CELLULAR
IMMUNOLOGY
16, 422-426 (1975)
SHORT COMMUNKATIONS Peripheral
Blood Lymphocyte
Subpopulations
in Sarcoidosis
K. RAMACHANDAR, S. D. DOUGLAS,L. E. SILTZBACH, AND R. N. TAUB Divisiom of Hcnzatoloyy alzd Thoracic Diseases, Department of Mcdicitte, Mount Sinai School of Medicine of The City University of Nezv York, New York, New York 10029 Received
October 8, 1974
We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sat-cc&& and 1.5
healthy controls. T cells were estimated from the number of lymphocytes forming rosettes 212vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total
lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreasedcellular immunity and increased reactivity of the antibody-forming systemas commonly seenin sarcoidosis. INTRODUCTION Sarcoidosis is so often accompanied by immunological aberrations that comprehension of their mechanisms may well shed light on its pathogenesis. In sarcoidosis there is a characteristic suppression of delayed-type skin hypersensitivity and decreased irt vitro responsiveness of cultured lymphocytes to mitogenic stimulation (1-5). Despite this reduction in cellular immune function, immunoglobulin levels and humoral antibody responses are normal or increased in most patients (6, 7). Over the past decade it has become evident that most immune responses are transacted by at least two distinct subpopulations of lymphocytes: the thymusderived or T cell is a crucial participant in cell-mediated immune reactions, while the bone marrow-derived or B lymphocytes develop into antibody-forming cells. Since alterations in cellular or humoral immune functions may be reflected in changes in these lymphocyte subpopulations (8)) we have investigated the numbers of T and B lymphocytes in the peripheral blood of patients with sarcoidosis. METHODS
AND MATERIALS
Subjects Twenty patients with known sarcoidosis confirmed by organ biopsy, a positive Kveim test or both, attending the Sarcoidosis Clinic of the Mount Sinai Hospital 422 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
SHORT
COMMUNICATIONS
423
were evaluated. Fourteen were black, 5 Puerto Kicau born, and 1 was Caucasian. Fifteen of the 20 patients were women. Ten were under 40 years old. Sarcoidosis had been present for less than 2 years in 5 patients (subacute), and 2-15 years in the remaining 15 patients, Thirteen patients had active lesions, while in 7 the disease was judged to have become stationary. In 14 patients, involvement was limited to intrathoracic locations, while in the other 6, extrathoracic lesions were present. The lesions were located in the peripheral lymph nodes in 2 patients, in the skin in 2. in the nervous system in 2 and in the eyes and parotid glands in 1 each. Tliese data, as well as tuberculin and Kveim reactivity. cliuical stage, and drug treatment \vitll steroids or chloroquine are summarized, in Table 1. Fifteen healthv volunteers 20SO vears old served as controls.
The absolute numbers of circulating lymphocytes were calculated from the white blood cell and differential counts of peripheral blood. Lymphocytes were separated by sedimentation of heparinized blood in a Ficoll-Hypaque mixture (9). B cells were enumerated by direct immunofluorescence ( 10, 11) using fluorescein-conjugated rabbit anti-human polyvalent immunoglobulin antiserum (Behring Diagnostics ) T cells were estimated by lymphocyte rosette formation with unsensitized sheep erythrocytes using the E rosette method of Bentwich et al. (12). We also used a rosette technique employing sheep red cells treated with beta-atiiinoeth~l isothiouronium bromide (AET) as described previously by Clark and Kaplan ( 13). RESULTS The total lymphocyte count of patients with sarcoidosis (1328 +- 614 SD) was significantly less (P < 0.00s) than that of normal control subjects (1876 * 359) (Table 1). In patients treated with corticosteroids the lymphocyte count was even lower (1035 2 729). The absolute numbers of T cells as reflected by the number of lymphocytes fot-ming E-binding rosettes (827 -+ 454) were significantly decreased (P < 0.001 ) in patients with sarcoidosis as compared to normals ( 1692 f 415 ) . Very similar values for T cells were obtained using either the E rosette (827 * 453) or AET rosette (865 2 456 j technique (correlation coefficient, K = 0.9462 ) . The absolute numbers of B cells were consistently increased (58X 2 200 ) iu patients with sarcoidosis even in the face of total lymphopenia (Table 1) The mean sum of T and B percentages obtained by the separately performed assays usually added up to over 100% (mean : 108.7 t 8.S). rendering it unIikeIy that a significant population of “null” cells were present. The changes in T and B lymphocytes did not show any consistent relationship to the clinical stage, tuberculin positivity. disease activity, duration, or drug treatment in our patients. DISClJSSION Our data (Table 1) indicate that patients with sarcoidosis show changes in two lymphocyte subpopulations : increased numbers of immunoglobulin-bearing peripheral lymphocytes (B cells), and a depletion of cells forming spontaneous rosettes with sheep erythrocytes (T cells). The factors which regulate the cell numbers
Sex/ race/age
F/B/46 F/B/40 F/B/26 F/B/?0 F/B/43 F/P/51 F/B/28 F/W/2? M/B/35 F/B/29 M/P/59 F/P/59 F/B/31 F/B/34 M/P/52 M/B/3? F/B/41 F/P/44 M/B/53 F/B/55
Patient
B.W.M. T.C. McL.B. P.E. P.S. T.G. H.S. P.E. W.J. S.H. A.M. P.M. A.H. W.C. M.P.
Status
Normal
controls
Sub Sub Sub Sub Sub Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr 120 Chr 144 Chr 156 Chr 216 Chr Sarcoidosis (20)
1 3 4 12 30 30 40 44 52 60 60 78 84 84 84
1
Duration (months)
(15)
1-active l-stat 3-active 3-active
2-stat
2-active l-active l-stat 2-active d-stat l-stat l-stat P-active
2-stat
l-active l-active l-active l-active 2-active 2-active
Stage and activity (active or stationary)
a Abbreviations used in the table: Sub, subacute (duration steroids ; Chlq, chloroquine ; stat, stationary ; stage 1, bilateral mottling only; I, intrathoracic involvement; E, extrathoracic
P
yY. T.C. L.B. WC. Mean &SD
TABLE
1
-t +
+
-If + -k
-t
-
PPD reaction
4.6 5.0
7.0
6.0 5.6 7.3 4.6 6.0 6.0 5.7 3.5 ND 7.4 10.2 5.2 8.8 7.4 3.2 4.0 5.1
Specific Kveim papule size (mm) None None None None Ster Ster None Ster Ster None Ster None None None Chlq None Chlq None Ster Ster
Rx
1530 672 2604 1364 1025 1300 1394 1540 2176 1140 325 1328 f614 1876 f359
IMMUNOLOGY
16, 422-426 (1975)
SHORT COMMUNKATIONS Peripheral
Blood Lymphocyte
Subpopulations
in Sarcoidosis
K. RAMACHANDAR, S. D. DOUGLAS,L. E. SILTZBACH, AND R. N. TAUB Divisiom of Hcnzatoloyy alzd Thoracic Diseases, Department of Mcdicitte, Mount Sinai School of Medicine of The City University of Nezv York, New York, New York 10029 Received
October 8, 1974
We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sat-cc&& and 1.5
healthy controls. T cells were estimated from the number of lymphocytes forming rosettes 212vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total
lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreasedcellular immunity and increased reactivity of the antibody-forming systemas commonly seenin sarcoidosis. INTRODUCTION Sarcoidosis is so often accompanied by immunological aberrations that comprehension of their mechanisms may well shed light on its pathogenesis. In sarcoidosis there is a characteristic suppression of delayed-type skin hypersensitivity and decreased irt vitro responsiveness of cultured lymphocytes to mitogenic stimulation (1-5). Despite this reduction in cellular immune function, immunoglobulin levels and humoral antibody responses are normal or increased in most patients (6, 7). Over the past decade it has become evident that most immune responses are transacted by at least two distinct subpopulations of lymphocytes: the thymusderived or T cell is a crucial participant in cell-mediated immune reactions, while the bone marrow-derived or B lymphocytes develop into antibody-forming cells. Since alterations in cellular or humoral immune functions may be reflected in changes in these lymphocyte subpopulations (8)) we have investigated the numbers of T and B lymphocytes in the peripheral blood of patients with sarcoidosis. METHODS
AND MATERIALS
Subjects Twenty patients with known sarcoidosis confirmed by organ biopsy, a positive Kveim test or both, attending the Sarcoidosis Clinic of the Mount Sinai Hospital 422 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
SHORT
COMMUNICATIONS
423
were evaluated. Fourteen were black, 5 Puerto Kicau born, and 1 was Caucasian. Fifteen of the 20 patients were women. Ten were under 40 years old. Sarcoidosis had been present for less than 2 years in 5 patients (subacute), and 2-15 years in the remaining 15 patients, Thirteen patients had active lesions, while in 7 the disease was judged to have become stationary. In 14 patients, involvement was limited to intrathoracic locations, while in the other 6, extrathoracic lesions were present. The lesions were located in the peripheral lymph nodes in 2 patients, in the skin in 2. in the nervous system in 2 and in the eyes and parotid glands in 1 each. Tliese data, as well as tuberculin and Kveim reactivity. cliuical stage, and drug treatment \vitll steroids or chloroquine are summarized, in Table 1. Fifteen healthv volunteers 20SO vears old served as controls.
The absolute numbers of circulating lymphocytes were calculated from the white blood cell and differential counts of peripheral blood. Lymphocytes were separated by sedimentation of heparinized blood in a Ficoll-Hypaque mixture (9). B cells were enumerated by direct immunofluorescence ( 10, 11) using fluorescein-conjugated rabbit anti-human polyvalent immunoglobulin antiserum (Behring Diagnostics ) T cells were estimated by lymphocyte rosette formation with unsensitized sheep erythrocytes using the E rosette method of Bentwich et al. (12). We also used a rosette technique employing sheep red cells treated with beta-atiiinoeth~l isothiouronium bromide (AET) as described previously by Clark and Kaplan ( 13). RESULTS The total lymphocyte count of patients with sarcoidosis (1328 +- 614 SD) was significantly less (P < 0.00s) than that of normal control subjects (1876 * 359) (Table 1). In patients treated with corticosteroids the lymphocyte count was even lower (1035 2 729). The absolute numbers of T cells as reflected by the number of lymphocytes fot-ming E-binding rosettes (827 -+ 454) were significantly decreased (P < 0.001 ) in patients with sarcoidosis as compared to normals ( 1692 f 415 ) . Very similar values for T cells were obtained using either the E rosette (827 * 453) or AET rosette (865 2 456 j technique (correlation coefficient, K = 0.9462 ) . The absolute numbers of B cells were consistently increased (58X 2 200 ) iu patients with sarcoidosis even in the face of total lymphopenia (Table 1) The mean sum of T and B percentages obtained by the separately performed assays usually added up to over 100% (mean : 108.7 t 8.S). rendering it unIikeIy that a significant population of “null” cells were present. The changes in T and B lymphocytes did not show any consistent relationship to the clinical stage, tuberculin positivity. disease activity, duration, or drug treatment in our patients. DISClJSSION Our data (Table 1) indicate that patients with sarcoidosis show changes in two lymphocyte subpopulations : increased numbers of immunoglobulin-bearing peripheral lymphocytes (B cells), and a depletion of cells forming spontaneous rosettes with sheep erythrocytes (T cells). The factors which regulate the cell numbers
Sex/ race/age
F/B/46 F/B/40 F/B/26 F/B/?0 F/B/43 F/P/51 F/B/28 F/W/2? M/B/35 F/B/29 M/P/59 F/P/59 F/B/31 F/B/34 M/P/52 M/B/3? F/B/41 F/P/44 M/B/53 F/B/55
Patient
B.W.M. T.C. McL.B. P.E. P.S. T.G. H.S. P.E. W.J. S.H. A.M. P.M. A.H. W.C. M.P.
Status
Normal
controls
Sub Sub Sub Sub Sub Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr 120 Chr 144 Chr 156 Chr 216 Chr Sarcoidosis (20)
1 3 4 12 30 30 40 44 52 60 60 78 84 84 84
1
Duration (months)
(15)
1-active l-stat 3-active 3-active
2-stat
2-active l-active l-stat 2-active d-stat l-stat l-stat P-active
2-stat
l-active l-active l-active l-active 2-active 2-active
Stage and activity (active or stationary)
a Abbreviations used in the table: Sub, subacute (duration steroids ; Chlq, chloroquine ; stat, stationary ; stage 1, bilateral mottling only; I, intrathoracic involvement; E, extrathoracic
P
yY. T.C. L.B. WC. Mean &SD
TABLE
1
-t +
+
-If + -k
-t
-
PPD reaction
4.6 5.0
7.0
6.0 5.6 7.3 4.6 6.0 6.0 5.7 3.5 ND 7.4 10.2 5.2 8.8 7.4 3.2 4.0 5.1
Specific Kveim papule size (mm) None None None None Ster Ster None Ster Ster None Ster None None None Chlq None Chlq None Ster Ster
Rx
1530 672 2604 1364 1025 1300 1394 1540 2176 1140 325 1328 f614 1876 f359
