Br. J. clin. Pharmac. (1990), 30, 150S-152S
Effect of lymphokines on 1-adrenoceptor function of human peripheral blood mononuclear cells A. J. M. VAN OOSTERHOUT & F. P. NIJKAMP Department of Pharmacology, Faculty of Pharmacy, University of Utrecht, Catharijnesingel 60, 3511 GH Utrecht, The Netherlands
Pathological induced changes in ,B-adrenoceptor function occur in diseases such as asthma and hypertension. The mechanism(s) of this dysfunction is at present unclear. In the present study, the effect of lymphokines on ,B-adrenoceptor agonist induced cAMP production in peripheral blood mononuclear cells (PBMC) is investigated. Pre-incubation of PBMC during 20 h with interleukin-2 (IL-2, 100 u ml-') and granulocyte/macrophagecolony stimulating factor (GM-CSF, 100 u ml-') significantly decreases ,B-adrenoceptor agonist induced cAMP production by 35 ± 8% and 37 ± 11% respectively. IL-3 and IL-4 do not affect P-adrenoceptor agonist induced cAMP production in PBMC. It can be concluded that IL-2 and GM-CSF, mediators derived from T-lymphocytes, can induce ,3adrenoceptor dysfunction in PBMC. Keywords f3-adrenoceptor cAMP lymphokines mononuclear cells Introduction Leucocytes isolated from the blood are often used to study P-adrenoceptor function in disease. This approach assumes that the ,1adrenoceptor status of leucocytes mirrors pathologically induced alterations in certain tissues. Although alterations of ,-adrenoceptor function in human essential hypertension is still controversial, it has been shown that 1-adrenoceptor dysfunction occurs in leucocytes and vascular smooth muscle in a variety of animal models of hypertension (Masuzawa et al., 1989; Michel et al., 1989). In patients with asthma, it has been shown that dysfunction of the 1adrenoceptor-adenylate cyclase complex occurs in leucocytes and airway smooth muscle (Lulich et al., 1988). Both in asthma and hypertension, the changes in 1-adrenoceptor function seem to be acquired rather than fundamental abnormalities. Recently, we demonstrated that the 1adrenoceptor function of airway and vascular smooth muscle and lymphocytes is decreased, 4 days after bacterial endotoxin administration to guinea pigs (Van Heuven-Nolsen et al., 1985;
Van Oosterhout et al., 1988a). This endotoxininduced 1-adrenoceptor dysfunction can be prevented by splenectomy or by administration of the T-lymphocyte selective immunosup-
pressive drug cyclosporin-A (Van Oosterhout et al., 1988a,b). Based on these observations, it can be suggested that mediators produced by lymphocytes may be involved in 3-adrenoceptor dysfunction in this animal model and possibly in human diseases as well. In the present study, peripheral blood mononuclear cells (PBMC) are used to study the influence of cell-line derived human interleukin2 (IL-2), recombinant human (rh) IL-3, rhIL-4 and rh granulocyte/macrophage colony stimulating factor (GM/CSF) on the 1-adrenoceptor function. Methods
PBMC were isolated from the blood of healthy volunteers or from buffy coats (Bloodbank, Utrecht, The Netherlands). Isolation was per-
Correspondence: Dr A. J. M. Van Oosterhout, Department of Pharmacology, Faculty of Pharmacy, University of Utrecht, Catharijnesingel 60, 3511 GH Utrecht, The Netherlands
150S
Lymphokines and 83-adrenoceptor function formed by centrifugation on a Ficoll-paque gradient (20 min, 1000 g). Cells at the interface were removed and washed three times with RPMI-1640 (10 min, 500 g). Finally, PBMC were suspended in RPMI-1640 at 3 x 107 cells/ ml and pre-incubated with or without IL-2 (Jurkat), rhIL-3, rhIL-4 and rhGM-CSF (all from Genzyme, Boston, USA). Pre-incubations were carried out at 370 C in a humidified atmosphere of 5% C02/95% air. After a preincubation period of 2 or 20 h, 3 x 106 PBMC were incubated for 5 min at 370 C in a shaking water bath, with or without 1 ,UM isoprenaline, in the presence of 0.5 mm 3-isobutyl-1-methylxanthine in a final volume of 200 ,ul. The reaction was terminated by means of heating the cellsuspensions in a boiling water bath for 5 min. Precipitated protein was centrifuged for 15 min at 1500 g and 50 ,ul of the supernatant was used to measure cAMP content. cAMP was determined by a competitive protein binding method using a cAMP assay kit (Amersham, Great Britain). Results
Pre-incubation of PBMC with lymphokines for 2 or 20 h did not affect basal cAMP content of the cells (results not shown). The increase in cAMP induced by the ,3-adrenoceptor agonist isoprenaline (1 FLM) was not changed when PBMC were pre-incubated with lymphokines for 2 h (results not shown). After a 20 h pre-incubation period, the isoprenaline-induced cAMP production was significantly decreased in PBMC incubated with IL-2 and rhGM-CSF at a concentration of 100 u ml-1 (Table 1). The reduction of the P-adrenoceptor agonist induced cAMP production amounted to 35 ± 8% (P < 0.01, paired t-test) and 37 ± 11% (P < 0.05) in case of IL-2 and rhGM-CSF respectively. Pre-incubation of PBMC with rhIL-3 and rhIL-4 during 20 h did
not influence ,-adrenoceptor function of the cells.
Discussion In the present study, we demonstrate that IL-2 and rhGM-CSF decrease 3-adrenoceptor agonist induced cAMP production in PBMC after a 20 h pre-incubation period. These results are in accordance with Beckner & Farrar (1987) who demonstrated that IL-2 immediately decreases adenylate cyclase activity in activated human T-lymphocytes. In contrast to freshly isolated lymphocytes, activated lymphocytes express a large number of high affinity IL-2 receptors. This may explain that in our experiments the decreased P-adrenoceptor function in PBMC only occurs after a 20 h and not after a 2 h pre-incubation period. GM-CSF is a T-lymphocyte glycoprotein that promotes the growth and differentiation of granulocytes and macrophages. It has been shown that rhGM-CSF (100 u ml-1) immediately decreases adenylate cyclase activity in human neutrophils (Coffey et al., 1988). The inhibition of adenylate cyclase activity was maximal after 30 min pre-incubation, thereafter, the activity slowly recovered. In our experiments, no decreased isoprenaline-induced cAMP production is observed after two h pre-incubation of PBMC with rhGM-CSF (100 u ml-1). However, when PBMC are incubated during 20 h with rhGM-CSF, the ,-adrenoceptor function is inhibited. An explanation for the delayed effect of rhGM-CSF can be that specific receptors have to be expressed on the cell membrane, analogous to IL-2 receptors. The 3-adrenoceptor function of leucocytes is thought to mirror pathologically induced alterations in tissues like airway or vascular smooth muscle in asthma and hypertension respectively.
Table 1 Isoprenaline induced cAMP production in human PBMC after pre-incubation with lymphokines during 20 h. Results are expressed as the percentage of the cAMP production by PBMC not incubated with lymphokine Concentration (u ml-') Control 1 10 100 P < 0.05 and test. *
**
151S
IL-2 (n =5)
rhIL-3 (n =5)
rhIL-4 (n 5)
100 21.3 105.8 ± 8.8 88.0 8.5 64.8 ± 7.6**
100 28.2 104.3 ± 8.4 96.0 8.6 79.6 ± 14.1
100 21.3 105.8 ± 10.7 107.1 20.3 97.6 ± 10.5
rhGM-CSF (n = 6)
100 ± 95.5 ± 88.2 ± 63.1 ±
18.1 2.0 9.1
10.7*
P < 0.01 as compared with control and determined with Student's paired t-
152S
A. J. M. Van Oosterhout & F. P. Nijkamp
Based on our findings, a potential role for mediators derived from lymphocytes in this 1Badrenoceptor dysfunction cannot be excluded. At present, little is known about the presence of lymphokine receptors on smooth muscle-, endothelial- and epithelial cells. Expression of IL-2 receptors was initially believed to be restricted to T-cells after their activation. However, it has been demonstrated that IL-2 receptor expression can be induced in B-lymphocytes, mast cells, monocytes and macrophages
(Hancock et al., 1987). In previous experiments, we did not observe a decreased isoprenalineinduced relaxation of guinea pig tracheal spirals after 2 h pre-incubation with IL-2 (100 u ml-', unpublished observation). More studies are needed to elucidate the effect of lymphokines on 3-adrenoceptor function of other cells such as airway and vascular smooth muscle. This study was supported by a research grant (87.29) of the Dutch Asthma Foundation.
References Beckner, S. K. & Farrar, W. L. (1987). Inhibition of adenylate cyclase by IL 2 in human T lymphocytes is mediated by protein kinase C. Can. Biochem. Biophys. Res. Comm., 145,176-182. Coffey, R. G., Davis, J. S. & Djeu, J. Y. (1988). Stimulation of guanylate cyclase activity and reduction of adenylate cyclase activity by granulocytemacrophage colony-stimulating factor in human neutrophils. J. Immunol., 140, 2695-2701. Hancock, W. W., Muller, W. A. & Cotran, R. S. (1987). Interleukin 2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis and are inducible by lymphokine treatment of normal human lung macrophages, blood monocytes, and monocyte cell lines. J. Immunol., 138, 185-191. Lulich, K. M., Goldie, R. G. & Paterson, J. W. (1988). Beta-adrenoceptor function in asthmatic bronchial smooth muscle. Gen. Pharmac., 19, 307-311. Masuzawa, K., Matsuda, T. & Asano, M. (1989). Decreased arterial responsiveness to multiple cyclic AMP-generating receptor agonists in spontaneously hypertensive rats. Br. J. Pharmac., 96, 227-235.
Michel, M. C., Galal, O., Stoermer, J., Bock, K. D. & Brodde, O-E. (1989). a- and 13-adrenoceptors in hypertension. II. Platelet a2- and lymphocyte 12adrenoceptors in children of parents with essential hypertension. A model for the pathogenesis of the genetically determined hypertension. J. cardiovasc. Pharmac., 13, 432-439. Van Heuven-Nolsen, D., De Wildt, D. J. & Nijkamp, F. P. (1985). Disturbed adrenergic regulation of coronary flow in the guinea-pig heart after endotoxin. Eur. J. Pharmwc., 118, 341-345. Van Oosterhout, A. J. M., Folkerts, G., Ten Have, G. A. M. & Nijkamp, F. P. (1988a). Involvement of the spleen in the endotoxin-induced deterioration of the respiratory airway and lymphocyte 3-adrenergic systems of the guinea pig. Eur. J. Pharmac., 147, 421-430. Van Oosterhout, A. J. M., Woutersen-Van Nijnanten, F. M. A. & Nijkamp, F. P. (1988b). Cyclosporin A prevents endotoxin-induced ,B-adrenoceptor impairment in lymphocytes. Eur. J. Pharmac., 149, 191-192.
Effect of lymphokines on 1-adrenoceptor function of human peripheral blood mononuclear cells A. J. M. VAN OOSTERHOUT & F. P. NIJKAMP Department of Pharmacology, Faculty of Pharmacy, University of Utrecht, Catharijnesingel 60, 3511 GH Utrecht, The Netherlands
Pathological induced changes in ,B-adrenoceptor function occur in diseases such as asthma and hypertension. The mechanism(s) of this dysfunction is at present unclear. In the present study, the effect of lymphokines on ,B-adrenoceptor agonist induced cAMP production in peripheral blood mononuclear cells (PBMC) is investigated. Pre-incubation of PBMC during 20 h with interleukin-2 (IL-2, 100 u ml-') and granulocyte/macrophagecolony stimulating factor (GM-CSF, 100 u ml-') significantly decreases ,B-adrenoceptor agonist induced cAMP production by 35 ± 8% and 37 ± 11% respectively. IL-3 and IL-4 do not affect P-adrenoceptor agonist induced cAMP production in PBMC. It can be concluded that IL-2 and GM-CSF, mediators derived from T-lymphocytes, can induce ,3adrenoceptor dysfunction in PBMC. Keywords f3-adrenoceptor cAMP lymphokines mononuclear cells Introduction Leucocytes isolated from the blood are often used to study P-adrenoceptor function in disease. This approach assumes that the ,1adrenoceptor status of leucocytes mirrors pathologically induced alterations in certain tissues. Although alterations of ,-adrenoceptor function in human essential hypertension is still controversial, it has been shown that 1-adrenoceptor dysfunction occurs in leucocytes and vascular smooth muscle in a variety of animal models of hypertension (Masuzawa et al., 1989; Michel et al., 1989). In patients with asthma, it has been shown that dysfunction of the 1adrenoceptor-adenylate cyclase complex occurs in leucocytes and airway smooth muscle (Lulich et al., 1988). Both in asthma and hypertension, the changes in 1-adrenoceptor function seem to be acquired rather than fundamental abnormalities. Recently, we demonstrated that the 1adrenoceptor function of airway and vascular smooth muscle and lymphocytes is decreased, 4 days after bacterial endotoxin administration to guinea pigs (Van Heuven-Nolsen et al., 1985;
Van Oosterhout et al., 1988a). This endotoxininduced 1-adrenoceptor dysfunction can be prevented by splenectomy or by administration of the T-lymphocyte selective immunosup-
pressive drug cyclosporin-A (Van Oosterhout et al., 1988a,b). Based on these observations, it can be suggested that mediators produced by lymphocytes may be involved in 3-adrenoceptor dysfunction in this animal model and possibly in human diseases as well. In the present study, peripheral blood mononuclear cells (PBMC) are used to study the influence of cell-line derived human interleukin2 (IL-2), recombinant human (rh) IL-3, rhIL-4 and rh granulocyte/macrophage colony stimulating factor (GM/CSF) on the 1-adrenoceptor function. Methods
PBMC were isolated from the blood of healthy volunteers or from buffy coats (Bloodbank, Utrecht, The Netherlands). Isolation was per-
Correspondence: Dr A. J. M. Van Oosterhout, Department of Pharmacology, Faculty of Pharmacy, University of Utrecht, Catharijnesingel 60, 3511 GH Utrecht, The Netherlands
150S
Lymphokines and 83-adrenoceptor function formed by centrifugation on a Ficoll-paque gradient (20 min, 1000 g). Cells at the interface were removed and washed three times with RPMI-1640 (10 min, 500 g). Finally, PBMC were suspended in RPMI-1640 at 3 x 107 cells/ ml and pre-incubated with or without IL-2 (Jurkat), rhIL-3, rhIL-4 and rhGM-CSF (all from Genzyme, Boston, USA). Pre-incubations were carried out at 370 C in a humidified atmosphere of 5% C02/95% air. After a preincubation period of 2 or 20 h, 3 x 106 PBMC were incubated for 5 min at 370 C in a shaking water bath, with or without 1 ,UM isoprenaline, in the presence of 0.5 mm 3-isobutyl-1-methylxanthine in a final volume of 200 ,ul. The reaction was terminated by means of heating the cellsuspensions in a boiling water bath for 5 min. Precipitated protein was centrifuged for 15 min at 1500 g and 50 ,ul of the supernatant was used to measure cAMP content. cAMP was determined by a competitive protein binding method using a cAMP assay kit (Amersham, Great Britain). Results
Pre-incubation of PBMC with lymphokines for 2 or 20 h did not affect basal cAMP content of the cells (results not shown). The increase in cAMP induced by the ,3-adrenoceptor agonist isoprenaline (1 FLM) was not changed when PBMC were pre-incubated with lymphokines for 2 h (results not shown). After a 20 h pre-incubation period, the isoprenaline-induced cAMP production was significantly decreased in PBMC incubated with IL-2 and rhGM-CSF at a concentration of 100 u ml-1 (Table 1). The reduction of the P-adrenoceptor agonist induced cAMP production amounted to 35 ± 8% (P < 0.01, paired t-test) and 37 ± 11% (P < 0.05) in case of IL-2 and rhGM-CSF respectively. Pre-incubation of PBMC with rhIL-3 and rhIL-4 during 20 h did
not influence ,-adrenoceptor function of the cells.
Discussion In the present study, we demonstrate that IL-2 and rhGM-CSF decrease 3-adrenoceptor agonist induced cAMP production in PBMC after a 20 h pre-incubation period. These results are in accordance with Beckner & Farrar (1987) who demonstrated that IL-2 immediately decreases adenylate cyclase activity in activated human T-lymphocytes. In contrast to freshly isolated lymphocytes, activated lymphocytes express a large number of high affinity IL-2 receptors. This may explain that in our experiments the decreased P-adrenoceptor function in PBMC only occurs after a 20 h and not after a 2 h pre-incubation period. GM-CSF is a T-lymphocyte glycoprotein that promotes the growth and differentiation of granulocytes and macrophages. It has been shown that rhGM-CSF (100 u ml-1) immediately decreases adenylate cyclase activity in human neutrophils (Coffey et al., 1988). The inhibition of adenylate cyclase activity was maximal after 30 min pre-incubation, thereafter, the activity slowly recovered. In our experiments, no decreased isoprenaline-induced cAMP production is observed after two h pre-incubation of PBMC with rhGM-CSF (100 u ml-1). However, when PBMC are incubated during 20 h with rhGM-CSF, the ,-adrenoceptor function is inhibited. An explanation for the delayed effect of rhGM-CSF can be that specific receptors have to be expressed on the cell membrane, analogous to IL-2 receptors. The 3-adrenoceptor function of leucocytes is thought to mirror pathologically induced alterations in tissues like airway or vascular smooth muscle in asthma and hypertension respectively.
Table 1 Isoprenaline induced cAMP production in human PBMC after pre-incubation with lymphokines during 20 h. Results are expressed as the percentage of the cAMP production by PBMC not incubated with lymphokine Concentration (u ml-') Control 1 10 100 P < 0.05 and test. *
**
151S
IL-2 (n =5)
rhIL-3 (n =5)
rhIL-4 (n 5)
100 21.3 105.8 ± 8.8 88.0 8.5 64.8 ± 7.6**
100 28.2 104.3 ± 8.4 96.0 8.6 79.6 ± 14.1
100 21.3 105.8 ± 10.7 107.1 20.3 97.6 ± 10.5
rhGM-CSF (n = 6)
100 ± 95.5 ± 88.2 ± 63.1 ±
18.1 2.0 9.1
10.7*
P < 0.01 as compared with control and determined with Student's paired t-
152S
A. J. M. Van Oosterhout & F. P. Nijkamp
Based on our findings, a potential role for mediators derived from lymphocytes in this 1Badrenoceptor dysfunction cannot be excluded. At present, little is known about the presence of lymphokine receptors on smooth muscle-, endothelial- and epithelial cells. Expression of IL-2 receptors was initially believed to be restricted to T-cells after their activation. However, it has been demonstrated that IL-2 receptor expression can be induced in B-lymphocytes, mast cells, monocytes and macrophages
(Hancock et al., 1987). In previous experiments, we did not observe a decreased isoprenalineinduced relaxation of guinea pig tracheal spirals after 2 h pre-incubation with IL-2 (100 u ml-', unpublished observation). More studies are needed to elucidate the effect of lymphokines on 3-adrenoceptor function of other cells such as airway and vascular smooth muscle. This study was supported by a research grant (87.29) of the Dutch Asthma Foundation.
References Beckner, S. K. & Farrar, W. L. (1987). Inhibition of adenylate cyclase by IL 2 in human T lymphocytes is mediated by protein kinase C. Can. Biochem. Biophys. Res. Comm., 145,176-182. Coffey, R. G., Davis, J. S. & Djeu, J. Y. (1988). Stimulation of guanylate cyclase activity and reduction of adenylate cyclase activity by granulocytemacrophage colony-stimulating factor in human neutrophils. J. Immunol., 140, 2695-2701. Hancock, W. W., Muller, W. A. & Cotran, R. S. (1987). Interleukin 2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis and are inducible by lymphokine treatment of normal human lung macrophages, blood monocytes, and monocyte cell lines. J. Immunol., 138, 185-191. Lulich, K. M., Goldie, R. G. & Paterson, J. W. (1988). Beta-adrenoceptor function in asthmatic bronchial smooth muscle. Gen. Pharmac., 19, 307-311. Masuzawa, K., Matsuda, T. & Asano, M. (1989). Decreased arterial responsiveness to multiple cyclic AMP-generating receptor agonists in spontaneously hypertensive rats. Br. J. Pharmac., 96, 227-235.
Michel, M. C., Galal, O., Stoermer, J., Bock, K. D. & Brodde, O-E. (1989). a- and 13-adrenoceptors in hypertension. II. Platelet a2- and lymphocyte 12adrenoceptors in children of parents with essential hypertension. A model for the pathogenesis of the genetically determined hypertension. J. cardiovasc. Pharmac., 13, 432-439. Van Heuven-Nolsen, D., De Wildt, D. J. & Nijkamp, F. P. (1985). Disturbed adrenergic regulation of coronary flow in the guinea-pig heart after endotoxin. Eur. J. Pharmwc., 118, 341-345. Van Oosterhout, A. J. M., Folkerts, G., Ten Have, G. A. M. & Nijkamp, F. P. (1988a). Involvement of the spleen in the endotoxin-induced deterioration of the respiratory airway and lymphocyte 3-adrenergic systems of the guinea pig. Eur. J. Pharmac., 147, 421-430. Van Oosterhout, A. J. M., Woutersen-Van Nijnanten, F. M. A. & Nijkamp, F. P. (1988b). Cyclosporin A prevents endotoxin-induced ,B-adrenoceptor impairment in lymphocytes. Eur. J. Pharmac., 149, 191-192.
