Comparison of the carbohydrate composition of alpha 2-macroglobulin from patients with cystic fibrosis and normal controls

The putative involvement of alpha 2-macroglobulin (alpha 2M) in the pathogenesis of cystic fibrosis has long been a subject of controversy. Previous reports have indicated that there are alterations in the carbohydrate moieties of alpha 2M in the disease. In the present study the carbohydrate composition of alpha 2M isolated from the plasma of six patients with CF was compared to that of alpha 2M from six age- and sex-matched normal controls. The carbohydrate composition of CF alpha 2M expressed as mean mumol carbohydrate +/- SD per 100 mg protein was: fucose 0.70 +/- 0.12; mannose 14.07 +/- 1.31; galactose 6.72 +/- 0.65; glucosamine 15.38 +/- 1.59; sialic acid 5.52 +/- 0.33 while that of normal control alpha 2M was: fucose 0.69 +/- 0.11; mannose 14.42 +/- 1.21; galactose 6.91 +/- 0.52; glucosamine 16.13 +/- 1.77; sialic acid 5.58 +/- 0.31. Therefore, contrary to previous reports, this paper demonstrates that there is no difference in the carbohydrate composition of alpha 2M in cystic fibrosis.     

A monoclonal antibody recognizes structural variation in cystic fibrosis alpha 2-macroglobulin

alpha 2-Macroglobulin (alpha 2M) is a major plasma protease inhibitor that has been studied because of its suggested role in the pathology of cystic fibrosis (CF). A panel of monoclonal antibodies specific for human alpha 2M were produced and screened for their ability to bind to a number of human alpha 2M samples. We have used these antibodies to characterize individual antigenic sites in this protein. alpha 2M was purified from plasma by polyethylene glycol precipitation followed by zinc chelate chromatography. A total of 23 alpha 2M samples in the native configuration, as well as the nucleophile-treated configuration, were screened by the panel of 18 monoclonal antibodies in an enzyme-linked immunosorbent assay procedure. Five of the samples tested were from individuals with cystic fibrosis. alpha 2M from family members of two of these patients was subsequently tested for reactivity with the monoclonal antibodies. One antibody, SAM94, exhibited a significant difference in binding to alpha 2M obtained from CF patients as compared with control individuals. This difference was particularly apparent in the binding of SAM94 to the nucleophile-treated CF alpha 2M; SAM94 showed significantly reduced binding to four of five unrelated CF individuals (p less than 0.005) and three of four cystic fibrosis obligate heterozygotes (p less than 0.005).     

Studies on cystic fibrosis using isoelectric focusing. II. Demonstration of deficient proteolytic cleavage of alpha2-macroglobulin in cystic fibrosis plasma.

A protein with an isoelectric point (pI) of 5.48 was found to be deficient in plasma from most cystic fibrosis (CF) homozygotes and obligate heterozygote carriers of CF as compared with normal control plasma. Purification of the protein with a pI of 5.48 from normal plasma was performed using ammonium sulfate precipitation, DEAE-cellulose and CM-cellulose chromatography, Sephadex G-200- gel filtration, starch block electrophoresis, and Sepharose 4B gel filtration. The purified protein migrated as a single band on polyacrylamide gel electrophoresis, and displayed a single arc on immunoelectrophoresis against polyvalent antiserum to whole human serum. Results from various techniques used in its characterization indicate that this protein is a fragment of alpha2-macroglobulin (alpha2M) which is derived from alpha2M by proteolytic cleavage of intact alpha2M subunits. Quantitation of alpha2M levels in plasma indicated no significant differences between levels of alpha2M in CF homozygote, obligate heterozygote carrier, or normal control plasma samples. Quantitation of arginine esterase activity in plasma treated with cloroform and ellagic acid indicated that both the total arginine esterase activity and that fraction of arginine esterase activity inhibited by soybean trypsin inhibitor (SBTI) were decreased in most CF homozygote and obligate heterozygote plasma samples relative to normal control values. The results of this study indicate that plasma samples from CF homozygotes and obligate heterozygote carriers for CF show deficient proteolytic cleavage of alpha2M as compared with normal control plasma, and suggest that a structural abnormality in alpha2M or a deficiency in plasma proteolytic activity may be responsible for this deficiency in proteolysis.     

Increased phagocytic cell chemiluminescence in patients with cystic fibrosis

The oxidative burst of polymorphonuclear cells and monocytes from patients with cystic fibrosis as measured by luminol-enhanced chemiluminescence was examined after in vitro activation of the cells. All patients were outpatients at the time of the assays; their median age was 25.5 years (range, 12 to 33 years) and normal controls were young healthy adults. Stimulation of polymorphonuclear cells with phorbol myristate acetate, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, and the calcium ionophore A23187 resulted in significantly greater chemiluminescence responses from the cells of patients than from the control cells. The monocyte response of patients to opsonized zymosan was also greater than that of controls. Thus, phagocytic cells from adolescents and young adults with cystic fibrosis have a greater chemiluminescence response to a variety of stimuli. This may result in tissue damage in the lungs of these patients and thus make them more susceptible to pulmonary infections.     

Antibodies to staphylococcal teichoic acid and alpha toxin in patients with cystic fibrosis

Enzyme-linked immunosorbent assay (ELISA) was used for IgG antibody determination to teichoic acid and alpha-toxin from Staphylococcus aureus in 65 patients with cystic fibrosis (CF). In patients chronically colonized with S. aureus, elevated titres to teichoic acid were found in 13/35 (37%) patients, to alpha-toxin in 12/35 (34%) and to either antigen in 18/35 (51%). Patients with elevated titres to teichoic acid had a significantly lower X-ray score than patients with normal titres. The highest titres against both teichoic acid and alpha-toxin were seen in patients not receiving optimal treatment. These findings suggest that staphylococci contribute to the tissue damage in CF and that the determination of antibodies especially to staphylococcal teichoic acid might be of value in the diagnosis and management of staphylococcal infections in patients with CF.     

Increased monocyte chemiluminescence in cystic fibrosis patients and in their parents

We examined the chemiluminescence response of peripheral blood monocytes from patients with cystic fibrosis (CF) and their asymptomatic parental carriers of the CF gene to three different types of stimulation. We found that monocytes from both patients and carriers have increased luminol-dependent chemiluminescence in the first 25 min after stimulation by adherence to glass. These results are consistent with the hypothesis that monocytes from both CF heterozygotes and homozygotes respond to adhesion with increased oxygen radical formation. The increased adherence-induced monocyte chemiluminescence of the parental carriers did not vary with age or length of exposure of the parents to a child with CF. Also, repeated exposure to medications and respiratory secretions of CF patients was not associated with an increase in adherence-induced monocyte chemiluminescence of their nonbiologically related caretakers. Thus, this observed increase in chemiluminescence is not simply secondary to the medications or respiratory dysfunction seen in the patients with CF. Patients with other types of obstructive lung disease did not show increased adherence-induced monocyte chemiluminescence. We conclude that increased early phase adherence-induced monocyte chemiluminescence occurs in patients with cystic fibrosis and the obligate carriers of the CF gene independent of environmental influences.     

Increased heparin binding in cystic fibrosis: a reflection of altered glycoprotein biosynthesis?

Some of the serum proteins which bind to heparin and contribute to the pH 5.57 "heparin binding capacity" of human serum are glycoproteins; those from cystic fibrosis serum were found to be 27% higher in fucose (methylpentose) content, 27% lower in sialic acid content, and 31% lower in hexose content when compared to heparin-precipitated serum glycoproteins from normal control subjects. Hexosamine content of the heparin-precipitated serum glycoproteins was the same. Results of this preliminary investigation indicate that altered carbohydrate composition in serum glycoproteins may affect significantly their heparin binding capacity.     

Trypsin-binding immunoglobulin G and associated antigen in cystic fibrosis

Trypsin-binding immunoglobulin G (TBIgG) is found in the sera of a high proportion of patients with cystic fibrosis. We previously reported that TBIgG preferentially binds human cationic trypsin rather than trypsin from other animal species. Binding affinity is enhanced by complex formation with bovine pancreatic trypsin inhibitor, which is known to induce characteristic conformational modifications in the active site region of the trypsin molecule. To identify the human trypsin-like antigen associated with TBIgG, we have studied the effects of conformational changes of cationic trypsin induced by limited proteolysis based on competitive binding studies. It is shown that the most likely TBIgG-related self-antigen is an 11,000-dalton fragment that is a cleavage product of the complex formed by trypsin and alpha 1-protease inhibitor. This result emphasizes the occurrence of circulating trypsinogen activation and is interpreted to be a consequence of the protease-antiprotease imbalance, which has been well documented by previous investigators in cystic fibrosis and also in other lung diseases associated with an inflammatory state.     

Glucose tolerance and insulin receptor binding to monocytes and erythrocytes in patients with cystic fibrosis

Cystic fibrosis (CF) is the most frequent life threatening hereditary disease in the Western World with an incidence of approximately 1:2000. Due to increasing survival rates the high frequency of abnormal glucose tolerance has become an important problem. We compared insulin concentrations during oral glucose tolerance test and insulin receptor binding to both monocytes and erythrocytes from 9 patients with CF, with results from 10 healthy controls of similar body weight. The insulin: glucose ratio was increased in the fasting state (p less than 0.05) in patients with CF compared to controls, indicating an increased insulin resistance in CF-patients. The total insulin secretion during oral glucose tolerance test as judged by the area beneath the insulin curve was similar in the two groups, but insulin secretion was significantly delayed in patients with CF. Insulin receptor binding to monocytes and the number of receptors were significantly increased (p less than 0.01 and 0.02, respectively) in patients with CF whereas the dissociation constant was similar in patients with CF and controls. No difference was observed in insulin receptor binding to erythrocytes between the two groups. No correlations were found between insulin receptor binding to monocytes or erythrocytes and glucose tolerance or insulin concentrations.     

Serum interleukin-1 alpha and soluble interleukin-2 receptor concentrations in cystic fibrosis

Interleukin (IL)-1 and IL-2 may participate in the systemic inflammatory response and hypergammaglobulinaemia observed in patients with cystic fibrosis. Thirty seven patients with cystic fibrosis were compared with 25 normal controls. High IgG and IgM concentrations were associated with more severe pulmonary disease. IL-1 alpha and soluble IL-2 receptor concentrations were higher in the cystic fibrosis group than in the controls and also correlated with concentrations of IgG and IgM. These results suggest that these cytokines may contribute to enhanced immunoglobulin synthesis and silent inflammatory activity in clinically stable patients with cystic fibrosis.     

Secretory immunoglobulin A (sIgA) in parotid saliva of patients with cystic fibrosis

Investigations of immunoglobulin concentrations--especially of secretory IgA (sIgA)--were performed in isolated parotid saliva samples by means of a modified Mancini technique considering the structural specialties of sIgA. The measurement of flow rates under continuous stimulation of parotid gland secretion by citric acid allowed calculations of glandular output. Cystic fibrosis patients showed decreased secretion rates of sIgA during gland secretion stimulation. Because parotid glands and bronchial glands are parts of BALT, we can conclude, that a functional sIgA deficiency is a pathogenetic factor under others in chronic bronchopulmonary infections of cystic fibrosis patients.     

Improvment of survival in cystic fibrosis patients from 1962 to 1971]

The survival rate of 42 patients with cystic fibrosis diagnosed during the five year period before institution of full prophylactic aerosol, mist tent and postural drainage therapy in 1967 was compared with that of 32 cases diagnosed in the five following years. The mean survival rate of the latter group was 81%, 81% and 73% at 1, 2 and 3 years respectivly, compared with 64%, 59% and 57% in the former group. The main difference was due to the significantly improved survival rate of cases diagnosed during the first year of life.     

Comparison of the structure and aspects of the proteinase-binding properties of cystic fibrotic alpha-macroglobulin with normal alpha 2-macroglobulin

Considerable attention has been focused recently on alpha 2-macroglobulin (alpha 2M), a major endopeptidase inhibitor in blood plasma, as a possible source of the primary defect in cystic fibrosis (CF). We report here studies designed to compare the structure of CF alpha 2M with normal alpha 2M to determine if there is a difference. The physicochemical properties of purified alpha 2M as revealed by various electrophoretic techniques, covalent proteinase binding properties, and primary structural studies on a variety of partial hydrolyzates of CF alpha 2M and normal alpha 2M are compared. These studies were carried out on eight different individual isolates of CF alpha 2M and three age-matched normal alpha 2M preparations and alpha 2M isolated from fetal cord blood. Three properties of CF alpha 2M were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): (1) the existence of four identically-sized subunits in the native molecule (10), (2) the cleavage of this subunit into fragments of approximately 100,000 daltons upon interaction with proteinases (10), and (3) the cleavage of an alkaline/heat sensitive bond to produce 120,000 and 60,000 dalton fragments (11). Both CF and normal alpha 2M were cleaved to the extent of 79-87%, CF alpha 2M behaves identically with normal alpha 2M with regard to all these properties. Salvesen and Barrett (24) have demonstrated that varying proportions of several [125I]-labeled proteinases form SDS-stable, non-reducible links to normal alpha 2M. Two of the CF alpha 2M preparations were studied to determine if similar covalent binding of proteinases occurred. The positions of the labeled and % of proteinase bound bands in SDS/reduced PAGE system were identical for normal alpha 2M and CF alpha 2M. These results indicate that CF alpha 2M behaves normally with regard to covalent binding of proteinases. Qualitative comparison of the peptide fragments separated by SDS-PAGE or isoelectric focusing of CF and normal alpha 2M produced by partial proteolysis with trypsin, chymotrypsin or Staphylococcus aureus V-8 proteinase did not reveal any difference unique to CF alpha 2M. The cyanogen bromide fragmentation studies and the cysteine cleavage studies also indicated that no major change in the positions of methionyl residues or cysteinyl/cystinyl residues has occurred in CF alpha 2M. The failure of all these different studies and those reported by others to demonstrate any differences between CF and normal alpha 2M makes it highly unlikely that there is a primary defect in alpha 2M in CF.     

RSV mediates Pseudomonas aeruginosa binding to cystic fibrosis and normal epithelial cells

Cystic fibrosis lung disease typically has a course of exacerbations and remissions, suggesting that external factors like viral infections can influence this course. Clinical data suggest synergism between respiratory syncytial virus (RSV) infections and Pseudomonas aeruginosa in cystic fibrosis (CF) lung disease. We studied the influence of RSV infection on adherence of P. aeruginosa to IB3-1, HEp-2, and A549 epithelial cell monolayers in vitro. RSV infection of epithelial cells as well as simultaneous addition of RSV and P. aeruginosa to noninfected cells both strongly enhanced the pseudomonal adherence to epithelial cells. The increased adherence varied from 1.2- to 8.2-fold in case of previous RSV infection, and from 1.7- to 16.1-fold in case of simultaneous addition. We observed direct binding of RSV to P. aeruginosa, and blocking of RSV with heparin eliminated the effect on increased adherence. This suggests that RSV possibly acts as a coupling agent between P. aeruginosa and epithelial cells. In conclusion, RSV enhances P. aeruginosa infection of respiratory epithelial cells. It suggests a role of specific viral-bacterial interactions in exacerbations of CF lung disease, which could have important implications on prevention and treatment.     

Serum interleukin-1 alpha and soluble interleukin-2 receptor concentrations in cystic fibrosis.

Interleukin (IL)-1 and IL-2 may participate in the systemic inflammatory response and hypergammaglobulinaemia observed in patients with cystic fibrosis. Thirty seven patients with cystic fibrosis were compared with 25 normal controls. High IgG and IgM concentrations were associated with more severe pulmonary disease. IL-1 alpha and soluble IL-2 receptor concentrations were higher in the cystic fibrosis group than in the controls and also correlated with concentrations of IgG and IgM. These results suggest that these cytokines may contribute to enhanced immunoglobulin synthesis and silent inflammatory activity in clinically stable patients with cystic fibrosis.     

Plasma arginine esterase in cystic fibrosis: kinetics of activation, identification as plasma kallikrein, reaction with alpha 2-macroglobulin and comparison with levels in normal plasma

Treatment of normal plasma with chloroform and ellagic acid yielded esterase activity against tosylarginine methyl ester, which reached a maximum within 2 h. After 2 h most or all of the activity as resistant to inhibition by soybean trypsin inhibitor (STI), but was still sensitive to the low molecular weight inhibitors di-isopropyl fluorophosphate, aprotinin and prolyl-phenylalanyl-arginyl chloromethane. The activity ran in gel chromatography with alpha 2 macroglobulin (ampha 2M), as if it were due to an alpha 2M proteinase complex. The generation of the arginine esterase activity by chloroform and ellagic acid was apparently dependent on the activation of factor XII, being blocked by Polybrene. In plasma pretreated with methylamine-HCl (an inactivator of alpha 2M), the arginine esterase was 95% sensitive to inhibition by STI. With regard to substrate specificity, inhibition characteristics, and gel chromatographic behaviour, it was indistinguishable from plasma kallikrein (EC 3.4.21.34, formerly 3.4.21.8). The chloroform and ellagic acid treatment of plasma resulted in a disappearance of prokallikrein simultaneous with the appearance of the arginine esterase. By these criteria, the arginine esterase activity was attributable entirely to plasma kallikrein either in its free form (methylamine-treated plasma) or bound to alpha 2M (buffer-treated plasma). Comparisons of STI-sensitive and STI-resistant arginine esterase activities of plasma samples from cystic fibrosis patients, obligate heterozyotes or other groups showed no significant differences in levels of activity, kinetics of activation or gel chromatographic behaviour. We conclude that cystic fibrosis is unrelated to any abnormality in plasma arginine esterase activity, contrary to some previous reports.