT-deficient individuals also reduced cathepsin B and MMP-2 activity in BAL fluid in vivo. Furthermore, A1AT-deficient patients had higher levels of SLPI and lactoferrin after A1AT augmentation therapy. Conclusion: These findings suggest a novel role for A1AT inhibition of NE-induced upregulation of MMP and cathepsin expression both in vitro and in vivo. [ABSTRACT FROM AUTHOR]