Immunobiol., vol. 181, pp. 22-30 (1990)
Department of Internal Medicine, 1 Division of Hematology, Oncology, Clin. Immunology, Division of Nephrology, University of Marburg, D-3550 Marburg, Federal Republic of Germany
2
Latent Inhibition of Granulocyte Function by Cyclosporine A GERALD KOLB\ ILSEBILLEcKLE\ KLAUsBITINER2 , THOMAS MULLER2 , KLAUS HAVEMANN 1, and HARALD LANGE 2 Received November 15, 1989 . Accepted in Revised Form March 30,1990
Abstract According to the literature, Cyclosporine A (CsA) is said to suppress specifically the activity of T and B cells. A significant influence on phagocyte function has been neglected. However, agravated courses of bacterial and fungal infections have been frequently reported under the treatment with CsA, suggesting that a latent depression of phagocytic activity may possibly occur under clinical circumstances. Therefore, this study set out to assess whether CsA can also change granulocyte function under therapy conditions or not. Thirty-seven patients, 3 months-l0 years after kidney transplantation being under immunosuppressive treatment with CsA + Prednisolone (n=25), Azathioprine + Prednisolone (n=6) and under Prednisolone alone (n=6) underwent the study. 18 healthy persons served as a normal control group. Granulocyte function was tested ex vivo by chemiluminescence (CL) after stimulation with phorbolmyristate acetate (PMA) and with zymosan (zym) activated autologous or poolserum. The obtained data were correlated to corresponding serum or plasma levels of CsA, human leukocyte elastase (HLE) and neopterin. Comparing the three therapy groups with the healthy control and with each other no differences could be seen in median CL values; but there was a significant (p = 0.05) negative correlation between CsA blood levels and maximum CL values of PMN. Such inhibition of CL could be calculated for zym but not for PMA stimulated PMN; suggesting that the CsA mediated inhibition of granulocyte function may be only partial and restricted to phagocytosis. In addition, a positive correlation between serum levels of human leukocyte elastase (HLE) and neopterin could be found. This indicates a simultaneous influence of CsA on both PMN and macrophages.
Introduction Cyclosporine A (CsA) is a cyclic endecapeptide of fungal origin, which is widely used in immunosuppressive treatment after organ transplantation (1-4). It has been shown that CsA acts as an inhibitor of both cell immunity and antibody formation (5, 6). In addition unresponsiveness of T cells to interleukins 1 and 2 (7) and suppression of synthesis of these lymphokines (8) have been reported.
Latent Inhibition of Granulocyte Function by Cyclosporine A . 23
Nevertheless, CsA seems to have less unspecific clinical side effects than other immunosuppressive drug regimens. Especially usage of corticosteroids in higher dosages, alone or in combination with cytotoxic agents, can predispose the patients to opportunistic bacterial infections (9). Fortunately CsA does not cause neutropenia (10) and has a steroid-sparing effect (11). However cases of deep-seated staphylococcal infections and aspergillosis were described (12), suggesting CsA may have a suppressive influence also on neutrophil function such as inhibition of phagocytosis, bacterial killing and/or peroxide production. Most of the previous in vitro studies failed to demonstrate an impressive inhibitory effect of CsA on neutrophils (13) apart from a slight inhibition of the early phase of phagocytosis and bacterial killing (14). But these results were not reproduced generally and varied depending on the different experimental in vitro conditions (13, 20) and CsA concentrations. We designed a combined in vitro and ex vivo study to answer the question: Whether CsA influences neutrophil activity dose and/or time dependently, and if this effect is cell and/or serum mediated? Additionally, we looked for possible correlations between CsA dosage, inhibition of neutrophil function and secondary phenomena like levels of acute phase reactants, human leucocyte elastase and macrophage derived neopterin.
Material and Methods Blood was drawn by venipuncture and anticoagulated with citrate. Neutrophils were isolated by ficoll separation and sedimentation of erythrocytes. According to a previously described modification (21) of BOYUM'S (15) method. Neutrophil oxidative metabolism was measured by luminol enhanced chemiluminescence (CL) with a 1251 luminometer by LKB, Bromma, Sweden, according to the method of EASMON et al. (16). CL was recorded of unstimulated cells and after addition of phorbolmyristate acetate (PMA) or zymosan activated serum. Serum was activated with zymosan (25 !-II serum + 200 !-II zymosan suspension: 12.5 mg/ml 30 min at 37°C) and the entire incubation mixture was applied for the stimulation of granulocytes. Assay mixture consisted of 50 !-II neutrophils (1.0X 107/ml) 100 !-Illuminol (200 !-1M), 50 !-II PMA (20 !-Ig/!-II) or 200 !-II zymosan activated serum in a final volume of 750 !-II Hanks balanced salt solution. CL was recorded for 35 min at 37°C. The drugs CsA (Sandimmun®, Sandoz AG, Niirnberg, W-Germany), Prednisolone (Solu-Decortin-H®, Merck, Darmstadt, W-Germany), and Azathioprine (Imurek®, Wellcome GmbH, Burgwedel, W-Germany) were diluted to the concentrations shown under the section results. Maximal CL values were taken for statistical evaluation by U-Test (WILCOXON, MANN and WHITNEY, 17) and SPEARMAN rank correlation test (18). CsA blood levels were measured by radioimmunoassay (IBL, Hamburg, W-Germany), C-reactive protein determination was performed by nephelometry (Behringwerke, Marburg, W-Germany). Neopterin levels were determined by radioimmunoassay (Kemzing Berlin GmbH, W-Germany). Elastase-alphatproteinaseinhibitor-complex was measured by enzymimmunoassay (Merck, Darmstadt, WGermany). Patients
Thirty-seven patients with chronic renal failure (13 male, 24 female, 21-69 years old) were selected and classified according to their immunosuppressive treatment following kidney transplantation 3 months to 4 years ago.
24 . G. KOLB, I. ECKLE, K. BITINER, T. MULLER, K. HAVEMANN, and H. LANGE All patients listed in this study were free of acute infections and without any signs of an acute rejection of the transplant. Values of creatinine and urea remained constant at time of study and during a follow-up of more than 6 months. Immunosuppressive treatment
Group
n
1 2 3
25
o
Cyclosporine A + prednisolone Azathioprine + prednisolone Azathioprine Healthy controls
6 6
18
Dosage of CsA was 1.9 to 6.1 mg/kg bodyweight, prednisolone 0.095 to 0.329 mg/kg bodyweight and azathioprine 50 to 150 mg/day.
Results
Patient and control neutrophils were isolated simultaneously and tested for luminol enhanced CL after stimulation with PMA or serum activated zymosan. Medians of the maximal CL values for both groups of neutrophils (patient neutrophils and the control group) are given in Table 1. Comparison of the group with U-test by WILCOXON, MANN, and WHITNEY showed no statistical significant differences between patients and controls. Analysis of patient plasma samples for human leucocyte elastase-alphat-proteinaseinhibitor-complex (HLE-UtPI), neopterin and CRP levels gave the values listed in Table 2, which shows also the ESR values. Table 1. Chemiluminescence of patient PMN after stimulation with PMA, zymosan and autologous serum (zym + AS) or zymosan and pool-serum (zym + PS) Median maximal CL (m V)
2
3
0
25
6
6
18
621 975 897
901 446 492
576 948 810
673 856 709
Group n Stimulus PMA zym + AS zym + PS
Table 2. Median values of human leucocyte-elastase-alphal-proteinaseinhibitor-complex (HLE-uIPI), neopterin and CRP in plasma samples of patient groups 1 to 3 and range of ESR Group HLE-uIPI (ng/ml plasma) Neopterin (ng/ml) CRP ESR
133
10-20 + 5/10-20/40
2
3
64
77
10-20 + 5/10-20/40
10 + 5/10-20/40
Latent Inhibition of Granulocyte Function by Cyclosporine A
25
Group 2 (n.6) G'oupl(n.22)
;;;
Department of Internal Medicine, 1 Division of Hematology, Oncology, Clin. Immunology, Division of Nephrology, University of Marburg, D-3550 Marburg, Federal Republic of Germany
2
Latent Inhibition of Granulocyte Function by Cyclosporine A GERALD KOLB\ ILSEBILLEcKLE\ KLAUsBITINER2 , THOMAS MULLER2 , KLAUS HAVEMANN 1, and HARALD LANGE 2 Received November 15, 1989 . Accepted in Revised Form March 30,1990
Abstract According to the literature, Cyclosporine A (CsA) is said to suppress specifically the activity of T and B cells. A significant influence on phagocyte function has been neglected. However, agravated courses of bacterial and fungal infections have been frequently reported under the treatment with CsA, suggesting that a latent depression of phagocytic activity may possibly occur under clinical circumstances. Therefore, this study set out to assess whether CsA can also change granulocyte function under therapy conditions or not. Thirty-seven patients, 3 months-l0 years after kidney transplantation being under immunosuppressive treatment with CsA + Prednisolone (n=25), Azathioprine + Prednisolone (n=6) and under Prednisolone alone (n=6) underwent the study. 18 healthy persons served as a normal control group. Granulocyte function was tested ex vivo by chemiluminescence (CL) after stimulation with phorbolmyristate acetate (PMA) and with zymosan (zym) activated autologous or poolserum. The obtained data were correlated to corresponding serum or plasma levels of CsA, human leukocyte elastase (HLE) and neopterin. Comparing the three therapy groups with the healthy control and with each other no differences could be seen in median CL values; but there was a significant (p = 0.05) negative correlation between CsA blood levels and maximum CL values of PMN. Such inhibition of CL could be calculated for zym but not for PMA stimulated PMN; suggesting that the CsA mediated inhibition of granulocyte function may be only partial and restricted to phagocytosis. In addition, a positive correlation between serum levels of human leukocyte elastase (HLE) and neopterin could be found. This indicates a simultaneous influence of CsA on both PMN and macrophages.
Introduction Cyclosporine A (CsA) is a cyclic endecapeptide of fungal origin, which is widely used in immunosuppressive treatment after organ transplantation (1-4). It has been shown that CsA acts as an inhibitor of both cell immunity and antibody formation (5, 6). In addition unresponsiveness of T cells to interleukins 1 and 2 (7) and suppression of synthesis of these lymphokines (8) have been reported.
Latent Inhibition of Granulocyte Function by Cyclosporine A . 23
Nevertheless, CsA seems to have less unspecific clinical side effects than other immunosuppressive drug regimens. Especially usage of corticosteroids in higher dosages, alone or in combination with cytotoxic agents, can predispose the patients to opportunistic bacterial infections (9). Fortunately CsA does not cause neutropenia (10) and has a steroid-sparing effect (11). However cases of deep-seated staphylococcal infections and aspergillosis were described (12), suggesting CsA may have a suppressive influence also on neutrophil function such as inhibition of phagocytosis, bacterial killing and/or peroxide production. Most of the previous in vitro studies failed to demonstrate an impressive inhibitory effect of CsA on neutrophils (13) apart from a slight inhibition of the early phase of phagocytosis and bacterial killing (14). But these results were not reproduced generally and varied depending on the different experimental in vitro conditions (13, 20) and CsA concentrations. We designed a combined in vitro and ex vivo study to answer the question: Whether CsA influences neutrophil activity dose and/or time dependently, and if this effect is cell and/or serum mediated? Additionally, we looked for possible correlations between CsA dosage, inhibition of neutrophil function and secondary phenomena like levels of acute phase reactants, human leucocyte elastase and macrophage derived neopterin.
Material and Methods Blood was drawn by venipuncture and anticoagulated with citrate. Neutrophils were isolated by ficoll separation and sedimentation of erythrocytes. According to a previously described modification (21) of BOYUM'S (15) method. Neutrophil oxidative metabolism was measured by luminol enhanced chemiluminescence (CL) with a 1251 luminometer by LKB, Bromma, Sweden, according to the method of EASMON et al. (16). CL was recorded of unstimulated cells and after addition of phorbolmyristate acetate (PMA) or zymosan activated serum. Serum was activated with zymosan (25 !-II serum + 200 !-II zymosan suspension: 12.5 mg/ml 30 min at 37°C) and the entire incubation mixture was applied for the stimulation of granulocytes. Assay mixture consisted of 50 !-II neutrophils (1.0X 107/ml) 100 !-Illuminol (200 !-1M), 50 !-II PMA (20 !-Ig/!-II) or 200 !-II zymosan activated serum in a final volume of 750 !-II Hanks balanced salt solution. CL was recorded for 35 min at 37°C. The drugs CsA (Sandimmun®, Sandoz AG, Niirnberg, W-Germany), Prednisolone (Solu-Decortin-H®, Merck, Darmstadt, W-Germany), and Azathioprine (Imurek®, Wellcome GmbH, Burgwedel, W-Germany) were diluted to the concentrations shown under the section results. Maximal CL values were taken for statistical evaluation by U-Test (WILCOXON, MANN and WHITNEY, 17) and SPEARMAN rank correlation test (18). CsA blood levels were measured by radioimmunoassay (IBL, Hamburg, W-Germany), C-reactive protein determination was performed by nephelometry (Behringwerke, Marburg, W-Germany). Neopterin levels were determined by radioimmunoassay (Kemzing Berlin GmbH, W-Germany). Elastase-alphatproteinaseinhibitor-complex was measured by enzymimmunoassay (Merck, Darmstadt, WGermany). Patients
Thirty-seven patients with chronic renal failure (13 male, 24 female, 21-69 years old) were selected and classified according to their immunosuppressive treatment following kidney transplantation 3 months to 4 years ago.
24 . G. KOLB, I. ECKLE, K. BITINER, T. MULLER, K. HAVEMANN, and H. LANGE All patients listed in this study were free of acute infections and without any signs of an acute rejection of the transplant. Values of creatinine and urea remained constant at time of study and during a follow-up of more than 6 months. Immunosuppressive treatment
Group
n
1 2 3
25
o
Cyclosporine A + prednisolone Azathioprine + prednisolone Azathioprine Healthy controls
6 6
18
Dosage of CsA was 1.9 to 6.1 mg/kg bodyweight, prednisolone 0.095 to 0.329 mg/kg bodyweight and azathioprine 50 to 150 mg/day.
Results
Patient and control neutrophils were isolated simultaneously and tested for luminol enhanced CL after stimulation with PMA or serum activated zymosan. Medians of the maximal CL values for both groups of neutrophils (patient neutrophils and the control group) are given in Table 1. Comparison of the group with U-test by WILCOXON, MANN, and WHITNEY showed no statistical significant differences between patients and controls. Analysis of patient plasma samples for human leucocyte elastase-alphat-proteinaseinhibitor-complex (HLE-UtPI), neopterin and CRP levels gave the values listed in Table 2, which shows also the ESR values. Table 1. Chemiluminescence of patient PMN after stimulation with PMA, zymosan and autologous serum (zym + AS) or zymosan and pool-serum (zym + PS) Median maximal CL (m V)
2
3
0
25
6
6
18
621 975 897
901 446 492
576 948 810
673 856 709
Group n Stimulus PMA zym + AS zym + PS
Table 2. Median values of human leucocyte-elastase-alphal-proteinaseinhibitor-complex (HLE-uIPI), neopterin and CRP in plasma samples of patient groups 1 to 3 and range of ESR Group HLE-uIPI (ng/ml plasma) Neopterin (ng/ml) CRP ESR
133
10-20 + 5/10-20/40
2
3
64
77
10-20 + 5/10-20/40
10 + 5/10-20/40
Latent Inhibition of Granulocyte Function by Cyclosporine A
25
Group 2 (n.6) G'oupl(n.22)
;;;
