Peripheral blood lymphocyte subpopulations in sarcoidosis

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

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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
570

1209 1920 784 1518 1008 976 952 2560

phocyte count (mm”)

Iym-

Absolute

40 29 61 35 62 38 40 37 36 52 65 34 45 35 42 49 55 55 62 35 45.3 All.3 1.5 f2.6
%

1197 707 114 588 f260.5 282.3 ~1~72.6
847~

484 557 478 531 625 371 381 947 205 795 436 885 613 3.59 546 683

Absolute No.

(Ig-bearing) B lymphocytes*

54 73 65 72 60 57 40 69 58 68 67 67 57 64 65 56 63.4 Zk8.1 80.1 fS.8
71

66 76 63

%

791 702 871 921 886 1382 741 184 865.2 zk4.56.6 1901 1408.3
E : z 5 s 2

864

8

z

6

269 1797

624 1843 342

798 1459 490 1078 549 708

Absolute No.

(Rosette-forming) T lymphocytesC

of disease less than 2 years) ; Chr, chronic (duration of disease more than 2 years) ; Ster, corticohilar adenopathy ; stage 2, bilateral hilar adenopathy and pulmonary mottling ; stage 3, pulmonary involvement; B, bone marrow-derived ; T, thymus-derived ; P, Puerto Rican.

I I I I I&E I I I I&E I I&E I I I I I&E I&E I I I&E

Intra- and extrathoracic involvement

CLINICAL FEATURES, ABSOLUTE LYMPHOCYTE COUNTS AND LYMPHOCYTE SUBPOPLXATIONS IN PATIENTS WITH SARCOIDOSIS”

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425

belonging to a given lymphocyte subpopulation and their apportionment between lymphoid tissues and blood are not well understood. Very likely, different regulatory influences operate on T and B lymphocytes. In mice (14), neonatal thymectomy, thoracic duct drainage, or a single dose of heterologous anti-lymphocyte serum will quickly deplete T cells from the blood, spleen, and lymph nodes without significantly affecting the number of circulating B lymphocytes. Similarly, patients with congenital thymic deficiency or absence (DiGeorge syndrome) show only a relative preponderance of B lymphocytes in the blood; the absolute B cell numbers are not increased (15). The mechanisms of T cell depletion in sarcoidosis as well as in some other conditions affecting lymph nodes such as lepromatous leprosy (16) and Hodgkin’s disease (17, 18) are unknown. It is possible that granulomatous or neoplastic infiltration interferes with the passage in the paracortical areas of these no&l; (J? lymphocytes recirculating from blood to lymph and that the cells are damage{1 or killed in these recirculatory areas. The decreased number of T cells in sarcoitlosis may in part explain previous observations of the reduced capacity of blood lymphocytes from sarcoidosis patients to respond to phytohemagglutinin in vitro (3, 4 1 ; such responsiveness is a function of the number of T cells in the culture (S). In addition to T cell depletion, patients with sarcoidosis show increased absolute numbers of B lymphocytes in the peripheral blood. A similar change has been observed in patients with lepromatous leprosy ( 19). In both conditions this increase may represent the result of B cell hyperplasia in the lymph node secondary to stimulation by the granuloma-producing agent. The peripheral blood B lvmphocytosis may also be related to increased humoral immune responsiveness, as evidenced 1)) high immunoglobulin levels, the presence of rheutnatoid factor, and increased antibody titers to EB (Epstein-Barr) virus as well as other viruses (20-23). Our finding that B lymphocytes are increased in patients with sarcoidosis contrasts with the data of Hedfors et al. (24)) who found that E rosette-forming T cells were reduced but that the total numbers of EAC rosettes ( a reflection of 13 cells) were not increased. It is possible that some B lymphocytes may react with anti-immunoglobulin antisera yet not form EAC rosettes. However, cells which did not form EAC rosettes were accounted for as T cells by E rosette formation 1~ Hedfors et al. (24). Since the B lymphocyte is known to have a shorter life span and greater turnover time than most T lymphocytes (14), it is possible that an increased number of circulating B lymphocytes may also represent an augmented rate of proliferation of B cells. Increased proliferative activity has been encountered in earlier studies which reported augmented incorporation of tritiated thymidine into lymphocytes from sarcoidosis patients, as compared to those of normal controls, even without mitegenie stimulation (25, 26). It may also explain the ease with which in z&o longterm lymphocyte-cultured cell lines may be established in sarcoidosis patients. Proliferating cells, especially B cells, are more susceptible to the entry of oncogenic viruses, including EB virus which may induce uncontrolled proliferation (21, 27-29). The changes in lymphocyte subpopulations bore no significant relationship to the clinical stage, duration, activity, or corticosteroid treatment, and it would be most important to ascertain if any clinical or pathologic feature of the disease does carrelate with the lymphocyte subpopulation changes. A prospective study is now being carried out to assess simultaneously cutaneous anergy, Kveim reactivity, in vitro

426

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COMMUNICATIONS

response to mitogens, lymph node pathology, peripheral blood of patients with sarcoidosis.

and T and B cell markers in the

ACKNOWLEDGMENTS Supported in part by National Institutes of Health, Research Grants CA14502 and HL13853, and by the American Lung Association, Chemotherapy Foundation, Inc., and National Foundation for Ileitis and Colitis, Inc. One of the authors (S.D.D.) is a recipient of Research Career Development Award lK04HL42575 from the National Heart and Lung Institute. Excellent technical assistance was provided by Miss Susan Dittmar and Mr. Steven Forman.

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