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Polycations Decrease the Transepithelial Resistance of Cultured Tracheal Epithelial Cells
LTA, Hydrolase of Human Airway Epithelial cells·
enzymatic activity was not demonstrated in this fraction, and evidence for a family of enzymes related to LTA.. hydrolase was not found by northern blot analysis.
Comparison with Neutrophils TImothy D. Bigby, M.D.
W
e have previously shown that human airway epithelial cells contain enzymatic activity which converts LTA 4 to LTB.., a potent proinflammatory lipid. Earlier studies suggested that this activity might be different than that found in inflammatory cells. The purpose ofthis investigation was to compare the properties of LTA4 hydrolase in cytosolic preparations of epithelial cells and neutrophils. Transformed human airway epithelial cells (BEAS-2B) were cultured to confluence in serum-free media (LHC-9). Human neutrophils were prepared from blood by density gradient centrifugation. Cells were sonicated, and cytosolic fractions were prepared by centrifugation at 15,000 x and 100,000 x g. Kinetic analysis was performed by incubating cytosolic fractions with LTA4 in the presence or absence of albumin. When epithelial cytosol was incubated with 15 IJ.M LTA.. in the presence of albumin, peak activity (440 pmoV mg protein) was observed at 30 min, whereas neutrophil peak activity (1,322 pmoVmg protein) was observed at 10 min. When fractions were incubated in the absence of albumin, time courses were similar. However, when doseresponse curves were constructed using LTA.. concentrations of 0 to 100 IJ.M, peak activity was observed between 40 and 60 IJ.M LTA.. for the neutrophil, but a continued doseresponse was observed in the epithelial cell without a plateau. Lineweaver-Burk analysis of neutrophil data revealed that they conformed to Michaelis-Menten kinetics whereas the epithelial cell data did not. The observed neutrophil Km was 7.1 IJ.M. The epithelial cell data were best described by two different regression lines corresponding to a Km of 0.9 and 9.2 IJ.M. Western blot analyses using a polyclonal antibody to the recombinant neutrophil LTA.. hydrolase revealed a single band (69 kDa) in the neutrophil and at least two bands in the epithelial cell (69 kDa and 110 to 115 kDa). Separation of these two proteins by size exclusion chromatography demonstrated that only fractions containing the 69 kDa band had enzymatic activity. Northern blot analysis of total cellular RNA from HL-60 cells and epithelial cells probed with a eDNA for the neutrophil hydrolase revealed a single positive band in both migrating at 2.4 kb. These data support a difference in kinetic behavior of LTA.. hydrolase in epithelial cells when compared to neutrophils. Western and northern blots strongly suggest that epithelial cells contain the same enzyme as inflammatory cells. Thus, differences in biochemical activity may be due to differences in cellular environments. The higher molecular weight protein (110 to 115 kDa) appears to share an immunoreactive epitope. However, this protein is not a likely explanation ofthe observed kinetic differences because *From the Department of Medicine, University of California, and the ~epartment ofVet~ran Affairs, San Diego. Rep~nt requests: Dr. Bigby, University of California SD, 9111J VA MedICal Center; La jolla, California 92161
Polycations Decrease the Transepithelial Resistance of Cultured Tracheal Epithelial cells· D. Uchida, M.D.; C. Ballowe, M.D.; G. Larsen, M.D.; C. lroin, M.D.; and G. Cott, M.D.
In asthma, the~ is eViden~e linking the development of hyperresponslveness to mflammation. Various inflammatory cells, including eosinophils and neutrophils, are capable of releasing cationic proteins. We have previously shown that instillation of major basic protein, a cationic eosinophil granule protein, as well as synthetic polycations into the airways of intact rats, increases airways responsiveness to inhaled methacholine. Since airway epithelial cells may modulate airways responsiveness, we sought to investigate the effect of polycations on these cells. Tracheal epithelial cells isolated from rabbits were cultured for 4 days on membranes, and transepithelial electrical resistance (Rt) was measured. Polyarginine (25 J.Lg!ml), polylysine (25 JLglml), heparin (30 unitslml), or polylysine plus heparin was then added to the apical or basolateral aspect of the membranes. Control membranes received no additions. The Rt was subsequently recorded at 10-min intervals for a total of 60 min. The control group showed no change in Rt over 60 min. Apical application of polylysine resulted in a decrease in Rt which was readily discernable at 10 min postexposure and reached a plateau at approximately 40 min postexposure (13% of baseline Rt at 40 min). Basolateral application of polylysine also resulted in a decrease in Rt with a similar time course (41% of baseline Rt at 40 min). Polyarginine had a comparable effect on Rt as polylysine when applied to either side. Apical or basolateral application of heparin or polylysine plus heparin resulted in no change in Rt over 60 min. These preliminary results suggest that apical or basolateral exposure of cultured tracheal epithelial cells to cationic proteins causes a decrease in Rt, implying a perturbation in the barrier properties of the epithelium. Cationic charge is likely a critical determinant of this effect since the anionic polysaccharide heparin had no effect on Rt and also blocked the effect of polylysine. Further, these results suggest that in a variety of airway diseases, the barrier function of the epithelium may be altered through the presence of cationic proteins from inflammatory cells within the airway lumen, the subepithelium, or both. *From the departments of Pediatrics and Medicine, National Jewish Ce~ter for Immunology and Respiratory Medicine, Denver. ThiS study was supported by grant H L 36577.
CHEST / 101 / 3 / MARCH, 1992 / Supplement
33S
enzymatic activity was not demonstrated in this fraction, and evidence for a family of enzymes related to LTA.. hydrolase was not found by northern blot analysis.
Comparison with Neutrophils TImothy D. Bigby, M.D.
W
e have previously shown that human airway epithelial cells contain enzymatic activity which converts LTA 4 to LTB.., a potent proinflammatory lipid. Earlier studies suggested that this activity might be different than that found in inflammatory cells. The purpose ofthis investigation was to compare the properties of LTA4 hydrolase in cytosolic preparations of epithelial cells and neutrophils. Transformed human airway epithelial cells (BEAS-2B) were cultured to confluence in serum-free media (LHC-9). Human neutrophils were prepared from blood by density gradient centrifugation. Cells were sonicated, and cytosolic fractions were prepared by centrifugation at 15,000 x and 100,000 x g. Kinetic analysis was performed by incubating cytosolic fractions with LTA4 in the presence or absence of albumin. When epithelial cytosol was incubated with 15 IJ.M LTA.. in the presence of albumin, peak activity (440 pmoV mg protein) was observed at 30 min, whereas neutrophil peak activity (1,322 pmoVmg protein) was observed at 10 min. When fractions were incubated in the absence of albumin, time courses were similar. However, when doseresponse curves were constructed using LTA.. concentrations of 0 to 100 IJ.M, peak activity was observed between 40 and 60 IJ.M LTA.. for the neutrophil, but a continued doseresponse was observed in the epithelial cell without a plateau. Lineweaver-Burk analysis of neutrophil data revealed that they conformed to Michaelis-Menten kinetics whereas the epithelial cell data did not. The observed neutrophil Km was 7.1 IJ.M. The epithelial cell data were best described by two different regression lines corresponding to a Km of 0.9 and 9.2 IJ.M. Western blot analyses using a polyclonal antibody to the recombinant neutrophil LTA.. hydrolase revealed a single band (69 kDa) in the neutrophil and at least two bands in the epithelial cell (69 kDa and 110 to 115 kDa). Separation of these two proteins by size exclusion chromatography demonstrated that only fractions containing the 69 kDa band had enzymatic activity. Northern blot analysis of total cellular RNA from HL-60 cells and epithelial cells probed with a eDNA for the neutrophil hydrolase revealed a single positive band in both migrating at 2.4 kb. These data support a difference in kinetic behavior of LTA.. hydrolase in epithelial cells when compared to neutrophils. Western and northern blots strongly suggest that epithelial cells contain the same enzyme as inflammatory cells. Thus, differences in biochemical activity may be due to differences in cellular environments. The higher molecular weight protein (110 to 115 kDa) appears to share an immunoreactive epitope. However, this protein is not a likely explanation ofthe observed kinetic differences because *From the Department of Medicine, University of California, and the ~epartment ofVet~ran Affairs, San Diego. Rep~nt requests: Dr. Bigby, University of California SD, 9111J VA MedICal Center; La jolla, California 92161
Polycations Decrease the Transepithelial Resistance of Cultured Tracheal Epithelial cells· D. Uchida, M.D.; C. Ballowe, M.D.; G. Larsen, M.D.; C. lroin, M.D.; and G. Cott, M.D.
In asthma, the~ is eViden~e linking the development of hyperresponslveness to mflammation. Various inflammatory cells, including eosinophils and neutrophils, are capable of releasing cationic proteins. We have previously shown that instillation of major basic protein, a cationic eosinophil granule protein, as well as synthetic polycations into the airways of intact rats, increases airways responsiveness to inhaled methacholine. Since airway epithelial cells may modulate airways responsiveness, we sought to investigate the effect of polycations on these cells. Tracheal epithelial cells isolated from rabbits were cultured for 4 days on membranes, and transepithelial electrical resistance (Rt) was measured. Polyarginine (25 J.Lg!ml), polylysine (25 JLglml), heparin (30 unitslml), or polylysine plus heparin was then added to the apical or basolateral aspect of the membranes. Control membranes received no additions. The Rt was subsequently recorded at 10-min intervals for a total of 60 min. The control group showed no change in Rt over 60 min. Apical application of polylysine resulted in a decrease in Rt which was readily discernable at 10 min postexposure and reached a plateau at approximately 40 min postexposure (13% of baseline Rt at 40 min). Basolateral application of polylysine also resulted in a decrease in Rt with a similar time course (41% of baseline Rt at 40 min). Polyarginine had a comparable effect on Rt as polylysine when applied to either side. Apical or basolateral application of heparin or polylysine plus heparin resulted in no change in Rt over 60 min. These preliminary results suggest that apical or basolateral exposure of cultured tracheal epithelial cells to cationic proteins causes a decrease in Rt, implying a perturbation in the barrier properties of the epithelium. Cationic charge is likely a critical determinant of this effect since the anionic polysaccharide heparin had no effect on Rt and also blocked the effect of polylysine. Further, these results suggest that in a variety of airway diseases, the barrier function of the epithelium may be altered through the presence of cationic proteins from inflammatory cells within the airway lumen, the subepithelium, or both. *From the departments of Pediatrics and Medicine, National Jewish Ce~ter for Immunology and Respiratory Medicine, Denver. ThiS study was supported by grant H L 36577.
CHEST / 101 / 3 / MARCH, 1992 / Supplement
33S