Veterinary Immunology and Immunopathology, 29 ( 1991 ) 295-312
295
Elsevier Science Publishers B.V., Amsterdam
Spontaneous and lipopolysaccharide-induced expression of procoagulant activity by equine lung macrophages in comparison with blood monocytes and blood neutrophils G. Griinig a'~, C. Hulliger a, C. Winder a, M. Hermann b, T.W. Jungi c and R. v o n F e l l e n b e r g a'2 aDivision of Applied Veterinary Physiology of the Institute of Veterina~ PhysioloD, and bDepartment of Veterinary Medicine, University of Zfirich, Winterthurerstrasse260, 805 7 Ziirich, Switzerland Clnstitute of Veterinary Virology, University of Berne, Berne, Switzerland (Accepted 26 November 1990)
ABSTRACT Griinig, G., Hulliger, C., Winder, C., Hermann, M., Jungi, T.W. and von Fellenberg, R., 1991. Spontaneous and lipopolysaccharide-induced expression of procoagulant activity by equine lung macrophages in comparison with blood monocytes and blood neutrophils. Vet. hnmunol, hmnunopathol., 29:295-312. The procoagulant activity (PCA) associated with equine bronchoalveolar lavage cells was determined and compared with that expressed by peripheral b!oo.d mononuclear cells and neutrophils. Lung cell preparations from horses affected with chronic pulmonary disease were included in all experiments and there was no difference in the qualitative type of response compared with lung cells which were obtained from healthy horses. Significant amounts of PCA were expressed by cells freshly procured from bronchoalveolar lavages of healthy and diseased horses. When adherent lung cells were kept in culture for some time, cell-associated PCA slightly decreased within 4 h, reached its lowest point after approximately 24 h and rose again during the second week of culture. In contrast, freshly isolated blood mononuclear cells or neutrophils expressed little PCA. Following culture for 24 h, mononuclear cells began to express increased PCA levels. Both cultivated lung cells (comprised mainly on alveolar macrophages) and blood mononuclear cells responded to LPS by dramatically increased PCA expression, whereas neutrophils showed a small augmentation of PCA on LPS stimulation. Fresh mononuclear cells and cultivated lung cells differed in their PCA response to LPS in several respects. Blood mononuclear cells were more sensitive to LPS than lung macrophages and responded to a 100fold lower LPS concentration than the latter. Mononuclear cell-associated PCA peaked 4 h after stimulation whereas that of cultured macrophages continued to increase up to 24 h after stimulation. Lung macrophages cultured in adherence responded to LPS stimulation with a much higher PCA increase than macrophages cultured in suspension, in teflon containers. However, the culture vessel did not influence the PCA expressed by unstimulated cells. PCA expression depended to a large extent on Ipresent address: J.A. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. 2Author to whom correspondence should be addressed.
0165-2427/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.
296
G. GRONIGET AL
transcription and translation, as evidenced by a 60-85% reduction of PCA in cycloheximide- or actinomycin D-treated, LPS-stimulated lung macrophages. PCA was largely cell-associated; only a small proportion of cell-associated PCA was shed into the medium. The PCA associated with mononuclear cells and with lung macrophages was tissue factor because of its dependence on clotting factor Vll and its independence from clotting factor VIII. The expression of PCA by freshly isolated cells, the lower sensitivity to LPS, and the loss of PCA in the first 24 h of cultivation are indicative of in vivo activation of lung macrophages. ABBREVIATIONS HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; PCA, procoagulant activity.
INTRODUCTION
Cell-free supernatants of equine respiratory secretions contain procoagulant activity (PCA) which is correlated with the severity of chronic pulmonary disease and with the quantity of neutrophils in the secretion specimens (Griinig et al., 1988). As in man and in experimental animals (Sitrin et al., 1983; Chapman et al., 1985), equine lung macrophages, cell-free bronchoalveolar lavage fluids (Griinig et al., 1990), and blood monocytes (Henry and Moore, 1988) express PCA. However, a kinetic analysis, a comparison between stimulated and unstimulated cells and between alveolar lavage cells and peripheral blood cells had not been performed in the equine species. In the present report, we assess the noninduced and the lipopolysaccharide (LPS)-induced PCA of freshly isolated and of cultured alveolar lavage cells, and we compare the PCA response with that of peripheral blood mononuclear ceils and neutrophiis immediately after isolation and after short-term culture. Particular attention is given to the LPS-induced PCA response by cells cultured under either adherent (polystyrene) or nonadherent (teflon) culture conditions, and it is confirmed that the cell-associated PCA represents, to a large extent, de novo synthesized tissue factor, as reported in a number of other species. The observation that lung macrophages from healthy horses and horses with chronic bronchitis express considerable levels of PCA suggests that these cells had been activated in some way in vivo. MATERIALS AND METHODS
Horses
Bronchoalveolar lavage cells were obtained from 24 medical or surgical patients referred to the veterinary clinic and from experimental horses. Seven animals had no signs of respiratory disease, and 17 horses were affected with chronic pulmonary disease. Blood cells were isolated from clinically healthy horses referred to the veterinary clinic.
PROCOAGULANT ACTIVITY OF EQUINE LUNG MACROPHAGES
297
Reagents Stock solutions of LPS from Escherichia coli 055 :B5 (Difco) were prepared in distilled water, aliquotted and kept frozen ( - 70°C) until use. Actinomycin D and cycloheximide were purchased from Sigma. Concentrations of LPS determined (Limusate test, Haemachem Inc., St. Luis) in buffers, media and sera were less than l 0 pg/ml.
Cell isolation Bronchoalveolar macrophages Thirty-four bronchoalveolar lavages were performed in 24 horses with two or three 250 ml aliquots of prewarmed (37°C) pyrogen-free 0.9% saline per lung using a modification (Griinig et al., 1990) of the technique described by Viel (1986).
PCA (activity of freshly isolated cells - 100) 3 1 0 0 -1
295
Elsevier Science Publishers B.V., Amsterdam
Spontaneous and lipopolysaccharide-induced expression of procoagulant activity by equine lung macrophages in comparison with blood monocytes and blood neutrophils G. Griinig a'~, C. Hulliger a, C. Winder a, M. Hermann b, T.W. Jungi c and R. v o n F e l l e n b e r g a'2 aDivision of Applied Veterinary Physiology of the Institute of Veterina~ PhysioloD, and bDepartment of Veterinary Medicine, University of Zfirich, Winterthurerstrasse260, 805 7 Ziirich, Switzerland Clnstitute of Veterinary Virology, University of Berne, Berne, Switzerland (Accepted 26 November 1990)
ABSTRACT Griinig, G., Hulliger, C., Winder, C., Hermann, M., Jungi, T.W. and von Fellenberg, R., 1991. Spontaneous and lipopolysaccharide-induced expression of procoagulant activity by equine lung macrophages in comparison with blood monocytes and blood neutrophils. Vet. hnmunol, hmnunopathol., 29:295-312. The procoagulant activity (PCA) associated with equine bronchoalveolar lavage cells was determined and compared with that expressed by peripheral b!oo.d mononuclear cells and neutrophils. Lung cell preparations from horses affected with chronic pulmonary disease were included in all experiments and there was no difference in the qualitative type of response compared with lung cells which were obtained from healthy horses. Significant amounts of PCA were expressed by cells freshly procured from bronchoalveolar lavages of healthy and diseased horses. When adherent lung cells were kept in culture for some time, cell-associated PCA slightly decreased within 4 h, reached its lowest point after approximately 24 h and rose again during the second week of culture. In contrast, freshly isolated blood mononuclear cells or neutrophils expressed little PCA. Following culture for 24 h, mononuclear cells began to express increased PCA levels. Both cultivated lung cells (comprised mainly on alveolar macrophages) and blood mononuclear cells responded to LPS by dramatically increased PCA expression, whereas neutrophils showed a small augmentation of PCA on LPS stimulation. Fresh mononuclear cells and cultivated lung cells differed in their PCA response to LPS in several respects. Blood mononuclear cells were more sensitive to LPS than lung macrophages and responded to a 100fold lower LPS concentration than the latter. Mononuclear cell-associated PCA peaked 4 h after stimulation whereas that of cultured macrophages continued to increase up to 24 h after stimulation. Lung macrophages cultured in adherence responded to LPS stimulation with a much higher PCA increase than macrophages cultured in suspension, in teflon containers. However, the culture vessel did not influence the PCA expressed by unstimulated cells. PCA expression depended to a large extent on Ipresent address: J.A. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. 2Author to whom correspondence should be addressed.
0165-2427/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.
296
G. GRONIGET AL
transcription and translation, as evidenced by a 60-85% reduction of PCA in cycloheximide- or actinomycin D-treated, LPS-stimulated lung macrophages. PCA was largely cell-associated; only a small proportion of cell-associated PCA was shed into the medium. The PCA associated with mononuclear cells and with lung macrophages was tissue factor because of its dependence on clotting factor Vll and its independence from clotting factor VIII. The expression of PCA by freshly isolated cells, the lower sensitivity to LPS, and the loss of PCA in the first 24 h of cultivation are indicative of in vivo activation of lung macrophages. ABBREVIATIONS HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; PCA, procoagulant activity.
INTRODUCTION
Cell-free supernatants of equine respiratory secretions contain procoagulant activity (PCA) which is correlated with the severity of chronic pulmonary disease and with the quantity of neutrophils in the secretion specimens (Griinig et al., 1988). As in man and in experimental animals (Sitrin et al., 1983; Chapman et al., 1985), equine lung macrophages, cell-free bronchoalveolar lavage fluids (Griinig et al., 1990), and blood monocytes (Henry and Moore, 1988) express PCA. However, a kinetic analysis, a comparison between stimulated and unstimulated cells and between alveolar lavage cells and peripheral blood cells had not been performed in the equine species. In the present report, we assess the noninduced and the lipopolysaccharide (LPS)-induced PCA of freshly isolated and of cultured alveolar lavage cells, and we compare the PCA response with that of peripheral blood mononuclear ceils and neutrophiis immediately after isolation and after short-term culture. Particular attention is given to the LPS-induced PCA response by cells cultured under either adherent (polystyrene) or nonadherent (teflon) culture conditions, and it is confirmed that the cell-associated PCA represents, to a large extent, de novo synthesized tissue factor, as reported in a number of other species. The observation that lung macrophages from healthy horses and horses with chronic bronchitis express considerable levels of PCA suggests that these cells had been activated in some way in vivo. MATERIALS AND METHODS
Horses
Bronchoalveolar lavage cells were obtained from 24 medical or surgical patients referred to the veterinary clinic and from experimental horses. Seven animals had no signs of respiratory disease, and 17 horses were affected with chronic pulmonary disease. Blood cells were isolated from clinically healthy horses referred to the veterinary clinic.
PROCOAGULANT ACTIVITY OF EQUINE LUNG MACROPHAGES
297
Reagents Stock solutions of LPS from Escherichia coli 055 :B5 (Difco) were prepared in distilled water, aliquotted and kept frozen ( - 70°C) until use. Actinomycin D and cycloheximide were purchased from Sigma. Concentrations of LPS determined (Limusate test, Haemachem Inc., St. Luis) in buffers, media and sera were less than l 0 pg/ml.
Cell isolation Bronchoalveolar macrophages Thirty-four bronchoalveolar lavages were performed in 24 horses with two or three 250 ml aliquots of prewarmed (37°C) pyrogen-free 0.9% saline per lung using a modification (Griinig et al., 1990) of the technique described by Viel (1986).
PCA (activity of freshly isolated cells - 100) 3 1 0 0 -1
