Neutrophil-Mediated Phagocytic Host Defense Defect in Myeloid Cftr-Inactivated Mice

Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr−/−) mice and the non-inactivated control (Cftr^fl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr−/− lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.     

RNA interference against CFTR affects HL60-derived neutrophil microbicidal function

Biosynthesis of hypochlorous acid, a potent antimicrobial oxidant, in phagosomes is one of the chief mechanisms employed by polymorphonuclear neutrophils to combat infections. This reaction, catalyzed by myeloperoxidase, requires chloride anion (Cl⁻) as a substrate. Thus, Cl⁻ availability is a rate-limiting factor that affects neutrophil microbicidal function. Our previous research demonstrated that defective CFTR, a cAMP-activated chloride channel, present in cystic fibrosis (CF) patients leads to deficient chloride transport to neutrophil phagosomes and impaired bacterial killing. To confirm this finding, here we used RNA interference against this chloride channel to abate CFTR expression in the neutrophil-like cells derived from HL60 cells, a promyelocytic leukemia cell line, with dimethyl sulfoxide. The resultant CFTR deficiency in the phagocytes compromised their bactericidal capability, thereby recapitulating the phenotype seen in CF patient cells. The results provide further evidence suggesting that CFTR plays an important role in phagocytic host defense.     

Evidence for a role of taurine in the in vitro oxidative toxicity of neutrophils toward erythrocytes

Production of hydrogen peroxide and secretion of myeloperoxidase by stimulated neutrophils resulted in myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl), the reaction of HOCl with taurine to yield taurine monochloramine (TauNHCl), and accumulation of TauNHCl in the extracellular medium. When erythrocytes were present, the yield of TauNHCl was lower as the result of uptake of TauNHCl into erythrocytes. The zwitterion taurine was not taken up, but the anion TauNHCl and other anionic oxidants including taurine dichloramine (TauNCl2) and L-alanine chloramines were transported into erythrocytes by the anion-transport system. Oxidation of intracellular components such as glutathione (GSH) by taurine chloramines resulted in reduction of the chloramines and trapping of taurine within erythrocytes. At high oxidant:erythrocyte ratios, TauNHCl also oxidized hemoglobin (Hb) and depleted ATP, but caused little lysis. TauNCl2 was much more effective as a lytic agent. At low oxidant:erythrocyte ratios, the chloramines caused net loss of GSH when no glucose was provided, but Hb was not oxidized and GSH content returned to normal when glucose was added. Therefore, anionic chloramines may mediate oxidative toxicity when the neutrophil:erythrocyte ratio is high. Under more physiologic conditions, chlorination of taurine by neutrophils and the uptake and reduction of TauNHCl by erythrocytes prevents accumulation of oxidants and may protect blood cells, plasma components, and tissues against oxidative toxicity.     

Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.     

Protein chlorination in neutrophil phagosomes and correlation with bacterial killing

Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produce hypochlorous acid (HOCl) following phagocytosis by measuring conversion of protein tyrosine residues to 3-chlorotyrosine. We also examined how varying chloride availability affects the relationship between HOCl formation in the phagosome and bacterial killing. Phagosomal proteins, isolated following ingestion of opsonized magnetic beads, contained 11.4 Cl-Tyr per thousand tyrosine residues. This was 12 times higher than the level in proteins from the rest of the neutrophil and ~6 times higher than previously recorded for protein from ingested bacteria. These results indicate that HOCl production is largely localized to the phagosomes and a substantial proportion reacts with phagosomal protein before reaching the microbe. This will in part detoxify the oxidant but should also form chloramines which could contribute to the killing mechanism. Neutrophils were either suspended in chloride-free gluconate buffer or pretreated with formyl-Met-Leu-Phe, a procedure that has been reported to deplete intracellular chloride. These treatments, alone or in combination, decreased both chlorination in phagosomes and killing of Staphylococcus aureus by up to 50%. There was a strong positive correlation between the two effects. Killing was predominantly oxidant and myeloperoxidase dependent (88% inhibition by diphenylene iodonium and 78% by azide). These results imply that lowering the chloride concentration limits HOCl production and oxidative killing. They support a role for HOCl generation, rather than an alternative myeloperoxidase activity, in the killing process.     

Estrogen and the cystic fibrosis gender gap

Cystic fibrosis (CF) is the most frequent inherited disease in Caucasian populations and is due to a defect in the expression or activity of a chloride channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutations in this gene affect organs with exocrine functions and the main cause of morbidity and mortality for CF patients is the lung pathology in which the defect in CFTR decreases chloride secretion, lowering the airway surface liquid height and increasing mucus viscosity. The compromised ASL dynamics leads to a favorable environment for bacterial proliferation and sustained inflammation resulting in epithelial lung tissue injury, fibrosis and remodeling. In CF, there exist a difference in lung pathology between men and women that is termed the “CF gender gap”. Recent studies have shown the prominent role of the most potent form of estrogen, 17β-estradiol in exacerbating lung function in CF females and here, we review the role of this hormone in the CF gender dichotomy.     

Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air–liquid but not liquid–liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr^{∆F508/∆F508} immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor–mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.     

CFTR: folding, misfolding and correcting the ΔF508 conformational defect

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (ΔF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the ΔF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote ΔF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of ΔF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Human neutrophils use the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate but not nitrate bacterial proteins during phagocytosis

The generation of extracellular oxidants by neutrophils has been widely investigated, but knowledge about the chemical reactions that occur in the phagolysosome, the cellular compartment that kills pathogens, is more limited. One important pathway may involve the production of potent halogenating agents such as hypochlorous acid (HOCl) by the myeloperoxidase-hydrogen peroxide-halide system. However, explorations of the oxidation chemistry of phagolysosomes have been hampered by the organelle's inaccessibility. To overcome this limitation, we recovered Escherichia coli that had been internalized by human neutrophils. We then analyzed the bacterial proteins for 3-chlorotyrosine, a stable marker of damage by HOCl. Mass spectrometric analysis revealed that levels of 3-chlorotyrosine in E. coli proteins increased markedly after the bacteria were internalized by human neutrophils. This increase failed to occur in E. coli exposed to neutrophils deficient in NADPH oxidase or myeloperoxidase, implicating H(2)O(2) and myeloperoxidase in the halogenation reaction. The extent of protein chlorination by normal neutrophils paralleled bacterial killing. Our observations support the view that the phagolysosome of human neutrophils uses the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate bacterial proteins. In striking contrast, human neutrophils failed to nitrate bacterial proteins unless the medium was supplemented with 1 mm nitrite, and the level of nitration was low. Protein chlorination associated with bacterial killing was unaffected by the presence of nitrite in the medium. Nitration required NADPH oxidase but appeared to be independent of myeloperoxidase, suggesting that neutrophils can nitrate proteins through a pathway that requires nitrite but is independent of myeloperoxidase.     

Annexin V is directly involved in cystic fibrosis transmembrane conductance regulator's chloride channel function

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-activated chloride channel, which is regulated by protein-protein interactions. The extent to which CFTR is regulated by these interactions remains unknown. Annexin V is overexpressed in cystic fibrosis (CF), and given the functional properties of annexin V and CFTR we considered whether they are associated and if so whether this has implications for CFTR function. Using co-immunoprecipitation and overlay experiments, we show that annexin V is associated with nucleotide-binding domain 1 (NBD1) of CFTR. Surface plasmon resonance (SPR) indicated different KD values in the absence and presence of both calcium and ATP, suggesting that this interaction is calcium- and ATP-dependent. Using an siRNA approach and overexpression, we showed that CFTR chloride channel function and its localization in the cell membranes were dependent on annexin V expression. We concluded that annexin V is necessary for normal CFTR chloride channel activity. Furthermore, we show that CFTR and annexin V are partially co-distributed in normal epithelial cells in human bronchi. In conclusion, we show for the first time that annexin V is associated with CFTR and is involved in its function.     

Functional characterization of a novel CFTR mutation P67S identified in a patient with atypical cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR serves as a cAMP-stimulated chloride channel in a wide range of epithelial tissues and its dysfunction is a hallmark of CF. Over 1400 mutations in the CFTR gene are known, but functional data exist only for a minority of the mutant channels. The aim of the present study was to functionally characterize a novel CFTR mutation identified in a patient with atypical CF. Full length sequencing of the patient's CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution. Mutant and wild-type CFTR were heterologously expressed in Xenopus laevis oocytes. CFTR whole-cell currents were studied using the two-electrode voltage-clamp technique. Channel surface expression was assessed by a chemiluminescence assay. Expression of P67S-CFTR resulted in functional CFTR chloride channels. However, the CFTR chloride conductance observed in oocytes expressing the mutant channel averaged only 24% of that in oocytes expressing wild-type CFTR. Similarly, surface expression of the mutant channel was reduced. In contrast, the mutation did not alter the anion selectivity of the channel, and Western blot analysis indicated a similar protein expression level of mutant and wild-type CFTR. Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression. The mild disease phenotype of the patient indicates that the residual function of the mutant channel is sufficient to prevent the development of severe CF symptoms.     

C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis. A novel class of mutation.

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.     

Reevaluating gel-forming mucins' roles in cystic fibrosis lung disease

The existence of mucus plugs, containing mucins, bacteria, and neutrophils, blocking the lower airways in the lung of cystic fibrosis (CF) patients has raised the possibility that production of "abnormal" mucins is a critical characteristic of this disease. The molecular nature, if any, of this abnormality is unknown. Recent studies suggest that CF lung disease progression is characterized by an early phase in which airway surface liquid (ASL) increased dehydration is accompanied by altered pH and levels of reduced glutathione (GSH). In a later phase, bacterial infection and neutrophil invasion lead to increased ASL of concentrations myeloperoxidase and hypochlorous acid (HOCl). Independent studies indicate that gel-forming mucins, the key components of airway mucus, form disulfide-linked polymers through a pH-dependent, likely self-catalyzed mechanism. In this article, we present the hypothesis that increased mucus concentration (dehydration) and altered pH, and levels of GSH, myeloperoxidase, and/or HOCl result in the extracellular formation of additional interchain bonds among airway mucins. These novel interactions would create an atypical mucin network with abnormal viscoelastic and adhesive properties.     

Early bronchial inflammation in cystic fibrosis

Cystic fibrosis (CF) is the most common genetic autosomal recessive disease in caucasian north-american and european populations. The CF gene codes for a transmembrane glycoprotein called CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), a chloride channel which regulates the luminal secretion of chloride and the active ion and water transport in the airway epithelial cells. Mutations of the CF gene lead to a dysregulation of chloride and sodium channel associated to airway mucus dehydration, neutrophil-dominated airway inflammation and chronic infection responsible for the morbidity and mortality of CF patients. Although a high number of studies has been devoted to the CFTR pleiotropic functions, the chronology of the physiopathological events leading to the airway inflammation linked to mutations of the CF gene is still an open question. The issue of whether airway inflammation takes place before infection or is a consequence of infection during CF pathogenesis is still controversial. It has been recently reported that in broncho-alveolar lavages collected in CF infants, there is an increased level of interleukin IL-8 and abnormal low level of IL-10. The decreased IL-10 production has been confirmed in peripheral blood monocytes as well as in airway cell lines. Under basal conditions, the increased expression of the pro-inflammatory IL-8 cytokine has also been recently observed in the airway liquid secreted by CF na?ve humanized airway xenografts and in the supernatant culture of CF human airway epithelial cells. These results suggest that CFTR dysfunction may result in a constitutive pro-inflammatory vs anti-inflammatory imbalance in CF disease. Recent data from the literature suggest that the failure of chloride transport, the maturation defect and mistraffricking of mutated CFTR, lead to its accumulation in the endoplasmic reticulum and activation of NF-kappa B, responsible for the imbalance in the CF airway cell cytokine production.     

Altered intracellular pH regulation in neutrophils from patients with cystic fibrosis

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.     

Pharmacological modulation of cGMP levels by phosphodiesterase 5 inhibitors as a therapeutic strategy for treatment of respiratory pathology in cystic fibrosis

The CFTR gene encodes a chloride channel with pleiotropic effects on cell physiology and metabolism. Here, we show that increasing cGMP levels to inhibit epithelial Na(+) channel in cystic fibrosis (CF) respiratory epithelial cells corrects several aspects of the downstream pathology in CF. Cell culture models, using a range of CF cell lines and primary cells, showed that complementary pharmacological approaches to increasing intracellular cGMP, by elevating guanyl cyclase activity though reduced nitric oxide, addition of cell-permeable cGMP analogs, or inhibition of phosphodiesterase 5 corrected multiple aspects of the CF pathological cascade. These included correction of defective protein glycosylation, bacterial adherence, and proinflammatory responses. Furthermore, pharmacological inhibition of phosphodiesterase 5 in tissues ex vivo or in animal models improved transepithelial currents across nasal mucosae from transgenic F508del Cftr(tm1Eur) mice and reduced neutrophil infiltration on bacterial aerosol challenge in Pseudomonas aeruginosa-susceptible DBA/2 mice. Our findings define phosphodiesterase 5 as a specific target for correcting a number of previously disconnected defects in the CF respiratory tract, now linked through this study. Our study suggests that phosphodiesterase 5 inhibition provides an opportunity for simultaneous and concerted correction of seemingly disparate complications in CF.     

Conformational and temperature-sensitive stability defects of the delta F508 cystic fibrosis transmembrane conductance regulator in post-endoplasmic reticulum compartments

Deletion of phenylalanine at position 508 (DeltaF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that DeltaF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous DeltaF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of DeltaF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued DeltaF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4-20-fold accelerated degradation rate between 37 and 40 degrees C is, most likely, due to decreased conformational stability of the rescued DeltaF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native DeltaF508 CFTR.     

Generation of hypochlorite-modified proteins by neutrophils during ischemia-reperfusion injury in rat liver: attenuation by ischemic preconditioning

Although it is well documented that neutrophils are critical for the delayed phase of hepatic ischemia-reperfusion injury, there is no direct evidence for a specific neutrophil-derived oxidant stress in vivo. Therefore, we used a model of 60 min of partial hepatic ischemia and 0-24 h of reperfusion to investigate neutrophil accumulation and to analyze biomarkers for a general oxidant stress [glutathione disulfide (GSSG) and malondialdehyde (MDA)] and for a neutrophil-specific oxidant stress [hypochlorite (HOCl)-modified epitopes] in rats. Plasma alanine transaminase activities and histology showed progressively increasing liver injury during reperfusion, when hepatic GSSG and soluble MDA levels were elevated. At that time, few neutrophils were present in sinusoids. However, the number of hepatocytes positively stained for HOCl-modified epitopes increased from 6 to 24 h of reperfusion, which correlated with the bulk of hepatic neutrophil accumulation and extravasation into the parenchyma. Consistent with a higher oxidant stress at later times, hepatic GSSG and protein-bound MDA levels further increased. Treatment with the NADPH oxidase inhibitor diphenyleneiodonium chloride attenuated postischemic oxidant stress (GSSG, protein-bound MDA, and hepatocytes positively stained for HOCl-modified epitopes) and liver injury at 24 h of reperfusion. Ischemic preconditioning suppressed all oxidant stress biomarkers, liver injury, and extravasation of neutrophils. In conclusion, extravasated neutrophils generate HOCl, which diffuses into hepatocytes and causes oxidative modifications of intracellular proteins during the neutrophil-mediated reperfusion injury phase. Ischemic preconditioning is an effective intervention for reduction of the overall inflammatory response and, in particular, for limitation of the cytotoxic activity of neutrophils during the later reperfusion period.