Lymphocyte suppressor activity in atopic eczema B. E. OGDEN, G. G. KRUEGER & H. R. HILL Howard Hughes Medical Institute Laboratories, Division of Clinical Immunology and Allergy, Department oj Pediatrics, the Division of Dermatology, Department of Medicine and Department of Pathology, University of Utah, Salt Lake City, Utah, USA (Received 22 May 1978)
SUMMARY
Previous studies have shown that patients with atopic eczema have depressed cell-mediated immunity. Whether this defect can be attributed to abnormal suppressor cell activity or to the presence of mediators of the allergic response has not been studied before. Lymphocyte transformation was found to be enhanced in patients with mild eczema and markedly depressed in patients with severe eczema, when compared with normal controls. Pre-incubation of cultures for 48 hr without mitogen prior to transformation studies restored normal lymphocyte thymidine uptake in cells from severe atopics, suggesting a labile suppressor cell population, or a labile suppressor substance. Since mononuclear cell supernatants from patients with severe eczema failed to suppress lymphocyte transformation more than supernatants from normals, it is unlikely that the depressed lymphocyte function seen in severe eczema is due to an abnormal suppressor cell population. The possibility that mediators of the allergic response may be acting as a labile suppressor substance was evaluated by adding various concentrations of histamine, cyclic-AMP, or prostaglandin E1 to lymphocytes undergoing mitogenesis. Histamine enhanced thymidine incorporation at low concentrations and depressed uptake at high concentrations; cyclic-AMP and prostaglandin E1 have similar effects on transformation. It is possible that the enhancement of transformation seen in mild eczema and the depression of this response in severe eczema may be related to the concentrations or degree of allergic mediator release.
INTRODUCTION Studies of patients with atopic eczema have shown several immune abnormalities including defective cell-mediated immunity (Buckley, Wray & Belmaker, 1972) and defective neutrophil chemotaxis (Hill & Quie, 1974; Hill et al., 1974). In most cases these defects have been associated with hyperimmunoglobulinaemia E suggesting that they may be a direct result of high IgE levels or indirectly, a result of IgE-mediated release of vasoactive amines. Since the overall levels of IgE are felt to be regulated by T lymphocytes (Okamura & Tada, 1971) it is possible that suppression of T cell function might lead to high IgE levels and atopic disease. Recent studies have shown depressed T lymphocyte function in patients with atopic disease, which correlates inversely with IgE levels (McGeady & Buckley, 1975; Rachelefsky et al., 1976). Depressed lymphocyte function is felt in many cases to be due directly to a population of suppressor T cells, or to the soluble suppressor products of such a population (Asherson & Zembala, 1976; Webb, Jamieson & Nowowiejski, 1976; Okamura & Tada, 1974). Other work suggests that some suppressor activity is a result of mediators produced by activated monocytes (Ogden, Krueger & Jederberg, 1977; Nelson, 1973; Keller, 1975; Wing & Remington, 1977). In the present study we have examined T lymphocyte function in atopic eczema and have looked closely for suppressor activity in these individuals. Suppressed function was observed in severe eczema which was abolished by 48 hr pre-incubation, suggesting the presence of a labile suppressor substance or population of cells. Correspondence: Dr B. E. Ogden, Division of Clinical Immunology and Allergy, Department of Pediatrics, University of Utah, Salt Lake City, Utah 84132, USA. 0099-9104/79/0020-0269$02.00 A) 1979 Blackwell Scientific Publications
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B. E. Ogden, G. G. Krueger & H. R. Hill MATERIALS AND METHODS
Subjects. Two of the ten atopic patients studied suffered from asthma, allergic rhinitis and eczema, two had asthma and eczema, four had allergic rhinitis and eczema, and two had eczema alone. Five of the patients had severe eczema ( > 500/o of the skin involved) and five patients had mild eczema (< 10% of the skin involved). Patients had not received daily corticosteroids for at least 9 months, or systemic or topical medication for at least 4 days prior to study. The patients' (n 10) ages ranged from 8 to 36 years with a mean-f s.e. of 25 + 3 while the age of normal controls (n = 12) ranged from 18 to 31 years with a mean + s.e. of 25 1. Control subjects were normal healthy young adults who were receiving no therapy. Age, sex and time variables were controlled by studying each patient simultaneously with a similarly aged and same sex control. IgE levels. IgE concentrations were measured in patient and control plasma samples by a radioimmunoabsorbent test using a Phadebas IgE test kit (Pharmacia Laboratories, Piscataway, New Jersey). T lymphocytes. T cell determinations were made by a modification of the E-rosette method described elsewhere (Jondal, Holm & Wigzell, 1972). An aliquot of mononuclear cells was harvested as described below, adjusted to 4 x 106 cells/ml and incubated at 37 C with an aliquot of fresh 0-50/ sheep erythrocytes for 5 min, centrifuged at 2000 g for 3 min, and then incubated at 4VC for 30 min before counting. Lymphocyte transformation. Lymphocyte transformation in response to several doses of the T cell mitogen, concanavalin A (Con A, Sigma Chemical Co., St. Louis, Missouri), was performed using mononuclear cells isolated from heparinized whole blood by Ficoll-Hypaque density gradient centrifugation, according to a modification of the technique published by Boyum (1968). Briefly, heparinized blood was spun at 100 g for 15 min and the plasma containing the majority of platelets was removed. Blood was then diluted in Hanks's balanced salt solution and layered over Ficoll-Hypaque for centrifugation at 250 g for 25 min. Mononuclear cells were harvested from the interface, washed twice in Hanks's and suspended in RPMI1640 tissue culture medium containing gentamicin and 10/o0 heat-inactivated foetal calf serum at a concentration of 105 T cells/ml. 0 2 ml of the cell suspension was then dispensed to each well of a microtitre plate. At this point 25 PI of media alone or media containing various doses of Con A was added and plates were incubated at 37 C, 5% CO2. For some experiments, a single dose of Con A (10 pg/ml) was used and 25 pl of various concentrations of histamine, cyclic-AMP or prostaglandin E, (Sigma Chemical Co., St. Louis, Missouri) was added at the beginning of the incubation period. After was 48 hr of incubation, 25 pl of 10 pCi/ml 3H-thymidine (sp. act. 6 7 Ci/mM, New England Nuclear, Boston, Massachusetts) added for a 24 hr pulse following which, plates were harvested with a multiple automated sample harvester and 'H-thymidine uptake was determined by liquid scintillation counting using a Beckman LS 8100 system (Beckman Instruments Inc., Irvine, California). For the suppressor cell studies, mononuclear cells were pre-incubated without mitogen for 48 hr, T lymphocyte concentration was readjusted to 105 T cells/ml and cells were plated for transformation as described above. Supernatants. Mononuclear cells were suspended in tissue culture flasks at 5 x 106 cells/ml. Fractionation was performed by allowing cells to adhere for 2 hr to glass and then decanting non-adherent cells after vigorous agitation. This technique routinely produced an adherent cell population containing 80-980% monocytes (by non-specific esterase stain; Yam, Li & Crosby, 1971) and a non-adherent population containing 10-20%o monocytes. Unfractionated mononuclear cell cultures routinely contained 20-40% monocytes. Three cell cultures (whole mononuclear, adherent and non-adherent fractions) were incubated with and without Con A (10 jig/ml) at 37 C, 500 CO2 for 48 hr, the cells were removed by centrifugation at 400g and the supernatants were frozen until use. In order to assess suppressor activity in the supernatants, 0 1 ml of supernatant was added to cultures set up for lymphocyte transformation as described above. All supernatants were tested in quadruplicate. Incubation, 'H-thymidine pulse, harvest and counting were carried out as described above. =
RESULTS IgE levels were measured in patients and controls. Patients with severe eczema had a mean~s.e. IgE concentration of 3792+1482 with a range of 260-7800 units. Patients with mild eczema had a mean~s.e. IgE level of 2118+ 1074 with a range of 5-5350 units. Control subjects had a mean+s.e. IgE level of 12±3 units with a range of 5-20 units. No correlation between plasma IgE concentration and lymphocyte response was observed. Lymphocyte transformation in response to various concentrations of the mitogen Con A was performed. As shown in Fig. 1, lymphocytes from patients with mild eczema demonstrate enhanced thymidine incorporation, while lymphocytes from patients with severe eczema had markedly depressed uptake when compared with that of controls. This effect was most noticeable at a Con A concentration of 10 pg/ml, where the difference between the response of normals and severe atopics was highly significant (P
SUMMARY
Previous studies have shown that patients with atopic eczema have depressed cell-mediated immunity. Whether this defect can be attributed to abnormal suppressor cell activity or to the presence of mediators of the allergic response has not been studied before. Lymphocyte transformation was found to be enhanced in patients with mild eczema and markedly depressed in patients with severe eczema, when compared with normal controls. Pre-incubation of cultures for 48 hr without mitogen prior to transformation studies restored normal lymphocyte thymidine uptake in cells from severe atopics, suggesting a labile suppressor cell population, or a labile suppressor substance. Since mononuclear cell supernatants from patients with severe eczema failed to suppress lymphocyte transformation more than supernatants from normals, it is unlikely that the depressed lymphocyte function seen in severe eczema is due to an abnormal suppressor cell population. The possibility that mediators of the allergic response may be acting as a labile suppressor substance was evaluated by adding various concentrations of histamine, cyclic-AMP, or prostaglandin E1 to lymphocytes undergoing mitogenesis. Histamine enhanced thymidine incorporation at low concentrations and depressed uptake at high concentrations; cyclic-AMP and prostaglandin E1 have similar effects on transformation. It is possible that the enhancement of transformation seen in mild eczema and the depression of this response in severe eczema may be related to the concentrations or degree of allergic mediator release.
INTRODUCTION Studies of patients with atopic eczema have shown several immune abnormalities including defective cell-mediated immunity (Buckley, Wray & Belmaker, 1972) and defective neutrophil chemotaxis (Hill & Quie, 1974; Hill et al., 1974). In most cases these defects have been associated with hyperimmunoglobulinaemia E suggesting that they may be a direct result of high IgE levels or indirectly, a result of IgE-mediated release of vasoactive amines. Since the overall levels of IgE are felt to be regulated by T lymphocytes (Okamura & Tada, 1971) it is possible that suppression of T cell function might lead to high IgE levels and atopic disease. Recent studies have shown depressed T lymphocyte function in patients with atopic disease, which correlates inversely with IgE levels (McGeady & Buckley, 1975; Rachelefsky et al., 1976). Depressed lymphocyte function is felt in many cases to be due directly to a population of suppressor T cells, or to the soluble suppressor products of such a population (Asherson & Zembala, 1976; Webb, Jamieson & Nowowiejski, 1976; Okamura & Tada, 1974). Other work suggests that some suppressor activity is a result of mediators produced by activated monocytes (Ogden, Krueger & Jederberg, 1977; Nelson, 1973; Keller, 1975; Wing & Remington, 1977). In the present study we have examined T lymphocyte function in atopic eczema and have looked closely for suppressor activity in these individuals. Suppressed function was observed in severe eczema which was abolished by 48 hr pre-incubation, suggesting the presence of a labile suppressor substance or population of cells. Correspondence: Dr B. E. Ogden, Division of Clinical Immunology and Allergy, Department of Pediatrics, University of Utah, Salt Lake City, Utah 84132, USA. 0099-9104/79/0020-0269$02.00 A) 1979 Blackwell Scientific Publications
269
270
B. E. Ogden, G. G. Krueger & H. R. Hill MATERIALS AND METHODS
Subjects. Two of the ten atopic patients studied suffered from asthma, allergic rhinitis and eczema, two had asthma and eczema, four had allergic rhinitis and eczema, and two had eczema alone. Five of the patients had severe eczema ( > 500/o of the skin involved) and five patients had mild eczema (< 10% of the skin involved). Patients had not received daily corticosteroids for at least 9 months, or systemic or topical medication for at least 4 days prior to study. The patients' (n 10) ages ranged from 8 to 36 years with a mean-f s.e. of 25 + 3 while the age of normal controls (n = 12) ranged from 18 to 31 years with a mean + s.e. of 25 1. Control subjects were normal healthy young adults who were receiving no therapy. Age, sex and time variables were controlled by studying each patient simultaneously with a similarly aged and same sex control. IgE levels. IgE concentrations were measured in patient and control plasma samples by a radioimmunoabsorbent test using a Phadebas IgE test kit (Pharmacia Laboratories, Piscataway, New Jersey). T lymphocytes. T cell determinations were made by a modification of the E-rosette method described elsewhere (Jondal, Holm & Wigzell, 1972). An aliquot of mononuclear cells was harvested as described below, adjusted to 4 x 106 cells/ml and incubated at 37 C with an aliquot of fresh 0-50/ sheep erythrocytes for 5 min, centrifuged at 2000 g for 3 min, and then incubated at 4VC for 30 min before counting. Lymphocyte transformation. Lymphocyte transformation in response to several doses of the T cell mitogen, concanavalin A (Con A, Sigma Chemical Co., St. Louis, Missouri), was performed using mononuclear cells isolated from heparinized whole blood by Ficoll-Hypaque density gradient centrifugation, according to a modification of the technique published by Boyum (1968). Briefly, heparinized blood was spun at 100 g for 15 min and the plasma containing the majority of platelets was removed. Blood was then diluted in Hanks's balanced salt solution and layered over Ficoll-Hypaque for centrifugation at 250 g for 25 min. Mononuclear cells were harvested from the interface, washed twice in Hanks's and suspended in RPMI1640 tissue culture medium containing gentamicin and 10/o0 heat-inactivated foetal calf serum at a concentration of 105 T cells/ml. 0 2 ml of the cell suspension was then dispensed to each well of a microtitre plate. At this point 25 PI of media alone or media containing various doses of Con A was added and plates were incubated at 37 C, 5% CO2. For some experiments, a single dose of Con A (10 pg/ml) was used and 25 pl of various concentrations of histamine, cyclic-AMP or prostaglandin E, (Sigma Chemical Co., St. Louis, Missouri) was added at the beginning of the incubation period. After was 48 hr of incubation, 25 pl of 10 pCi/ml 3H-thymidine (sp. act. 6 7 Ci/mM, New England Nuclear, Boston, Massachusetts) added for a 24 hr pulse following which, plates were harvested with a multiple automated sample harvester and 'H-thymidine uptake was determined by liquid scintillation counting using a Beckman LS 8100 system (Beckman Instruments Inc., Irvine, California). For the suppressor cell studies, mononuclear cells were pre-incubated without mitogen for 48 hr, T lymphocyte concentration was readjusted to 105 T cells/ml and cells were plated for transformation as described above. Supernatants. Mononuclear cells were suspended in tissue culture flasks at 5 x 106 cells/ml. Fractionation was performed by allowing cells to adhere for 2 hr to glass and then decanting non-adherent cells after vigorous agitation. This technique routinely produced an adherent cell population containing 80-980% monocytes (by non-specific esterase stain; Yam, Li & Crosby, 1971) and a non-adherent population containing 10-20%o monocytes. Unfractionated mononuclear cell cultures routinely contained 20-40% monocytes. Three cell cultures (whole mononuclear, adherent and non-adherent fractions) were incubated with and without Con A (10 jig/ml) at 37 C, 500 CO2 for 48 hr, the cells were removed by centrifugation at 400g and the supernatants were frozen until use. In order to assess suppressor activity in the supernatants, 0 1 ml of supernatant was added to cultures set up for lymphocyte transformation as described above. All supernatants were tested in quadruplicate. Incubation, 'H-thymidine pulse, harvest and counting were carried out as described above. =
RESULTS IgE levels were measured in patients and controls. Patients with severe eczema had a mean~s.e. IgE concentration of 3792+1482 with a range of 260-7800 units. Patients with mild eczema had a mean~s.e. IgE level of 2118+ 1074 with a range of 5-5350 units. Control subjects had a mean+s.e. IgE level of 12±3 units with a range of 5-20 units. No correlation between plasma IgE concentration and lymphocyte response was observed. Lymphocyte transformation in response to various concentrations of the mitogen Con A was performed. As shown in Fig. 1, lymphocytes from patients with mild eczema demonstrate enhanced thymidine incorporation, while lymphocytes from patients with severe eczema had markedly depressed uptake when compared with that of controls. This effect was most noticeable at a Con A concentration of 10 pg/ml, where the difference between the response of normals and severe atopics was highly significant (P
