Clin. exp. Immunol. (1992) 89, 347-350
Protective effects of cyclophosphamide, cyclosporin A and FK506 against antigen-induced lung eosinophilia in guinea-pigs A. A. NORRIS, D. M. JACKSON & R. P. EADY Department of Pharmacology, Fisons plc, Loughborough, UK
(Acceptedfor publication 1I June 1992)
SUMMARY A close association has been recognized between activated T cells and eosinophils in asthma, albeit circumstantial. The present study attempted to investigate this relationship in an animal model of lung eosinophilia using the new generation of T cell-selective immunosuppressants, cyclosporin A and FK506, compared with the myelotoxic immunosuppressive agent cyclophosphamide. Antigen challenge of ovalbumin-sensitized guinea-pigs resulted in a lung eosinophilia which was assessed by bronchoalveolar lavage. All three agents caused a marked suppression of lung eosinophilia at 24 h post-challenge when the compounds were administered at the time of sensitization but not when administered for 3 days before lavage. However, the lung eosinophilia at 72 h post-challenge was reduced significantly by FK506 and by cyclophosphamide, but not by cyclosporin A, when the drugs were administered for 3 days, before lavage. These results strongly suggest the involvement of T cells in antigen-induced late phase (72 h) eosinophilia in guinea-pigs but not at 24 h. The effects of cyclophosphamide were always associated with a reduction in circulating white cell counts, whereas cyclosporin A and FK506 showed no myelotoxic properties. These results suggest the potential therapeutic use of selective, non-cytotoxic immunosuppressive agents in asthma.
Keywords cyclophosphamide cyclosporin A FK506 eosinophils lung
INTRODUCTION sarcoidosis, asthma and alveolitis [15-18]. However, there is little information available on their effects on eosinophil cell T cell-derived cytokines are capable of exerting control over numbers in these clinical studies. With the use of a new effector cells in allergic inflammation such as basophils, eosinogeneration of T cell-selective, non-myelotoxic immunosuppresphils and mast cells [1]. Activated T cells are a rich source of sive agents, cyclosporin A (CsA) [19] and FK506 [20], it is now GM-CSF, IL-3 and IL-5 [2-5] and these growth factors can possible to define precisely the role of T cells in animal models of influence responses such as cell maturation, activation and lung eosinophilia. For comparative purposes, cyclophosphachemotaxis. In vivo evidence for activated T cells in the mide, an immunosuppressve agent with myelotoxic properties, circulation [6] and in bronchial biopsies of asthmatics [7] has led has also been investigated in the present study of lung eosinophithese authors to believe that inappropriately activated T cells lia in ovalbumin-challenged guinea-pigs. may be responsible for both initiation and maintenance of chronic allergic inflammation. Furthermore, an improvement in asthma after treatment with corticosteroids has been shown to MATERIALS AND METHODS be accompanied by a reduction in activated CD25+/CD4+ T lymphocytes [8]. The role of eosinophils as final effector cells in Reagents allergic inflammation, which includes bronchial asthma, has Unless otherwise stated, all chemicals were purchased from attracted much interest [9-1 1] and there is growing evidence that Fisons Scientific Equipment (Loughborough, UK). Cyclophosan association may exist between CD4+ T cells and eosinophils phamide, mepyramine maleate, and ovalbumin (grade III) were in allergic inflammation [12,13]. Recent studies in asthmatics [7] obtained from Sigma Chemical Co. (Poole, UK). Sodium and in antigen-challenged guinea-pigs [14] lend support to this pentobarbitone was from May and Baker (Dagenham, UK). hypothesis with the observations of close correlations between CsA and FK506 were generous gifts from Fujisawa (Osaka, relative numbers of CD3+ T cells and activated (EG2+) Japan). Olive oil BP was from Evans Medical Ltd (Horsham, eosinophils in the airways. UK). Numerous studies have been carried out with immunosupSestzto pressive agents in various inflammatory lung disorders such as an.nie hlneo unapg Correspondence: A. A. Norris, Department of Pharmacology, Male, Dunkin-Hartley guinea-pigs (250-300 g) (in groups of 6 Fisons plc, Bakewell Road, Loughborough, Leics LE1 1 ORH, UK. to 10 animals) were sensitized to ovalbumin given intraperito347
A. A. Norris, D. M. Jackson & R. P. Eady
348
neally and subcutaneously at 100 mg/ml in saline in a volume of 1 ml per site. Approximately 21 days later, animals were challenged with aerosolized ovalbumin (2%) for 2 min in a perspex chamber, 30 mi after pretreatment with mepyramine maleate (10 mg/kg intraperitoneally). Non-sensitized guineapigs were also challenged with ovalbumin and these animals served as controls. Aerosols were generated using a Wright's nebulizer operated from a flow of compressed air (138 kPa) at a rate of 15 I/min. This method is similar to that descnibed by Hutson et al [21]. Animals were killed either 24 h or 72 h after exposure to antigen using sodium pentobarbitone (200 mg/kg) intraperitoneally. Bronchoalveolar ravage Bronchoalveolar lavage (BAL) was carried out via a tracheal cannula using three aliquots (5 ml) of PBS. The BAL fluid was recovered by suction and was centrifuged in plastic tubes at 1000 rev/min for 10 min. The cell pellet was resuspended in 1 ml Hanks' balanced salt solution (HBSS) and an aliquot diluted with five volumes of Kimura's stain [22]. Total and differential BAL cell counts were carried out under a microscope using a modified Fuchs-Rosenthal haemocytometer.
Blood counts Blood samples (approximately 0 1 ml) were taken from the saphenous vein of guinea-pigs on the final day of the experiment. Total leucocyte counts were measured using a Coulter Counter (Model ZF) (Coulter Electronics) and differential counts were carried out on Wright's stained blood smears, using morphological criteria. Drug treatments FK506 was prepared in distilled water and Tween 80 (0 05%) for oral administration. CsA was prepared by dissolving the solid compound in a small volume of ethanol and mixing with olive oil BP for subcutaneous administration. Cyclophosphamide was dissolved in normal saline for i.p. administration. Drug treatment took place once per day only. Animals were dosed according to one of the following regimens: R1, one day after sensitization for 3 days; R2, for 3 days before the lavage at 24 h post-antigen challenge; R3, for 3 days before the lavage at 72 h post-antigen challenge, as shown in Fig. 1.
Statistical analysis Data referring to cell counts are expressed as geometric means and s.e.m. Differences were compared using a Mann-Whitney U-test and were considered significant if P < 0 05.
RESULTS Cell content of BALfluid in ovalbumin-challenged guinea-pigs Antigen challenge produced a consistent lung eosinophilia, detected in BAL fluid at 24 h and 72 h post-challenge. Eosinophils represented approximately 30% of total cells at 24 h and almost 50% of total cells in BAL at 72 h post-challenge rarely (aled1). forNeuthano more than 3%S of lymphces total cells at tothe both time points. accounted Cyclophosphamide (50 mg/kg for 3 days), FK506 (10 mg/kg for 3 days) and .sA (30 mg/kg for 3 days) all inhibited BAL eosinophilia and reduced macrophage numbers when given immediately after sensitization (RI regimen) (Table 2) whereas Table 1. Effects of antigen challenge of guinea-pigs on cell content of BAL fluid
Time of lavage
23 24 ~~~~~~~22
1 t 1 R2
C
Day
t
II
RtRI t
25
B
(+ 24 h)
(+72 h) Sensitized (+24 h)
(+ 72 h)
Fig. 1. Drug dosing regimens for studies using ovalbumin-sensitized guinea-pigs. S, Sensitization; C, antigen challenge; B, bronchoalveolar lavage; R, drug administration (once daily); Ri, on days 2-4 inclusive; R2, on days 20-22 inclusive; R3, on days 22-24 inclusive.
48 0
32
42.1
(42 2-55 4)
(18-4-8)
(38-9-46-0)
57 1 (52 6-62-4)
56 (4-9-6-4)
47.9 (42-4-53 3)
115.9* (106 6-124 8) 95 6* (87 4-104-0)
37 7t (29 9-45 1) 42 6t (37 3-48 3)
71 0* (63 8-79 5) 48.5 (40 4-56 8)
Table 2. Effects of drug treatments on cell content of BAL fluid of antigen-challenged guinea-pigs
treatment Vehicle control Cyclophosphamide
Eosinophils
Macrophages
(22-8-33-0)
27.8
61 3 (55 0-68 3)
(1015)
31.4*
35.7
629
(30 7-41 6)
(49-9-791) 40 5
Cyclosporin A
6.9* (4-7-10-3)
Vehicle control
420
88-9
(36 1-52-4)
(74 0-105 5)
B
?
Macrophages (x 104/ml)
Values represent geometric mean and s.e.m. * P < 0 05 compared with corresponding non-sensitized value. 1 P < 0 01 compared with corresponding non-sensitized value.
FK506
t t
Eosinophils (X 104/ml)
Non-sensitized
Vehicle control 1
Total cells (x 104/ml)
6.1*
(4-3-8-4)
(32-2-51-4) 36 9t (24-9-54-7)
Values represent geometric mean + s~em. All drug treatments were carried out according to regimen Ri. * P
Protective effects of cyclophosphamide, cyclosporin A and FK506 against antigen-induced lung eosinophilia in guinea-pigs A. A. NORRIS, D. M. JACKSON & R. P. EADY Department of Pharmacology, Fisons plc, Loughborough, UK
(Acceptedfor publication 1I June 1992)
SUMMARY A close association has been recognized between activated T cells and eosinophils in asthma, albeit circumstantial. The present study attempted to investigate this relationship in an animal model of lung eosinophilia using the new generation of T cell-selective immunosuppressants, cyclosporin A and FK506, compared with the myelotoxic immunosuppressive agent cyclophosphamide. Antigen challenge of ovalbumin-sensitized guinea-pigs resulted in a lung eosinophilia which was assessed by bronchoalveolar lavage. All three agents caused a marked suppression of lung eosinophilia at 24 h post-challenge when the compounds were administered at the time of sensitization but not when administered for 3 days before lavage. However, the lung eosinophilia at 72 h post-challenge was reduced significantly by FK506 and by cyclophosphamide, but not by cyclosporin A, when the drugs were administered for 3 days, before lavage. These results strongly suggest the involvement of T cells in antigen-induced late phase (72 h) eosinophilia in guinea-pigs but not at 24 h. The effects of cyclophosphamide were always associated with a reduction in circulating white cell counts, whereas cyclosporin A and FK506 showed no myelotoxic properties. These results suggest the potential therapeutic use of selective, non-cytotoxic immunosuppressive agents in asthma.
Keywords cyclophosphamide cyclosporin A FK506 eosinophils lung
INTRODUCTION sarcoidosis, asthma and alveolitis [15-18]. However, there is little information available on their effects on eosinophil cell T cell-derived cytokines are capable of exerting control over numbers in these clinical studies. With the use of a new effector cells in allergic inflammation such as basophils, eosinogeneration of T cell-selective, non-myelotoxic immunosuppresphils and mast cells [1]. Activated T cells are a rich source of sive agents, cyclosporin A (CsA) [19] and FK506 [20], it is now GM-CSF, IL-3 and IL-5 [2-5] and these growth factors can possible to define precisely the role of T cells in animal models of influence responses such as cell maturation, activation and lung eosinophilia. For comparative purposes, cyclophosphachemotaxis. In vivo evidence for activated T cells in the mide, an immunosuppressve agent with myelotoxic properties, circulation [6] and in bronchial biopsies of asthmatics [7] has led has also been investigated in the present study of lung eosinophithese authors to believe that inappropriately activated T cells lia in ovalbumin-challenged guinea-pigs. may be responsible for both initiation and maintenance of chronic allergic inflammation. Furthermore, an improvement in asthma after treatment with corticosteroids has been shown to MATERIALS AND METHODS be accompanied by a reduction in activated CD25+/CD4+ T lymphocytes [8]. The role of eosinophils as final effector cells in Reagents allergic inflammation, which includes bronchial asthma, has Unless otherwise stated, all chemicals were purchased from attracted much interest [9-1 1] and there is growing evidence that Fisons Scientific Equipment (Loughborough, UK). Cyclophosan association may exist between CD4+ T cells and eosinophils phamide, mepyramine maleate, and ovalbumin (grade III) were in allergic inflammation [12,13]. Recent studies in asthmatics [7] obtained from Sigma Chemical Co. (Poole, UK). Sodium and in antigen-challenged guinea-pigs [14] lend support to this pentobarbitone was from May and Baker (Dagenham, UK). hypothesis with the observations of close correlations between CsA and FK506 were generous gifts from Fujisawa (Osaka, relative numbers of CD3+ T cells and activated (EG2+) Japan). Olive oil BP was from Evans Medical Ltd (Horsham, eosinophils in the airways. UK). Numerous studies have been carried out with immunosupSestzto pressive agents in various inflammatory lung disorders such as an.nie hlneo unapg Correspondence: A. A. Norris, Department of Pharmacology, Male, Dunkin-Hartley guinea-pigs (250-300 g) (in groups of 6 Fisons plc, Bakewell Road, Loughborough, Leics LE1 1 ORH, UK. to 10 animals) were sensitized to ovalbumin given intraperito347
A. A. Norris, D. M. Jackson & R. P. Eady
348
neally and subcutaneously at 100 mg/ml in saline in a volume of 1 ml per site. Approximately 21 days later, animals were challenged with aerosolized ovalbumin (2%) for 2 min in a perspex chamber, 30 mi after pretreatment with mepyramine maleate (10 mg/kg intraperitoneally). Non-sensitized guineapigs were also challenged with ovalbumin and these animals served as controls. Aerosols were generated using a Wright's nebulizer operated from a flow of compressed air (138 kPa) at a rate of 15 I/min. This method is similar to that descnibed by Hutson et al [21]. Animals were killed either 24 h or 72 h after exposure to antigen using sodium pentobarbitone (200 mg/kg) intraperitoneally. Bronchoalveolar ravage Bronchoalveolar lavage (BAL) was carried out via a tracheal cannula using three aliquots (5 ml) of PBS. The BAL fluid was recovered by suction and was centrifuged in plastic tubes at 1000 rev/min for 10 min. The cell pellet was resuspended in 1 ml Hanks' balanced salt solution (HBSS) and an aliquot diluted with five volumes of Kimura's stain [22]. Total and differential BAL cell counts were carried out under a microscope using a modified Fuchs-Rosenthal haemocytometer.
Blood counts Blood samples (approximately 0 1 ml) were taken from the saphenous vein of guinea-pigs on the final day of the experiment. Total leucocyte counts were measured using a Coulter Counter (Model ZF) (Coulter Electronics) and differential counts were carried out on Wright's stained blood smears, using morphological criteria. Drug treatments FK506 was prepared in distilled water and Tween 80 (0 05%) for oral administration. CsA was prepared by dissolving the solid compound in a small volume of ethanol and mixing with olive oil BP for subcutaneous administration. Cyclophosphamide was dissolved in normal saline for i.p. administration. Drug treatment took place once per day only. Animals were dosed according to one of the following regimens: R1, one day after sensitization for 3 days; R2, for 3 days before the lavage at 24 h post-antigen challenge; R3, for 3 days before the lavage at 72 h post-antigen challenge, as shown in Fig. 1.
Statistical analysis Data referring to cell counts are expressed as geometric means and s.e.m. Differences were compared using a Mann-Whitney U-test and were considered significant if P < 0 05.
RESULTS Cell content of BALfluid in ovalbumin-challenged guinea-pigs Antigen challenge produced a consistent lung eosinophilia, detected in BAL fluid at 24 h and 72 h post-challenge. Eosinophils represented approximately 30% of total cells at 24 h and almost 50% of total cells in BAL at 72 h post-challenge rarely (aled1). forNeuthano more than 3%S of lymphces total cells at tothe both time points. accounted Cyclophosphamide (50 mg/kg for 3 days), FK506 (10 mg/kg for 3 days) and .sA (30 mg/kg for 3 days) all inhibited BAL eosinophilia and reduced macrophage numbers when given immediately after sensitization (RI regimen) (Table 2) whereas Table 1. Effects of antigen challenge of guinea-pigs on cell content of BAL fluid
Time of lavage
23 24 ~~~~~~~22
1 t 1 R2
C
Day
t
II
RtRI t
25
B
(+ 24 h)
(+72 h) Sensitized (+24 h)
(+ 72 h)
Fig. 1. Drug dosing regimens for studies using ovalbumin-sensitized guinea-pigs. S, Sensitization; C, antigen challenge; B, bronchoalveolar lavage; R, drug administration (once daily); Ri, on days 2-4 inclusive; R2, on days 20-22 inclusive; R3, on days 22-24 inclusive.
48 0
32
42.1
(42 2-55 4)
(18-4-8)
(38-9-46-0)
57 1 (52 6-62-4)
56 (4-9-6-4)
47.9 (42-4-53 3)
115.9* (106 6-124 8) 95 6* (87 4-104-0)
37 7t (29 9-45 1) 42 6t (37 3-48 3)
71 0* (63 8-79 5) 48.5 (40 4-56 8)
Table 2. Effects of drug treatments on cell content of BAL fluid of antigen-challenged guinea-pigs
treatment Vehicle control Cyclophosphamide
Eosinophils
Macrophages
(22-8-33-0)
27.8
61 3 (55 0-68 3)
(1015)
31.4*
35.7
629
(30 7-41 6)
(49-9-791) 40 5
Cyclosporin A
6.9* (4-7-10-3)
Vehicle control
420
88-9
(36 1-52-4)
(74 0-105 5)
B
?
Macrophages (x 104/ml)
Values represent geometric mean and s.e.m. * P < 0 05 compared with corresponding non-sensitized value. 1 P < 0 01 compared with corresponding non-sensitized value.
FK506
t t
Eosinophils (X 104/ml)
Non-sensitized
Vehicle control 1
Total cells (x 104/ml)
6.1*
(4-3-8-4)
(32-2-51-4) 36 9t (24-9-54-7)
Values represent geometric mean + s~em. All drug treatments were carried out according to regimen Ri. * P
