Clin. exp. Immunol. (1975) 22, 438-445.
Lymphocyte subpopulations, serum IgE and total eosinophil counts in patients with bronchial asthma S. GUPTA,* R. FRENKEL, M. ROSENSTEIN & M. H. GRIECO The R. A. Cooke Institute of Allergy, The Roosevelt Hospital, Columbia University, New York, U.S.A.
(Received 5 May 1975) SUMMARY
Absolute T and B lymphocytes, total eosinophil counts (TEC), and serum IgE levels were studied in twenty-three asthmatic subjects. Compared to a mean peripheral Tlymphocyte count of 1620+486/mm3 in twenty control subjects, the mean value in the asthmatic patients was depressed significantly at 1192 + 412 (P< 0005). T lymphopenia was observed in ten of twenty-three patients (430%). The serum IgE was elevated above the upper limits of 507 yg/ml in six of seventeen patients. TEC above 250/mm3 was observed in twelve of seventeen subjects. T lymphopenia and hyperimmunoglobulinaemia E were each associated with hypereosinophilia. No correlation was observed between T lymphopenia and hyperimmunoglobulinaemia E.
INTRODUCTION Kaufman & Hobbs (1970) have demonstrated immunoglobulin deficiencies in an atopic population and suggested that individuals with 'backward' immunological memory are prone to develop atopy. Selective IgA deficiency is associated with increased incidence of atopic manifestations (Taylor et al., 1973). A defect in the defence barrier across mucosal surfaces with more 'efficient' contact between antigen and immunologically competent cells has been proposed as one hypothesis for the development of atopic states (Leskowitz, Salvaggio & Schwartz, 1972). Eosinophilia is a characteristic finding in patients with atopic disorders, including dermatitis, rhinitis and asthma (Stickney & Heck, 1944). The mechanisms involved are unclear but lymphocytes have been implicated (Basten & Beeson, 1970). At least four chemotactic factors have been identified. Two of these factors attract eosinophils as well as other peripheral cells and include: complement derived products resulting from the interaction of guinea-pig sera treated with antigen-antibody complexes (Kay, 1970); and mediators present in culture filtrates of bacteria grown in medium 199 (Ward, 1969). Recently Kay & Austen (1971) have demonstrated the antigenic release of an eosinophil chemotactic factor of anaphylaxis (ECF-A) from sensitized human lung. ECF-A, a preformed 500-600 Dalton * Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, U.S.A. Correspondence: Dr M. H. Grieco, R. A. Cooke Institute of Allergy, The Roosevelt Hospital, 428 West
59th Street, New York, New York 10019, U.S.A.
438
Lymphocyte subpopulations in asthma
439
peptide associated with mast cell granules, is selectively chemotactic for eosinophils in vitro and leads to deactivation as well. A fourth factor, also antigen specific, has been demonstrated by Turisu et al. (1973) to arise from sensitized guinea-pig lymphocytes after challenge with DNP-BGG. This diffusible factor from lymphocytes has been referred to as ECFp. Total eosinophil counts (TEC) have been regarded by Lowell (1967) as useful in diagnosis and even in management of allergic diseases. He reports that patients with obstructive pulmonary disease and eosinophilia failing to improve and having persistent eosinophilia in the face of corticosteroid therapy are receiving inadequate therapeutic dosage. He suggests increased dosage until the TEC falls at least below 100/mm3 and preferably below 50/mm3. Patients with atopic disorders have been shown to have increased serum IgE levels (Johansson, 1969). Depressed cell-mediated immunity (Gottlieb & Hanifin, 1974) and increased serum IgE levels (Juhlin et al., 1969) have been demonstrated in patients with atopic dermatitis. Despite the frequent occurrence of eosinophilia in atopic disorders and its usefulness as a therapeutic guide, there is remarkably little known about the immunological events occurring in asthmatic subjects. T-lymphocyte deficiency has been reported in association with eosinophilia and increased IgE (Goldman et al., 1974). The purpose of this study was to examine the circulating T and B lymphocytes, total eosinophil counts and levels of IgE in patients with bronchial asthma. MATERIALS AND METHODS Subjects. The twenty-three asthmatic subjects studied included twenty-one attending the R. A. Cooke Institute of Allergy Clinic as well as two physicians. The mean age was 37-1 ± 13-4 years (s.d.), fifteen were women, eight were men. In sixteen patients the aetiology was considered both intrinsic and extrinsic while seven were classified as predominantly extrinsic. Only subject no. 1 was receiving prednisone at the time of the study. The remainder were taking catecholamine and theophylline-containing preparations and all but eight subjects were receiving long-term hyposensitization therapy. The controls for the lymphocyte studies were twenty healthy hospital employees. They were 295± 6-8 years of age and included six women and fourteen men. They were not taking any medications at the time of the study. Lymphocyte studies. Ten millilitres of heparinized venous blood were obtained. Lymphocytes were isolated on Ficoll-Hypaque medium by centrifugation at 400 g for 40 min. The interface ring contained mononuclear cells (approximately 80-85O lymphocytes and 15-20% monocytes). These cells were washed three times in Hanks's balanced salt solution. About 80% of lymphocytes were recovered. Almost 100% of cells were viable as tested by trypan blue dye exclusion. Monocytes were labelled by ingestion of latex particles. The lymphocytes were adjusted in medium TC 199 to a concentration of 1 x 107 cells/ml. B-cell rosettes. B cells were assayed for their C3 receptors by a technique described previously (Gupta, Grieco & Cushman, 1974). One hundred microlitre aliquots of lymphocyte suspension (concentration 2 x 106 cells/ml) were added to three plastic tubes containing 100 ,ul of 0 5% trypsinized sheep erythrocytes coated with anti-sheep haemolysin and fresh mouse serum. The mixture was centrifuged for 5 min at 200 g followed by incubation at 37°C for 30 min. The pellet was suspended and lymphocytes were counted in triplicate and rosettes expressed both as percentage and absolute number. A positive rosette was defined as a lymphocyte surrounded by three or more sheep erythrocytes. T-cell rosettes. T lymphocytes were enumerated by sheep erythrocyte rosette formation as described previously (Gupta & Grieco, 1974). Twenty-five-microlitre aliquots of a lymphocyte suspension of 1 x 107 cells/ml initial concentration were added to three plastic tubes containing 25 ,l of foetal calf serum (heatinactivated and absorbed with sheep red blood cells). Fifty-microlitre aliquots of 2% sheep erythrocytes
440
S. Gupta et al.
TABLE 1. Peripheral rosette-forming lymphocytes, absolute eosinophil count and serum IgE level and enumeration of twenty-three subjects with bronchial asthma
Lymphocytes T
B
TEC
Subject Age
2 3 4 S 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
DT RH JB MS FF JDA ED LR LI WT BD DT VA JB PL EJ RL EV LB CF HL MR JW
36 59 19 50 58 35 19 22 27 20 50 19 53 33 50 44 36 48 29 54 33 29 30
Sex
Asthmat No./mm3
F F M M M M F F M F F F F F F F M F F F M M F
Mixed Mixed Extrinsic Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Extrinsic Mixed Extrinsic Extrinsic Extrinsic Extrinsic Extrinsic
194 512 289 97 89 151 240 94 232 283 239 519
360 100 142 199 326 79 149 136 124 213 165
No./mm3
%
(No./mm3)
(12 4) (28 3) (12 3) (7-7) (8 5) (12 0) (12 2) (4 7) (10 3) (10 0) (10 0) (12 8) (12 3) (6 7) (8 7) (12 6) (11-7) (4 7) (10 0) (8 3) (14 6) (11 3) (9 6)
953* 835* 793* 778* 853* 923* 1459 1662 1552 1484 1744 1969 1915 1025 1079 1010 1848 1176 836* 906* 669* 1005
(60 1) (46 0) (34 2) (61 0) (76 5) (73*3) (73 4) (77 7) (69 0) (52 3) (72 7) (48 7) (65 5) (68 2) (65 7) (64 0) (660) (69 2) (55-3) (60 0) (78-6) (53 3) (56 6)
832 496 845 915 968 634 1291 422 370 264 704 464 105 176 246 229 194 n.d. n.d. n.d. n.d. n.d. n.d.
50 67 700 700 200 215 2300 1200 600 520 219 150 195 260 300 67 250 n.d. n.d. n.d. n.d. n.d. n.d.
539 + 340
455 + 547
214+ 123
Mean and s.d. 371 + 34
(10-9 + 4
Control subjects (20) F-distribution Significance Student's 't'-test
%
IgE (u/ml)
6%)
250+ 74 (108+ 16%)
949*
1192 + 412
(62-9 +11 1%) 1620 + 486 N < 250/mm3 N (71 4+ 6 2%)
n.s.
Lymphocyte subpopulations, serum IgE and total eosinophil counts in patients with bronchial asthma S. GUPTA,* R. FRENKEL, M. ROSENSTEIN & M. H. GRIECO The R. A. Cooke Institute of Allergy, The Roosevelt Hospital, Columbia University, New York, U.S.A.
(Received 5 May 1975) SUMMARY
Absolute T and B lymphocytes, total eosinophil counts (TEC), and serum IgE levels were studied in twenty-three asthmatic subjects. Compared to a mean peripheral Tlymphocyte count of 1620+486/mm3 in twenty control subjects, the mean value in the asthmatic patients was depressed significantly at 1192 + 412 (P< 0005). T lymphopenia was observed in ten of twenty-three patients (430%). The serum IgE was elevated above the upper limits of 507 yg/ml in six of seventeen patients. TEC above 250/mm3 was observed in twelve of seventeen subjects. T lymphopenia and hyperimmunoglobulinaemia E were each associated with hypereosinophilia. No correlation was observed between T lymphopenia and hyperimmunoglobulinaemia E.
INTRODUCTION Kaufman & Hobbs (1970) have demonstrated immunoglobulin deficiencies in an atopic population and suggested that individuals with 'backward' immunological memory are prone to develop atopy. Selective IgA deficiency is associated with increased incidence of atopic manifestations (Taylor et al., 1973). A defect in the defence barrier across mucosal surfaces with more 'efficient' contact between antigen and immunologically competent cells has been proposed as one hypothesis for the development of atopic states (Leskowitz, Salvaggio & Schwartz, 1972). Eosinophilia is a characteristic finding in patients with atopic disorders, including dermatitis, rhinitis and asthma (Stickney & Heck, 1944). The mechanisms involved are unclear but lymphocytes have been implicated (Basten & Beeson, 1970). At least four chemotactic factors have been identified. Two of these factors attract eosinophils as well as other peripheral cells and include: complement derived products resulting from the interaction of guinea-pig sera treated with antigen-antibody complexes (Kay, 1970); and mediators present in culture filtrates of bacteria grown in medium 199 (Ward, 1969). Recently Kay & Austen (1971) have demonstrated the antigenic release of an eosinophil chemotactic factor of anaphylaxis (ECF-A) from sensitized human lung. ECF-A, a preformed 500-600 Dalton * Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, U.S.A. Correspondence: Dr M. H. Grieco, R. A. Cooke Institute of Allergy, The Roosevelt Hospital, 428 West
59th Street, New York, New York 10019, U.S.A.
438
Lymphocyte subpopulations in asthma
439
peptide associated with mast cell granules, is selectively chemotactic for eosinophils in vitro and leads to deactivation as well. A fourth factor, also antigen specific, has been demonstrated by Turisu et al. (1973) to arise from sensitized guinea-pig lymphocytes after challenge with DNP-BGG. This diffusible factor from lymphocytes has been referred to as ECFp. Total eosinophil counts (TEC) have been regarded by Lowell (1967) as useful in diagnosis and even in management of allergic diseases. He reports that patients with obstructive pulmonary disease and eosinophilia failing to improve and having persistent eosinophilia in the face of corticosteroid therapy are receiving inadequate therapeutic dosage. He suggests increased dosage until the TEC falls at least below 100/mm3 and preferably below 50/mm3. Patients with atopic disorders have been shown to have increased serum IgE levels (Johansson, 1969). Depressed cell-mediated immunity (Gottlieb & Hanifin, 1974) and increased serum IgE levels (Juhlin et al., 1969) have been demonstrated in patients with atopic dermatitis. Despite the frequent occurrence of eosinophilia in atopic disorders and its usefulness as a therapeutic guide, there is remarkably little known about the immunological events occurring in asthmatic subjects. T-lymphocyte deficiency has been reported in association with eosinophilia and increased IgE (Goldman et al., 1974). The purpose of this study was to examine the circulating T and B lymphocytes, total eosinophil counts and levels of IgE in patients with bronchial asthma. MATERIALS AND METHODS Subjects. The twenty-three asthmatic subjects studied included twenty-one attending the R. A. Cooke Institute of Allergy Clinic as well as two physicians. The mean age was 37-1 ± 13-4 years (s.d.), fifteen were women, eight were men. In sixteen patients the aetiology was considered both intrinsic and extrinsic while seven were classified as predominantly extrinsic. Only subject no. 1 was receiving prednisone at the time of the study. The remainder were taking catecholamine and theophylline-containing preparations and all but eight subjects were receiving long-term hyposensitization therapy. The controls for the lymphocyte studies were twenty healthy hospital employees. They were 295± 6-8 years of age and included six women and fourteen men. They were not taking any medications at the time of the study. Lymphocyte studies. Ten millilitres of heparinized venous blood were obtained. Lymphocytes were isolated on Ficoll-Hypaque medium by centrifugation at 400 g for 40 min. The interface ring contained mononuclear cells (approximately 80-85O lymphocytes and 15-20% monocytes). These cells were washed three times in Hanks's balanced salt solution. About 80% of lymphocytes were recovered. Almost 100% of cells were viable as tested by trypan blue dye exclusion. Monocytes were labelled by ingestion of latex particles. The lymphocytes were adjusted in medium TC 199 to a concentration of 1 x 107 cells/ml. B-cell rosettes. B cells were assayed for their C3 receptors by a technique described previously (Gupta, Grieco & Cushman, 1974). One hundred microlitre aliquots of lymphocyte suspension (concentration 2 x 106 cells/ml) were added to three plastic tubes containing 100 ,ul of 0 5% trypsinized sheep erythrocytes coated with anti-sheep haemolysin and fresh mouse serum. The mixture was centrifuged for 5 min at 200 g followed by incubation at 37°C for 30 min. The pellet was suspended and lymphocytes were counted in triplicate and rosettes expressed both as percentage and absolute number. A positive rosette was defined as a lymphocyte surrounded by three or more sheep erythrocytes. T-cell rosettes. T lymphocytes were enumerated by sheep erythrocyte rosette formation as described previously (Gupta & Grieco, 1974). Twenty-five-microlitre aliquots of a lymphocyte suspension of 1 x 107 cells/ml initial concentration were added to three plastic tubes containing 25 ,l of foetal calf serum (heatinactivated and absorbed with sheep red blood cells). Fifty-microlitre aliquots of 2% sheep erythrocytes
440
S. Gupta et al.
TABLE 1. Peripheral rosette-forming lymphocytes, absolute eosinophil count and serum IgE level and enumeration of twenty-three subjects with bronchial asthma
Lymphocytes T
B
TEC
Subject Age
2 3 4 S 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
DT RH JB MS FF JDA ED LR LI WT BD DT VA JB PL EJ RL EV LB CF HL MR JW
36 59 19 50 58 35 19 22 27 20 50 19 53 33 50 44 36 48 29 54 33 29 30
Sex
Asthmat No./mm3
F F M M M M F F M F F F F F F F M F F F M M F
Mixed Mixed Extrinsic Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Extrinsic Mixed Extrinsic Extrinsic Extrinsic Extrinsic Extrinsic
194 512 289 97 89 151 240 94 232 283 239 519
360 100 142 199 326 79 149 136 124 213 165
No./mm3
%
(No./mm3)
(12 4) (28 3) (12 3) (7-7) (8 5) (12 0) (12 2) (4 7) (10 3) (10 0) (10 0) (12 8) (12 3) (6 7) (8 7) (12 6) (11-7) (4 7) (10 0) (8 3) (14 6) (11 3) (9 6)
953* 835* 793* 778* 853* 923* 1459 1662 1552 1484 1744 1969 1915 1025 1079 1010 1848 1176 836* 906* 669* 1005
(60 1) (46 0) (34 2) (61 0) (76 5) (73*3) (73 4) (77 7) (69 0) (52 3) (72 7) (48 7) (65 5) (68 2) (65 7) (64 0) (660) (69 2) (55-3) (60 0) (78-6) (53 3) (56 6)
832 496 845 915 968 634 1291 422 370 264 704 464 105 176 246 229 194 n.d. n.d. n.d. n.d. n.d. n.d.
50 67 700 700 200 215 2300 1200 600 520 219 150 195 260 300 67 250 n.d. n.d. n.d. n.d. n.d. n.d.
539 + 340
455 + 547
214+ 123
Mean and s.d. 371 + 34
(10-9 + 4
Control subjects (20) F-distribution Significance Student's 't'-test
%
IgE (u/ml)
6%)
250+ 74 (108+ 16%)
949*
1192 + 412
(62-9 +11 1%) 1620 + 486 N < 250/mm3 N (71 4+ 6 2%)
n.s.
