Interleukin 8 secretion from monocytes of subjects heterozygous for the deltaF508 cystic fibrosis transmembrane conductance regulator gene mutation is altered

Patients with cystic fibrosis (CF) exhibit an excessive host inflammatory response. The aim of this study was to determine (i) whether interleukin 8 (IL-8) secretion is increased from monocytes from subjects heterozygous as well as homozygous for cystic fibrosis transmembrane conductance regulator (CFTR) mutations and (ii) whether this is due to increased cell surface lipopolysaccharide (LPS) receptors or, alternatively, increased activation of mitogen-activated protein kinases (MAPK). The basal level of IL-8 secretion was higher from monocytes from CF patients than from monocytes from healthy controls (P = 0.02) and obligate heterozygotes (parents of the CF patients). The 50% effective concentrations for LPS-induced IL-8 production for monocytes from both CF patients and obligate heterozygotes were 100-fold lower than those for monocytes from healthy controls (P < 0.05). No differences in the levels of IL-1beta production were seen between these groups. Expression of the LPS surface receptors CD14 and Toll-like receptor 4 were not different between CF patients and healthy controls. In contrast, phosphorylation of the MAPKs p38 and ERK occurred at lower doses of LPS in monocytes from patients heterozygous and homozygous for CFTR mutations. These results indicate that a single allelic CFTR mutation is sufficient to augment IL-8 secretion in response to LPS. This is not a result of increased LPS receptor expression but, rather, is associated with alterations in MAPK signaling.     

Reduced IL-10 secretion by CD4+ T lymphocytes expressing mutant cystic fibrosis transmembrane conductance regulator (CFTR)

Expression of the CFTR protein is thought to be physiologically important only in exocrine epithelial cells. However, chronic respiratory inflammation and infection remain unexplained phenomena in disease pathogenesis. Non-transformed, antigen-responsive CD4⁺ T cells cloned from healthy controls and CF patients homozygous or heterozygous for the δF508 mutation transcribed CFTR mRNA and expressed immunoreactive cytoplasmic CFTR protein. T cell clones (TCC) from controls and CF patients displayed equivalent Ca²⁺-mediated Cl⁻ current; however, TCC from patients with CF but not controls displayed defective cAMP-mediated Cl⁻ current. Although CF-derived TCC preserved mitogen and antigen proliferative responses and specificity to tetanus toxoid epitopes, they selectively secreted ≈ 45% less IL-10 compared with control TCC after activation with concanavalin A (Con A) (624 ± 101 versus 1564 ± 401 pg/ml per 10⁶ cells, respectively; P = 0.04) or anti-CD3/phorbol ester (5148 ± 1634 versus 11 788 ± 2390 pg/ml; P = 0.05). This difference was independent of atopy. Secretion of interferon-gamma, IL-2, and IL-4 was comparable in CF and control TCC after both forms of activation, while IL-5 was reduced in CF TCC following anti-CD3/phorbol myristate acetate (PMA) but not after Con A. We conclude that expression of mutant CFTR in human TCC is accompanied by ion channel dysfunction characteristic of the CF phenotype, and is accompanied by a reduction in IL-10 secretion after polyclonal activation. It is possible that disruption of IL-10-mediated anti-inflammatory homeostasis may contribute to early onset sustained inflammation in CF airways.     

Selective Up-Regulation of Chemokine IL-8 Expression in Cystic Fibrosis Bronchial Gland Cells in Vivo and in Vitro

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the ΔF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1β and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured ΔF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl⁻ concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl⁻ concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.     

mGluR1 interacts with cystic fibrosis transmembrane conductance regulator and modulates the secretion of IL-10 in cystic fibrosis peripheral lymphocytes

Cystic fibrosis (CF) is caused by the mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. CFTR dysfunction in T cells could lead directly to aberrant immune responses. The action of glutamate on the secretion of IL-8 and IL-10 by lymphocytes derived from healthy subjects and cystic CF patients, as well as the expression of metabotropic glutamate receptor subtype 1 (mGluR1) in the membrane fractions of lymphocytes was investigated. Our results have shown that CF-derived T-cells in the presence of IL-2 produce more IL-8 and IL-10, than T-cell from healthy control. However, only in normal lymphocytes a significant increase (144%) in the IL-10 secretion during exposure to high concentration of glutamate (10(-4)M) was detected. Glutamate-dependent secretion of IL-10 was not inhibited either by NMDA-receptor (NMDAR), or by AMPA-receptor (AMPAR) antagonist. Only mGluR1 antagonist, LY367385, strongly decreases the production of IL-10. Furthermore, the content of mGluR1, as well as cystic fibrosis transmembrane conductance regulator-associated ligand (CAL), Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), was analyzed in plasma membrane of lymphocytes after immunoprecipitation of CFTR. We have found that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with metabotropic mGluR1, but the level of surface exposed mGluR1 in CF-lymphocytes was much lower than in normal cells. Besides, our results have shown that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with NHERF-1 and CAL; however in lymphocytes with CFTR mutation the amount of cell-surface expressed CFTR-CAL complex was greatly decreased. We have concluded that CFTR and mGluR1 could compete for binding to CAL, which in turn downregulates the post-synthetic trafficking of mGluR1 and decreases the synthesis of IL-10.     

Heterogeneity of phenotype in two cystic fibrosis patients homozygous for the CFTR exon 11 mutation G551D.

In the heterozygous state, the cystic fibrosis transmembrane conductance regulator (CFTR) exon 11 mutation G551D has been described as "severe," causing pancreatic insufficiency. Two cystic fibrosis (CF) patients homozygous for this mutation showed a mild rather than severe pancreatic phenotype and a variable pulmonary phenotype.     

Cystic fibrosis transmembrane conductance regulator gene screening and clinical correlation in Taiwanese males with congenital bilateral absence of the vas deferens

BACKGROUND: In Taiwan, an area with a very low incidence of cystic fibrosis (CF), we first screened for the most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and looked for clinical correlations in 27 patients with clinically diagnosed congenital bilateral absence of the vas deferens (CBAVD). METHODS AND RESULTS: The clinical results showed that none of the 27 patients had CF symptoms. We did not detect any definite renal anomaly ultrasonographically. Mutation analysis was carried out on these 27 cases and 46 normal fertile males as controls. No mutations of Delta F508 or R117H were identified in any of the samples analysed. In the screening of IVS8-poly T, five of the 27 CBAVD patients showed the homozygous genotype for 5T/5T, 14 showed the heterozygous genotype for 5T/7T and eight showed the homozygous genotype for 7T/7T. The frequency of 5T alleles was 44.4%, which was significantly higher than in the 46 normal fertile males, for which there was a 5T frequency of 5.4%. CONCLUSIONS: The absence of major mutations of CFTR genes could be related to the much lower CF incidence in Taiwan. Further investigations into differences in the mutation spectrum of other CFTR genes are needed for a better understanding of the development of Taiwanese-Oriental CBAVD.     

Frequency and clinical significance of the S1235R mutation in the cystic fibrosis transmembrane conductance regulator gene: results from a collaborative study

More than 900 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been reported to the cystic fibrosis (CF) consortium. A missense mutation, S1235R, was originally reported in a CF patient with a second mutation (G628R) on the same chromosome. The clinical significance of S1235R was not clear. S1235R is not among the commonly reported mutations, and it is not routinely screened for in most laboratories. However, we have detected the S1235R allele at a frequency that is significantly higher than that of many other CF mutations. Among more than 3,000 patients tested for either a possible diagnosis of CF or to determine CF carrier status, we identified 51 patients heterozygous for S1235R. No patients were homozygous for S1235R. Five patients were compound heterozygotes for a second CFTR mutation: two cases (one family) were N1303K/S1235R and three unrelated cases were deltaF508/S1235R. Our data suggest that S1235R, when combined with a second CF mutation, may be pathogenic, although phenotypic manifestations appear to be variable. The possibility that this represents a rare polymorphism cannot be discounted completely. Genetic counseling is difficult when S1235R is identified, even in the presence of a second known mutation, especially in prenatal cases.     

Increased monocyte oxidase activity in cystic fibrosis heterozygotes and homozygotes.

Freshly isolated monocytes from cystic fibrosis (CF) heterozygotes and homozygotes had significantly increased oxygen uptake and superoxide formation after surface glycoprotein stimulation than did monocytes from age- and sex-matched controls. Lack of differences among the genotypes in inhibition by simple sugars of the concanavalin A-stimulated superoxide production and lack of differences in concanavalin A-binding surface proteins suggested that different regulation of the oxidase pathway produced the increased oxygen uptake and superoxide formation in CF patients and carriers. This regulatory role is consistent with the predicted structure of the CF gene product. The results support the hypothesis that the mononuclear phagocytes of CF heterozygotes have a significantly increased ability to kill intracellular microbes and may confer a selective advantage to the host.     

Clinical features of cystic fibrosis patients with rare genotypes.

We describe the clinical features of seven cystic fibrosis patients from southern Italy who bear rare genotypes: (1) a patient homozygous for the 2183 AA-->G mutation who was affected by a very early pulmonary form of cystic fibrosis, and five patients who were compound heterozygotes either for the 2183 AA-->G mutation or for the I148T mutation, in both instances with the delta F508 mutation; and (2) a patient homozygous for the early nonsense R553X mutation who showed only a moderately severe form of cystic fibrosis. Our results confirm that environmental or genetic factors unrelated to the CF disease contribute significantly to the development of the phenotype.     

Nasal epithelial ion transport and genetic analysis of infertile men with congenital bilateral absence of the vas deferens

It has been suggested that congenital bilateral absence of thevas deferens (CBAVD), an important cause of male infertility,is a variant of cystic fibrosis (CF). This study describes adefect in chloride conductance across the nasal epithelium ofsubjects with CBAVD which is dissimllar to that found in patientswith CF. It also demonstrates normal sodium transport acrossthe nasal epithelium in these men, in contrast to patients withCF who exhibit increased sodium absorption. The increased frequencyof CFTR mutations in these men implicates the CFTR gene in thepathogenesis of this disorder. Genetic analysis of men withCBAVD who were heterozygous for a known CFTR mutation failedto identify a second mutation within any of the exons or intronsof the CFTR gene. These results demonstrate that most men presentingwith CBAVD are not compound heterozygotes for mutations withinthe CFTR gene and can be distinguished from individuals withatypical or asymptomatic CF on the basis of the bioelectricproperties of their nasal epithelium. We postulate that mutationsin the promoter region or at other regulatory sites of the CFTRgene may be reponsibie for the CBAVD phenotype in a proportionof cases.     

Cystic fibrosis genotype and bacterial infection: a possible connection

Patients with cystic fibrosis (CF) have repeated bacterial infection of the airways, which can lead to chronic infection. There is evidence that disease severity is determined by the genetic mutations present. This study aims to establish if CF genotype is related to the frequency and types of airway bacterial infection. Adult patients attending the regional CF unit are followed for two years and assigned to one of three groups depending on whether they are chronically infected with Burkholderia cepacia complex (BCC) organisms, Pseudomonas aeruginosa, or neither of these organisms. Genotype analysis is performed on all patients to determine which of the cystic fibrosis transmembrane regulator (CFTR) gene mutations are present. The numbers and types of organism with the CFTR mutations isolated from sputum are identified. Data are available on 59 patients: 15 colonised with BCC, 24 colonised with P. aeruginosa, and 20 not colonised with either organism. Twenty patients were homozygous for deltaF508, 25 were heterozygous, and the deltaF508 mutation was not present in the remaining 14 patients. Patients homozygous or heterozygous for the deltaF508 mutation had an increased likelihood of colonisation with BCC or P. aeruginosa, an increased number of positive sputum cultures and a higher frequency of multiple infecting organisms. Cystic fibrosis mutational analysis identified seven patients who had the R117H mutation. These patients were less likely to be colonised with BCC or P. aeruginosa. In conclusion, patients homozygous or heterozygous for the deltaF508 deletion are more likely to suffer airway colonisation with BCC or P. aeruginosa, with increased numbers of positive sputum cultures and infecting organisms. Those with the R117H mutation are less likely to be colonised by Gram-negative organisms.     

Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.     

Mutation spectrum of the CFTR gene in Taiwanese patients with congenital bilateral absence of the vas deferens

BACKGROUND: Clinically affected cystic fibrosis (CF) patients present a spectrum of genital phenotypes ranging from normal fertility to moderately impaired spermatogenesis and congenital bilateral absence of vas deferens (CBAVD). Little is known about the CF incidence in the Taiwanese population. It has been shown that the CBAVD in men without clinical evidence of CF is associated with a high incidence of mutated CFTR (cystic fibrosis transmembrane conductance regulator) alleles. In order to understand the involvement of the CFTR gene in the aetiology of Asian/Taiwanese male infertility, we screened the entirety of the CFTR gene in 36 infertile males with CBAVD. METHODS: Temporal temperature gradient gel electrophoresis (TTGE) followed by direct DNA sequencing was used. RESULTS: Five mutations, p.V201M, p.N287K, c.-8G > C (125G > C), p.M469I and p.S895N, were found in five of the patients. p.N287K occurred in the first transmembrane-spanning domain, p.M469I in the first ATP-binding domain and p.S895N in the second transmembrane-spanning domain, were novel. In addition, seven homozygous and seven heterozygous 5T alleles in the intron 8 poly(T) tract were found. The overall frequency of CFTR mutant alleles in Taiwanese CBAVD males was 26 out of 72 = 36%. This finding was lower than the published frequency of CFTR mutations in other ethnic CBAVD patients (ranging from 50 to 74%). The frequency of p.M470V in Taiwanese CBAVD patients is not significantly different from that in the general population (P = 0.12). CONCLUSIONS: The results of this study add to the short list of Taiwanese/Asian CFTR mutations. Unlike Caucasian patients, the CFTR mutations cannot account for the majority of Taiwanese CBAVD. This is consistent with the low incidence of CF in the Asian/Taiwanese population. Furthermore, the mutation spectrum of CFTR in CBAVD patients does not overlap with the Caucasian CFTR mutation spectrum.     

Increased elastase secretion by peripheral blood monocytes in cystic fibrosis patients.

Morbidity and mortality in cystic fibrosis (CF) is predominantly due to destruction of pulmonary tissue. The host immune response may, in part, play a pathogenic role in pulmonary destruction in these patients. To further understand host immune response in CF, we examined the state of activation of peripheral blood monocytes in CF. Baseline elastase activity was 2.2-fold greater in the CF monocytes than in controls. Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) and high molecular weight polysaccharide (HMP) increased elastase activity in both control and CF monocytes, with a greater absolute increase in the CF monocytes. There was no difference in baseline or MEP-stimulated secretion of interleukin-1 (IL-1) or interleukin-6 (IL-6) between CF and control monocytes. Ibuprofen enhanced both MEP and HMP-stimulated elastase activity, whereas dexamethasone suppressed both baseline and stimulated elastase activity greater than 20% in both CF and control monocytes. These results suggest that circulating monocytes in CF are stimulated in vivo, resulting in a remarkably elevated elastase activity in vitro. Elevated elastase release by peripheral blood monocytes as they enter the lung in response to chemotactic stimuli may contribute to lung destruction in CF.     

Macrophages from high-risk HLA-DQB1*0201/*0302 type 1 diabetes mellitus patients are hypersensitive to lipopolysaccharide stimulation

Levels of nonantigen-induced pro-inflammatory cytokines and prostaglandin in macrophages isolated from human leucocyte antigen (HLA)-matched type 1 diabetes mellitus patients, first-degree relatives and healthy controls were determined. We hypothesize that monocytes isolated from patients are sensitized or preactivated and therefore, have an altered response to in vitro stimulus compared with control groups as measured by levels of pro- and anti-inflammatory mediators. In this study, peripheral blood monocytes were differentiated to macrophages with macrophage-colony stimulating factor (M-CSF) to determine lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-12 and prostaglandin E-2 (PGE-2) secretion from hetero- or homozygous HLA DQB1*0201 and *0302 type 1 diabetes mellitus patients, first-degree relatives and homozygous HLA DQB1*0602 healthy controls. LPS-stimulated secretion of TNF-alpha, IL-1beta and IL-6 was immediate and markedly higher in the HLA-DQB1*0201/*0302 type 1 diabetes patients compared with all other groups including HLA-matched healthy first-degree relatives. In DQB1*0201/*0302 diabetes patients PGE-2 secretion was delayed but increased by LPS stimulation compared with HLA-matched healthy relatives. IL-12 was not detected at any condition. These data suggest that macrophages from DQB1*0201/*0302 type 1 diabetes patients are sensitized to secrete both cytokines and PGE-2 following nonantigenic stimulation. Sensitized macrophages may be important to high-risk DQB1*0201/*0302-associated type 1 diabetes.     

The CFTR-mediated protein secretion defect: pharmacological correction

The cystic fibrosis transmembrane conductance regulator (CFTR) mediates secretion of mucins and serous proteins. The aim was to correct pharmacologically the CFTR defect in protein secretion in airway gland cells and so to correct the viscous mucous secretions in cystic fibrosis (CF) airways and lungs. The strategies tested included direct activation of CFTR, bypass of CFTR-mediated protein secretion and movement of the mutated form of CFTR (DeltaF(508)-CFTR) to the cell membrane. Compounds related to 3-isobutyl-1-methylxanthine (IBMX), including a selective type-IV phosphodiesterase inhibitor and the adenosine receptor antagonists 8-cyclopentyltheophylline (CPT) and 8-cyclopentyl-1,3-dipropylxanthine (CPX), corrected the defective beta-adrenergic stimulation of mucin secretion in CFTR antibody-inhibited submandibular gland cells. CPT also corrected lactoferrin secretion in DeltaF(508)/DeltaF(508)-CFTR nasal gland cells. The data suggest that correction of CFTR protein secretion activity is not mediated by excessive increase in cyclic AMP, involves direct interaction with CFTR but does not require increase in CFTR Cl(-) channel activity. Regulated glycoprotein secretion was characterised in the airway gland cell line Calu-3 to investigate whether a CFTR bypass is present. Studies of DeltaF(508)-CFTR trafficking using confocal imaging showed that some DeltaF(508)-CFTR colocalised with the apical membrane protein CD59; however a large amount was mislocalised within the cell. The results showing pharmacological correction of the defective CFTR-mediated protein secretion afford promise for the development of a rational drug therapy for CF patients.     

Enhancement of lipopolysaccharide-induced tumor necrosis factor secretion by hyperimmune serum from chronic infected patients

Patients with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa lung infection have a very high load of endotoxins in their lungs. However, sepsis practically never occurs in this group of patients and the presence of tumor necrosis factor (TNF)- (one of the mediators of septic shock) in serum from chronically infected CF patients is contentious. The purpose of this study was to investigate the effect of hyperimmune serum from patients with CF on lipopolysaccharide (LPS, endotoxin)-induced TNF secretion from human peripheral blood mononuclear cells (PBMC). PBMC were purified from healthy donors and stimulated with a mixture of purified LPS from P. aeruginosa and serum from chronically infected CF patients or healthy controls. TNF in the cell supernatants was detected by an enzyme-linked immunosorbent assay method. CF sera showed a pronounced potentiating effect on TNF secretion from human PBMC induced by LPS from P. aeruginosa. In comparison, serum from healthy controls did not have this effect. By contrast, CF serum and serum from healthy controls showed only little potentiating effect when using LPS from Salmonella abortus equi at concentrations above 0.01 g/ml per 2 × 10⁶ PBMC. This indicates a specific interaction between P. aeruginosa LPS and CF serum which enhances TNF secretion. The TNF responses varied depending on the sera used in the preincubation with LPS, and correlated positively to the specific IgG, IgA, and IgM anti-P. aeruginosa LPS titers of the sera. However, since TNF is hardly detectable in sera from these patients another LPS- and/or TNF-inhibitory activity may be present in these sera.     

Nitric oxide has no beneficial effects on ion transport defects in cystic fibrosis human nasal epithelium

Nitric oxide (NO) has been reported to activate Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) and inhibit epithelial Na+ absorption mediated by amiloride-sensitive epithelial Na+ channels (ENaC). These ion transport systems are defective in cystic fibrosis (CF): Cl- secretion by CFTR is impaired and Na+ absorption by ENaC is dramatically increased. By activating CFTR and depressing ENaC, NO is a potentially beneficial therapeutic agent for ion transport defects in human CF respiratory epithelia. To assess the effects of NO on human respiratory epithelial cells, the NO donors sodium nitroprusside (SNP) and spermine NONOate were applied to primary cultured nasal cells, surgically obtained from non-CF and CF patients. Measurements of transepithelial short-circuit current (ISC) showed that NO has no inhibitory potency against amiloride-sensitive nasal ENaC (nENaC) or amiloride-insensitive Na+-absorbing mechanisms in non-CF and CF epithelia. Furthermore, NO had no stimulatory effect on Cl- secretion by CFTR or any other Cl- conductance pathway in either tissue. Although NO elevated the intracellular Ca2+ concentration, we did not detect any activation of Ca2+-dependent Cl- channels. These results demonstrate that NO has no beneficial effect on CF epithelial cells of the upper airways.