IL-8 and IFN-gamma in tear fluid of patients with cystic fibrosis.

Cystic fibrosis (CF) is inherited as an autosomal recessive disorder. It is caused by mutations in the protein-coding gene of chromosome 7, resulting in chronic pulmonary disease and pancreatic insufficiency. The disease affects all secretory epithelia, including the eye. The pathogenesis of ocular changes in CF is still unknown, but the involvement of immunologic processes in patients with CF has been studied in recent years. We measured interleukin-8 (IL-8) and interferon-gamma (IFN-gamma) levels in tears in a group of patients and a group of normal controls to determine if the levels of these cytokines are elevated in CF. The levels of these cytokines in tears and the clinical severity of CF and eye disease were compared. Tear samples were collected from 24 patients with CF at the department of pediatric diseases, Medical University of Bialystok, Poland. Cytokine levels were determined by ELISA. Ophthalmic examinations, including tests for keratoconjunctivitis sicca (dry eye), were used to study the ocular surface. The tear levels of IL-8 and IFN-gamma in the CF patients were significantly higher than those in controls. The clinical severity of CF correlated significantly with the IL-8 and IFN-gamma levels. We found positive correlation between the tear levels of IFN-gamma and dry eye findings in CF patients. Our results suggest that the inflammatory cytokines IL-8 and IFN-gamma may play key roles in the regulation of ocular surface inflammation and the immunologic reaction in patients with CF. The tear levels of IL-8 and IFN-gamma may be candidate markers for evaluation of the clinical status of CF and eye disease. These findings help to provide a new insight into the pathogenesis of dry eye in patients with CF and provide potential targets for therapy.     

Elevated serum IgG4 levels in cystic fibrosis patients

The quantitative measurement of the IgG subclass composition of the sera from sixteen patients with cystic fibrosis has revealed grossly elevated levels of IgG4 in seven patients. The possible significance of this observation is discussed in relation to recent reports of a high incidence of immediate-type hypersensitivity in such patients.     

Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis.

To determine the role of chemokines in the pathogenesis of systemic sclerosis (SSc), we examined serum levels, spontaneous production by peripheral blood mononuclear cells (PBMC), and histological distribution in the affected skin, of MCP-1, MIP-1alpha and MIP-1beta in SSc patients. Serum levels of these chemokines were examined by ELISA in 58 patients with SSc and 20 normal controls. The levels of these chemokines in culture supernatants from PBMC were also measured by ELISA. Serum levels and spontaneous production levels by PBMC of MCP-1, MIP-1alpha, and MIP-1beta were significantly elevated in patients with SSc compared with normal controls. Elevated serum levels of MCP-1 and MIP-1alpha significantly correlated with the presence of pulmonary fibrosis. MCP-1 expression in the skin of SSc was immunohistochemically examined using anti-MCP-1 MoAb. MCP-1 was strongly expressed in the epidermis, inflammatory mononuclear cells, and vascular endothelial cells in the sclerotic skin of SSc patients, but not expressed in any control skin. Furthermore, the MCP-1 expression in inflammatory mononuclear cells and endothelial cells significantly correlated with earlier onset of SSc. Thus, MCP-1, MIP-1alpha and MIP-1beta may be involved in the disease process, possibly by augmenting leucocyte migration into the affected tissues in SSc. Furthermore, MCP-1 and MIP-1alpha may play an important role in the development of pulmonary fibrosis in SSc.     

MIP-1alpha expression in tissues from patients with hemophagocytic syndrome

Hemophagocytic syndrome (HPS) is a clinicopathologic syndrome that can be precipitated by a variety of causes and is characterized by a systemic activation of macrophages, which are induced to undergo phagocytosis. Chemokines play an important role in the inflammatory cell recruitment into tissues. We examined the expression of chemokines and cytokines in tissues exhibiting histologic evidence of HPS in a variety of settings: peripheral T-cell lymphoma, three patients; nasal T/NK cell lymphoma, one patient; subcutaneous panniculitis-like T-cell lymphoma, one patient; and chronic EBV infection, one patient. Compared with control tissues, we found elevated macrophage inflammatory protein-1alpha (MIP-1alpha) and interferon-gamma (IFN-gamma) expression, but not macrophage-derived chemotactic factor (MDC) or TNF-alpha, in tissues of patients with HPS irrespective of the cause or setting. MIP-1alpha can promote macrophage chemotaxis and IFN-gamma promotes macrophage activation. Elevated expression of IP-10 and monokine induced by IFN-gamma (Mig) was also detected in tissues exhibiting features of HPS, providing an explanation for the occurrence of chemoattraction of T-cells and NK cells. Immunohistochemical analysis of tissues with evidence of phagocytic activity in that site showed MIP-1alpha characteristically localized to endothelial cells of blood vessels and splenic sinuses, lymphocytes, and macrophages. These results provide evidence for MIP-1alpha chemokine expression in tissues from patients with HPS and suggest that MIP-1alpha may play an important role in the pathogenesis of the hemophagocytic syndrome.     

Increased circulating levels of plasma ATP in cystic fibrosis patients.

Recent studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette (ABC) transporter whose mutations are responsible for cystic fibrosis (CF), permeates ATP. However, little information is available concerning extracellular ATP concentrations in CF patients. Thus, the goal of this preliminary study was to determine the circulating levels of plasma ATP in CF patients. Circulating levels of plasma ATP were determined by the luciferin-luciferase assay in both CF patients and healthy volunteer control subjects. The two groups were compared using an analysis of variance. CF genotype and age, which ranged from 7 to 56 years, were also used to compare data by single-blind analysis. With comparable sample numbers, CF patients had statistically higher levels of circulating ATP (34%, P<0.01) when compared by analysis of covariance with the age of the subjects as the cofactor. The CF patients bearing the DeltaF508 genotype had a 54% (n=33, P<0.01) higher plasma ATP concentration compared to controls, while patients bearing other CF genotypes were similar to controls (n=10, P<0.4). We conclude that CF patients have higher circulating levels of ATP when compared to controls. Increased levels of plasma ATP, which is an important autocrine/paracrine hormone in many cell types, may be associated with chronic manifestations of the disease.     

Carcinoembryonic antigen, alpha 1-antitrypsin, and alpha-fetoprotein in the pancreas of patients with cystic fibrosis

The ductular accumulation of "abnormal mucus" is the key histologic feature in cystic fibrosis. This material is periodic acid-Schiff positive and diastase resistant, suggesting that it is glycoprotein in nature. We used the avidin-biotin-peroxidase method to identify this material using antibodies to the serum glycoproteins carcinoembryonic antigen, alpha 1-antitrypsin, and alpha-fetoprotein on paraffin sections of pancreas obtained from a total of 21 patients: 9 with cystic fibrosis, 5 with chronic pancreatitis, and 7 controls. The control patients had normal pancreatic histologic findings, no alpha 1-antitrypsin or alpha-fetoprotein was demonstrated, and only the ductular epithelium reacted weakly for carcinoembryonic antigen. The pancreas in pancreatitis showed fibrosis, acinar atrophy, and ectasia of the ducts that contained only a small amount of periodic acid-Schiff-positive material. This material reacted weakly for carcinoembryonic antigen and alpha 1-antitrypsin. The appearance of the pancreas in cystic fibrosis was similar to that in chronic pancreatitis. However, the ducts contained a greater amount of periodic acid-Schiff-positive material, mostly in the form of globules that reacted strongly for carcinoembryonic antigen and alpha 1-antitrypsin and weakly for alpha-fetoprotein, as did the ductular epithelium. This study shows that the periodic acid-Schiff-positive material in cystic fibrosis contains at least the three serum glycoproteins and that the accumulation may represent a possible defect in cellular synthesis, assembly, or transport of glycoproteins in the ducts.     

Elevated chemokine levels in bronchoalveolar lavage fluid of patients with eosinophilic pneumonia

BACKGROUND: Allergic lung inflammation is caused by accumulation and activation of different leukocyte subsets, such as eosinophils and T lymphocytes, in the lung. The chemokines are a large group of chemotactic cytokines that regulate leukocyte trafficking and may play an important role in allergic lung inflammation. OBJECTIVE: The purpose of this study was to evaluate the role of various chemokines, including eotaxin, RANTES, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1beta, and IL-8 in the pathogenesis of eosinophilic pneumonia (EP). METHODS: The concentrations of eotaxin, RANTES, MCP-1, MIP-1beta, and IL-8 in bronchoalveolar lavage fluid (BALF) were measured by using ELISA in 15 patients with EP, 10 with idiopathic pulmonary fibrosis, 10 with sarcoidosis, and 11 healthy volunteers. RESULTS: Eotaxin in BALF was high only in patients with EP, and its level correlated significantly with the number of eosinophils in BALF of patients with EP and healthy volunteers. MCP-1 and MIP-1beta in BALF were preferentially increased in patients with EP. There was a significant correlation between MCP-1 levels and the number of macrophages in BALF of patients with EP and healthy volunteers. CONCLUSION: Our findings suggest that these CC chemokines contribute to the pathogenesis of EP through the specific recruitment of leukocyte subsets in the lung.     

Elevated IL-5 levels in pleural fluid of patients with paragonimiasis westermani

To investigate the pathogenic mechanisms of eosinophilic pleural effusion in patients with paragonimiasis, we measured the levels of IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) in pleural effusions. Samples were obtained from 11 patients with Paragonimus westermani infection. In addition, samples from 12 patients with pleural transudates, 16 with tuberculous pleurisy, seven with empyema and 20 with lung cancer were also examined. Eosinophilia was remarkable in peripheral blood (range 4-34%, median 23.4%) and pleural fluid (range 0-95%, median 71%) of paragonimiasis patients. IL-5 concentrations in pleural effusions of paragonimiasis were markedly higher than those in other groups. Although marked elevation of GM-CSF and IFN-gamma levels was observed in pleural effusion of empyema and tuberculosis patients, it was marginal in the pleural effusion of paragonimiasis patients. In paragonimiasis patients, IL-5 levels in the pleural effusion correlated well with the percentage of eosinophils in peripheral blood and pleural fluid. Such a correlation was not observed between GM-CSF levels in pleural effusion and percentages of eosinophils in pleural fluid or peripheral blood. Our findings suggest that in paragonimiasis IL-5 in the local inflammatory site is particularly important in mediating eosinophilia in peripheral blood and pleural effusion.     

Prediction of mortality in patients with cystic fibrosis.

BACKGROUND. The majority of patients with cystic fibrosis die in early adulthood of lung disease. Lung transplantation is a treatment option for patients with advanced pulmonary disease, although the waiting period for organs may be as long as two years. Our purpose was to determine whether the risk of death due to respiratory failure could be predicted one or two years in advance on the basis of pulmonary function, blood gas levels, and nutritional status. METHODS. The study cohort consisted of 673 patients followed between 1977 and 1989. In each patient, pulmonary function, blood gas levels, nutritional status, and vital status were assessed between 1977 and 1987. Cox proportional-hazards regression analysis was used to compute the relative risk of death within one or two years after particular measurements. The effects of age and sex on mortality were also included in the analysis. RESULTS. One hundred ninety patients (28 percent) died during the study period. Overall, patients with a forced expiratory volume in one second (FEV1) less than 30 percent of the predicted value, a partial pressure of arterial oxygen below 55 mm Hg, or a partial pressure of arterial carbon dioxide above 50 mm Hg had two-year mortality rates above 50 percent. Among the laboratory measurements, the FEV1 was the most significant predictor of mortality, but age and sex were also significant in predicting risk. After adjustment for age and sex, the relative risk of death within two years was 2.0 (95 percent confidence interval, 1.9 to 2.2) for each decrement in the FEV1 of 10 percent below the predicted value. Among patients with the same FEV1, the relative risk of death was 2.0 (95 percent confidence interval, 1.5 to 2.6) in patients 10 years younger than other patients, and 2.2 (1.6 to 3.1) in female patients as compared with male patients. CONCLUSIONS. Patients with cystic fibrosis should be considered candidates for lung transplantation when the FEV1 falls below 30 percent of the predicted value. Female patients and younger patients may need to be considered for transplantation at an earlier stage.     

Polymorphisms of TGF-beta1 in cystic fibrosis patients

There is a significant phenotypic variance among cystic fibrosis (CF) patients. Due to the role of TGF-beta1 in fibrotic processes we investigated its role in CF pathogenesis. TGF-beta 1 codons 10 and 25 were genotyped in 118 Czech CF patients and 268 controls by PCR-ARMS. Difference between CF and controls was found at codon 10, lower frequency of T/T homozygotes, and codon 25, higher frequency of G/C heterozygotes. We did not prove the association of TGF-beta1 polymorphisms and lung function in CF, however, the TT (codon 10)/GG (codon 25) genotype was preferentially associated with CF-related liver disease and diabetes. Independent of the TGF-beta1 genotype, production of cytokine was higher in patients than in controls with the notable exception of very low levels in Burkholderia cepacia complex colonized patients. In CF, both extremes, highest or lowest TGF-beta 1 production, were associated with impaired lung function.     

Increased PIVKA-II concentrations in patients with cystic fibrosis.

Serum vitamin K concentrations and prothrombin induced by absence of vitamin K (PIVK-II) concentrations were assayed in 43 patients with cystic fibrosis. Twenty nine showed a normal PIVKA-II and vitamin K concentrations; 14 showed an increased PIVKA-II concentration, in one of whom serum vitamin K was decreased. Although their vitamin K concentrations were normal, some patients with cystic fibrosis still had an increased PIVKA-II. There was a significant correlation between PIVKA-II concentrations and the administration of antibiotics, a factor which has not previously been considered responsible for an increase in PIVKA-II.     

Elevated interleukin-18 levels in bronchoalveolar lavage fluid of patients with eosinophilic pneumonia

BACKGROUND: Interleukin (IL)-18 can induce Th2 cytokine production particularly in collaboration with IL-2. Accumulation of Th2 cells and increased levels of Th2 cytokines are found in bronchoalveolar lavage fluid (BALF) from patients with eosinophilic pneumonia (EP). To evaluate the role of IL-18 in the pathogenesis of EP, we measured the concentration of IL-2, IL-12, IL-18, and Th2 cytokines in BALF from patients with EP. METHODS: The concentrations of interferon (IFN)-gamma, IL-2, IL-5, IL-10, IL-12, IL-13, and IL-18 in BALF were measured in patients with idiopathic acute eosinophilic pneumonia (AEP), with idiopathic chronic eosinophilic pneumonia (CEP), with sarcoidosis and healthy volunteers (HV). RESULTS: The BALF concentrations of Th2 cytokines, IL-5, IL-10, and IL-13, were higher in patients with EP than in sarcoidosis and control. The IL-2 level in BALF was higher in EP than in sarcoidosis and control. The IL-18 and IL-12 (p40 + p70) levels were higher in patients with EP than sarcoidosis, while the level of IL-12 (p70) was below the detection limit in patients with EP. There was a significant correlation between IL-2 level and both IL-5 and IL-13 in BALF of patients with EP. CONCLUSIONS: Our findings suggest that IL-18 may contribute to Th2 cytokine-dominant responses in patients with EP in collaboration with IL-2.     

Recovery of mycobacteria from patients with cystic fibrosis.

Despite decontamination, overgrowth by pseudomonads renders cultural isolation of mycobacteria from respiratory specimens of patients with cystic fibrosis (CF) difficult or impossible. We performed a prospective study by comparing levels of reduction of overgrowth and recovery of mycobacteria using either pretreatment with N-acetyl-L-cysteine (NALC)-NaOH alone or pretreatment with NALC-NaOH and then with oxalic acid. From 406 specimens of 148 CF patients, 11 specimens were positive for mycobacteria, 5 of which grew mycobacteria after decontamination by either procedure. Three specimens grew mycobacteria only after decontamination with NALC-NaOH, whereas three specimens grew mycobacteria only after treatment with NALC-NaOH followed by oxalic acid but were overgrown after decontamination with NALC-NaOH. Thus, inactivation of mycobacteria by the more aggressive oxalic acid treatment offsets its beneficial effect of reducing the proportion of cultures overgrown with microorganisms other than mycobacteria.     

Microbiology of airway disease in patients with cystic fibrosis.

Individuals with cystic fibrosis have abbreviated life spans primarily due to chronic airway infection. A limited number of types of organisms are responsible for these infections, with Staphylococcus aureus and Pseudomonas aeruginosa being of primary importance. In the pre-antibiotic era, greater than 90% of deaths due to infection were caused by S. aureus and death usually occurred in the first 2 years of life. With the advent of effective antistaphylococcal therapy, life spans increased and P. aeruginosa became the pathogen of primary importance. P. aeruginosa isolates recovered from patients with cystic fibrosis have a unique phenotypic characteristic referred to as "mucoid." The mucoid phenotype is due to the production of a mucoid exopolysaccharide. A mucoid exopolysaccharide is believed to play a central role in the establishment of chronic pseudomonal lung infection in these patients. A third organism, Pseudomonas cepacia, has recently been detected in the airways of older patients with cystic fibrosis and is associated with increased mortality. The virulence of P. cepacia is not understood, but the organism is extremely refractory to antimicrobial therapy. Other bacteria, including Haemophilus influenzae and members of the family Enterobacteriaceae, appear to play a secondary role in airway infection. Aspergillus fumigatus is the most important fungal agent causing allergic bronchopulmonary disease. The role of viruses has only recently been examined. At least in some patients with cystic fibrosis, respiratory syncytial virus may be important in predisposing to subsequent bacterial infections.     

The effect of the decoy molecule PA401 on CXCL8 levels in bronchoalveolar lavage fluid of patients with cystic fibrosis

Background

The chemokine interleukin-8 (CXCL8) is a key mediator of inflammation in airways of patients with cystic fibrosis (CF). Glycosaminoglycans (GAGs) possess the ability to influence the chemokine profile of the CF lung by binding CXCL8 and protecting it from proteolytic degradation. CXCL8 is maintained in an active state by this glycan interaction thus increasing infiltration of immune cells such as neutrophils into the lungs. As the CXCL8-based decoy PA401 displays no chemotactic activity, yet demonstrates glycan binding affinity, the aim of this study was to investigate the anti-inflammatory effect of PA401 on CXCL8 levels, and activity, in CF airway samples in vitro.

Methods

Bronchoalveolar lavage fluid (BALF) was collected from patients with CF homozygous for the ΔF508 mutation (n = 13). CXCL8 in CF BALF pre and post exposure to PA401 was quantified by ELISA. Western blot analysis was used to determine PA401 degradation in CF BALF. The ex vivo chemotactic activity of purified neutrophils in response to CF airway secretions was evaluated post exposure to PA401 by use of a Boyden chamber-based motility assay.

Results

Exposure of CF BALF to increasing concentrations of PA401 (50–1000 pg/ml) over a time course of 2–12 h in vitro, significantly reduced the level of detectable CXCL8 (P < 0.05). Interestingly, PA401 engendered release of CXCL8 from GAGs exposing the chemokine susceptible to proteolysis. Subsequently, a loss of PA401 was observed (P < 0.05) due to proteolytic degradation by elastase like proteases. A 25% decrease in neutrophil chemotactic efficiency towards CF BALF samples incubated with PA401 was also observed (P < 0.05).

Conclusion

PA401 can disrupt CXCL8:GAG complexes, rendering the chemokine susceptible to proteolytic degradation. Clinical application of a CXCL8 decoy, such as PA401, may serve to decrease the inflammatory burden in the CF lung in vivo.     

Elevated serum D-arabinitol levels in patients with sarcoidosis.

D-Arabinitol has been found in the serum of patients with candidiasis in an incidence varying from 38 to 82%. While screening serum by gas chromatography for the presence of this sugar, we observed elevated concentrations in several patients with sarcoidosis. In an attempt to determine the significance of this chance observation, we tested serum from additional patients with sarcoidosis along with serum from patients with other clinical conditions known to be associated with elevated D-arabinitol levels. Of 53 patients with sarcoidosis, 27 (51%) had elevated concentrations of this compound. Only one of these patients had decreased renal function (creatinine, 2.5 mg/dl). We were unable to correlate elevated values with extent of the disease. Although the significance of this finding is not clear, it may represent a clue to the pathogenesis of sarcoidosis.     

Pseudomonas aeruginosa flagellar antibodies in patients with cystic fibrosis.

An enzyme-linked immunosorbent assay specific for flagellum type (a or b) of Pseudomonas aeruginosa was used to detect serum immunoglobulin antibodies in 98 random outpatients and 14 colonized cystic fibrosis patients. Antibodies were detected to both types of flagella in addition to M-2 lipopolysaccharide. Titers to both flagellar antigens (FlAg) were 10 to 100 times higher in cystic fibrosis patients than in random outpatients of a comparable age group. Mean antibody titers against b-type FlAg were 454 for outpatients (ages newborn to 21 years), whereas the mean titer for cystic fibrosis patients (ages 6 to 21 years) was 51,520. Titers against a-type FlAg were generally lower, with mean outpatient titers of 68 and mean cystic fibrosis patient titers of 34,323. Differences were also seen in antibody titer against M-2 lipopolysaccharide, but these differences did not correspond to M-2 FlAg titers. In 98 random outpatients (ages newborn to 86 years), FlAg titers generally increased with age. To demonstrate further specificity of the enzyme-linked immunosorbent assay for flagellum antibody, Western blots were performed with selected high-titer cystic fibrosis patient sera. Sera that had a high titer (greater than 25,600) for b- or a-type FlAg showed a corresponding reactive band. These results demonstrate that flagellum antibodies are produced in humans in response to P. aeruginosa infection.     

Immediate hypersensitivity in patients with cystic fibrosis

Twenty-three out of thirty patients with cystic fibrosis gave strong immediate skin hypersensitivity reaction to a wide variety of allergens. Seventy-five per cent of these had a markedly elevated serum IgE concentration whereas those patients who had negative Type 1 immediate skin reactions also had normal levels of serum IgE. The sputum of those patients with immediate skin reactivity also had positive precipitins to a variety of antigens.