CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
5,
133-141 (1976)
Immunological Characterization of Subpopulations of Mononuclear Cells in Tissue and Peripheral Blood from Patients with Sarcoidosisl HYMAN
TANNENBAUM,~‘~
S. PINKUS,
GERALDINE
AND
PETER
H.
SCHUR
Department of Medicine, Montreal General Hospital, Montreal, Canada, Robert B. Brigham Hospital, Boston,Massachusetts 02120, and the Department of Pathology, Peter Bent Brigham Hospital, Boston, Massachusetts, 02115
Received July 25, 1975 The immunological characteristics of the mononuclear cells comprising sarcoid granulomas were studied in cryostat sections of eight lymph nodes and one lung biopsy. In eight of the nine tissues, the histiocytes in the center of the sarcoid granuloma had surface receptors for 7S y-globulin. Surrounding these histiocytes was a mantle of B lymphocytes as identified by their complement receptors. The percentage of B and T lymphocytes in the circulation was normal in 24 patients with sarcoidosis, but a diminution in the absolute number of B and T cells was present due to the relative lymphopenia observed in the sarcoidosis subjects compared with the control group. It is postulated that the anatomic distribution of the various immunocompetent cells within the sarcoid granuloma may play an important role in the pathogenesis of sarcoidosis.
INTRODUCTION
Human lymphocytes may be classified, based upon animal experiments and studies in patients with immunodeficiency states, into at least two groups, thymicdependent lymphocytes, T cells, and thymic-independent or bone marrow-derived B cells (l-5). T cells are thought to mediate reactions of cellular immunity whereas B cells are primarily concerned with humoral immunity. Different types of mononuclear cells may be recognized in peripheral blood, in cell suspensions and in tissue sections because of differences in cell membrane receptors (6-10). Differentiation between B lymphocytes, T lymphocytes, histiocytes and plasma cells within pathologic lesions has now become possible. Sheep erythrocytes (E) may be reacted with antibody and complement to form complement-coated EAC cells which can recognize B lymphocytes by virtue of the complement receptor on these cells. B lymphocytes also have surface receptors for anti-immunoglobulins and the Fc portion of IgG. Monocytes, histiocytes and macrophages also have complement receptors but differ from B lymphocytes in that they have a receptor for cytophilic antibody which will bind E cells coated with IgG (EA-7s) (9, 10). Recent work has suggested that histiocytes in lymph nodes are similar to blood monocytes in that both cells have a receptor for EAC and EA-7S, * Supported in part by U.S. Public Health Service grants, No. AM 11414, AM 12051, AM 05577, and FR 05669. Presented in part at the Seventh International Conference on Sarcoidosis and other Granulomatous Disorders, New York City. Oct. 6-10, 1975. * Formerly Fellow of the Medical Research Council of Canada. 3 Address reprint requests to: Dr. H. Tannenbaum, Room 7116, The Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec, Canada. 133 Copyright All rights
@ 1976 by Academic Press, Inc. of reproduction in any form reserved.
134
1 .~ivNENR.S~‘M.
I’lYKlS
\.\I)
SC t-1I.K
whereas histiocytes in splenic tissue have only a receptor for EA-7S (10). 7 lymphocytes can be readily identified by formation of nonimmune rosettes with E cells in cell suspensions but have not been detected in tissue sections with this method by most investigators (9, 10). Plasma cells can be recognized by the presence of cytoplasmic granules of immunoglobulin detected by fluorescein tagged anti-immunoglobulin antisera. Immunological characterization of the mononuclear cells within the noncaseating granuloma observed in sarcoidosis may contribute to a better understanding of the pathogenesis of this disease. We have studied tissue sections and cell suspensions of lymph node and lung and peripheral blood lymphocytes from patients with sarcoidosis in an attempt to clarify the nature and the in sits interrelationships of the infiltrating mononuclear cells present within sarcoid granuloma. METHODS
AND MATERIALS
Patient population. Fresh lymph nodes and lung tissues were obtained at the Peter Bent Brigham and/or New England Deaconess Hospitals from eight patients undergoing biopsy procedures as part of their diagnostic evaluation. Nine specimens (four scalene lymph nodes, four hilar lymph nodes and one lung biopsy) diagnostic of sarcoidosis were examined by cryostat section. and cell suspensions were made from four of these tissues (Tables 1 and 2). Peripheral blood lymphocytes were obtained from three of these patients as well as from 21 other patients with sarcoidosis (Table 3). Peripheral blood lymphocytes from 27 normal healthy volunteers comprised the control group (Table 3). In addition, histologically normal lymph nodes and spleen tissue obtained at surgery from patients with other diseases, e.g., cirrhosis, idiopathic thrombocytopenic purpura, and reactive follicular hyperplasia, served as controls for the in sitrr binding characteristics of the various sheep erythrocyte reagents. Preparation of .sheep red blood cell (SRBC’) recrgcnts and rlntiserurn. Complement-coated SRBC were made fresh daily by a modification of the method
Histiocytes” Patient
Biopsy
site
PC AH WH IM RE LL BK JM
Hilar node Lung Hilar node Hilar node Scalene node Scalene node Scalene node Scalene node Hilar node
e Intensity complement; reactive with
of EAC and EA-7S graded on a O-4+ EA-753, sheep erythrocytes coated with fluorescein-conjugated anti-immunoglobulin;
Lymphocytes” --_____ _____~
EAC
EA-7S
I!4
EAC
0 1+ 0 3+ 1+ I+ 3+ I+ 0
2-c 2+
0 0 0 0 0 0 0 0 0
4+ 1+ 4t 4+ 3t 4t 3t 3t 4+
434t 24
40 2+
EA-7S
k
0
3+ ?A 73 -+ 3t 3+ 3t it 3+
0 0 I 4 0 I -0 0 0
scale. EAC, sheep erythrocytes coated with rabbit 7s immunoglobulin; Ig, number of cells graded on O-4+ scale.
MONONUCLEAR
CELL
SUBPOPULATIONS
TABLE PERIPHERAL
BLOOD
STUDIES
AND
IN
135
SARCOIDOSIS
2 CELL
SUSPENSION
EXPERIMENTS
Percentage of Cells Patient
Source
PC
Hilar node Lung Peripheral blood Hilar node Peripheral blood Hilar node Peripheral blood
AH WH
EAC
EA-7S
E
53 14 18
ND ND ND
45 36 69
40 16
4
59 326
ND 11 ND
30
11
56 76
a ND, not done. b Received 60 mg of prednisone per day for 3 days prior to test. TABLE PERIPHERAL
Cells Lymphocytes (No./mm3) B lymphs (%) T lymphs (%) B lymphs (No./mm3) T lymphs (No./mm3)
BI.OOD
LYMPHOCYTE
STUDIES
3 IN PATIENTS
WITH
SARCOIDOSIS
Sarcoidosis (N = 24)
Control (N = 27)
Significance
1521 rt 673
1964 + 588
P < 0.02
16.0 +
16.2 + 66.7 k
NS NS P < 0.01
4.9
61.6 + 11.7 231.7 + 112.6 951.4
2 484.9
3.6 6.5
316.9 + 113.7 1292.3
k 397.8
P < 0.01
described by Shevach et al. (11). A 2% suspension of washed SRBC in Verona1 buffer, pH 7.4, containing 0.1% gelatin, Mg2+ (5 x 10-4M) and Ca2+ (1.5 x lo-“M) salts (GVB2+) was incubated with equal volumes of a l/2000 dilution of a rabbit IgM antibody to SRBC (Cordis Lab, Miami, Fla.) for 10 min at 23°C to form EA-19S cells. Pooled mouse serum as a source of complement previously frozen at -70°C was diluted l/10 with GVB2+ and then incubated with an equal volume of 1% EA-19s cells for 30 min at 37°C forming EAC cells. The cell suspension was then washed three times in tissue culture medium 199 with Hanks’ balanced salt solution (TCM 199) (Microbiological Associates, Bethesda, Md.) and adjusted to a 0.5% suspension. EA-7S cells were formed by incubating SRBC with a l/100 dilution of a rabbit IgG antibody directed against SRBC (Cordis Lab) for 10 min at 23°C. The EA-7S and unsensitized SRBC (E) were washed three times in TCM 199 and adjusted to a final concentration of 0.5%. Monospecific fluorescein conjugated anti-immunoglobulin antisera to IgG, IgM, and IgA were prepared as previously described (12). Isolation of mononuclear cells from peripheral blood and tissues. Twenty milliliters of peripheral blood were collected by venipuncture in plastic syringes containing 1000 U of heparin (Organon, West Orange, N.J.) and 2 ml of 6% dextran (Pharmacia Fine Chemicals, Piscataway, N.J.). The blood was allowed to sediment for 45-60 min at 37°C and the leukocyte-rich plasma was removed and centrifuged.
136
TANNENBAUM,
PINKUS
AND
SCHUR
The residual plasma was discarded and the sedimented cells were then resuspended in 4 ml ofTCM 199 supplemented with 10% heat-inactivated (56°C. 30 mitt) fetal calf serum (FCS) (Gibco, Grand Island, N.Y.). This suspension was layered carefully over 3 ml of a solution composed of 24 parts of 9% Ficoll (Pharmacia Fine Chemicals) and 10 parts of 34% Hypaque (Winthrop Laboratories, New York, N.Y.) and centrifuged at 4008 for 35 min at 23°C. The lymphocyte-rich layer at the interface was aspirated with a Pasteur pipet, washed twice in TCM containing FCS, and centrifuged at 200g for 10 min at 23°C. The final cell suspension consisted of lymphocytes (2 x lOVm1) contaminated with a small number (less than 15%) of monocytes and polymorphonuclear cells. Identification of these later cells in the final suspension could readily be made by use of the special staining techniques outlined below. Cell suspensions were prepared from three lymph nodes and one lung biopsy by placing a portion of the tissue in a petri dish containing TCM with FCS. The lymph node cells were gently teased apart with a small forceps, centrifuged over a Ficoll/Hypaque gradient at 4008 for 35 min, washed twice in TCM with FCS and adjusted to a final concentration of 2 x lo6 cells/ml. EAC and EA-7s rosette formation in ceil suspensions. A 0.4ml portion of a 0.5% suspension of EAC or EA-7S was incubated with an equal volume of mononuclear cells at 2 x lo6 cells/ml at 37°C for 30 min. A drop of Euchrysine 3RX, lo-* g/ml, (Chroma-Gesellschaft, Schmid & Co., Stuttgart-Unterturkheim, West Germany4) in TCM was added to this suspension, and the cells were examined by ultraviolet light. The cytoplasmic lysosomes in monocytes and polymorphonuclear leukocytes take on a red fluorescence with this supravital stain (13). The lymphocyte nucleus appears green and there is a conspicuous absence of any red cytoplasmic fluorescence due to the sparse number of lysosomes. This stain thus readily differentiates monocytes from lymphocytes. The number of EAC or EA-7S rosettes (minimum of three SRBC per mononuclear cell) forming between the mononuclear cells was enumerated by using simultaneous ultraviolet and white light. E rosette formation in cell suspension. Two-tenths of a milliliter of lymphocytes was mixed with an equal volume of 0.5% E cells, centrifuged at 2008 for 5 min at 23°C and then placed on ice for 90 min (14). The cell button was gently resuspended with a Pasteur pipet, and one drop of this suspension was placed on a microscope slide previously coated with 0.1% toluidine blue in 90% methanol; the slides were allowed to evaporate to dryness prior to being used. Differentiation between polymorphonuclear leukocytes and lymphocytes present in the cell suspension could readily be observed by this staining procedure. This staining procedure did not alter the number of E rosettes formed with lymphocytes, and no E rosettes were formed by polymorphonuclear leukocytes. Each E, EAC, and EA-7S test was done in duplicate and a minimum of 400 mononuclear cells was counted. Cell viability was assessed by trypan blue exclusion. Identification of lymphocytes, histiocytes and plasma cells in frozen tissue sections. Tissue specimens were quick frozen in cryostat and embedded in OCT (Ames Co., Elkart, Ind.). Sections 6-microns-thick were cut and stored at -20°C until used. When ready for testing the sections were air dried for 30 min at room tempera’ Distribution
by Roboz
Surgical
Instrument
Co.,
810 18th Street.
N.W.,
Washington,
D.C.
MONONUCLEAR
CELL
SUBPOPULATIONS
IN
SARCOIDOSIS
137
ture, washed three times in cold GVB2+ and then overlaid with the various SRBC reagents for 30 min at 23°C (EA-7s) or at 37°C (EAC and EA-19s). Incubation with E cells was performed for 1 hr at 23°C or 1 hr at 37°C followed by overnight incubation at 4°C. After incubation the slides were gently washed by dipping up and down in a dish containing GVB2+ until there were no SRBC visibly coming off from the tissue section. The tissue was then fixed with 3% glutaraldehyde, stained with hematoxylin and eosin (H & E) and examined by light microscopy. The density of SRBC adherent to a given area of tissue was scored on an arbitrarily defined O-4+ scale. Adjacent cryostat sections were air dried for 30 min, fixed in acetone, washed twice in cold 0.01 M phosphate-buffered saline, pH 7.2 (PBS), and then covered with fluorescein-conjugated anti-immunoglobulin for 30 min at room temperature. The tissue was then washed twice in PBS, mounted in glycerol and observed by means of a Leitz Orthoplan microscope with an Osram HBO 200 mercury-arc lamp and Ploem vertical illuminator (15). Peripheral
RESULTS Blood Lymphocytes
A normal percentage of circulating B and T lymphocytes was observed in the sarcoidosis patients (Table 3). However, the absolute number of circulating B and T cells was significantly diminished in the sarcoidosis group due to the relative lymphopenia of these patients compared with the control subjects. Localization of B Cells, Histiocytes Immunopathology of Normal Lymph
and Plasma Cells in Tissue Sections Node and Spleen Frozen Tissue Sections
In control experiments with histologically normal-appearing human lymph node and spleen tissue, the EAC reagent was observed to adhere to lymphoid follicles, with strongest binding (4+) being present in the germinal centers. This reagent did not react with the paracortex of lymph nodes or with the splenic periarteriolar lymphoid sheath regions, which represent the thymic-dependent zones. The EA-7S reagent reacted with sinusoidal histiocytes of lymph nodes and histiocytes in the splenic red pulp. The EA-19s reagent, as anticipated, always gave negative results as a heterologous IgM receptor and has not been identified on human cells (10). The unsensitized SRBC(E) also did not react with the tissues. Zmmunopathology on Sarcoidosis Frozen Tissue Sections Routine histology. Sections from the nine tissues were stained with hematoxylin
and eosin and each showed multiple noncaseating granulomas comprising between 75 and 95% of the biopsy specimen. Zmmunopathology. 1. Histiocytes. The EA-7S cells reacted with the histiocytic cells located within the center of the noncaseating granuloma (Fig. 1) in eight of nine tissues (Table 1). EAC cells reacted with the central histiocytic cells in six of nine tissues. This reaction was of 3+ intensity in two specimens (WH,LL) and of l+ intensity in the remaining four specimens (PC, IM,RE,BK). The histiocytic cells did not react with either E or EA-19s cells or with fluorescein-conjugated antiimmunoglobulin antisera (Table 1). 2. Lymphocytes. The mantle of lymphocytes observed surrounding the central collection of histiocytes reacted with EAC (Fig. 2) in all specimens (Table 1). These lymphocytes did not react with E or EA-19s cells, and in only two instances was a
FIG:. 1,
Lymph
node
from
PC, demonstrating
reaction
of histiocytes
Nith
EA-7S
cell\.
The
the figure is identical to the left-hand side except that it was taken under conditions microscopy which causes the eosin-stained sheep red Mood cells to appear as white dots. EA-7S cells are confined only to the granuloma and do not react with the lymphocytes upper nght-hand corner of the photograph. Hematoxylin and eo\in stain. 1 140
right
side
of
of dark field Note that the located in the
weak reaction with EA-7S observed (WH,RE). The surface membrane of approximately 50-60% of these cells reacted with fluorescein-conjugated anti-IgG (Fig. 3). The surface membrane of approximately 35% of lymphocytes reacted with anti-IgM and only scattered cells reacted with anti-IgA. Frequent plasma cells were identified by cytoplasmic fluorescence among these lymphocytes but were never observed among the central collection of histiocytes. Cell Suspensions
from Tissue
Involved
with
Sarcoidosis
Cell suspensions were made from four sarcoid tissues (three lymph nodes, one lung biopsy (Table 2). E rosettes formed with 36-59% of the isolated mononuclear cells. EAC rosettes formed with 14-53% and EA-7S rosettes formed with 4-l 1% of the isolated mononuclear cells (Table 2). Many of the mononuclear cells observed in the cell suspension of lung tissue were much larger than lymphocytes, contained numerous lysosomes, and did not react with either EAC or E cells. These cells may have represented lung histiocytes. Testing with EA-7S was not performed on these cells. T lymphocytes in our experiments could not be identified in situ by their reactions with E cells. However, cell suspensions from lymph nodes involved with sarcoidosis indicate that T cells constitute approximately 4S-59% of the isolatable mononuclear cells from these abnormal glands (Table 2). As most of the nodes were involved by the granulomatous process, it is highly unlikely that the residual or normal lymphocytes of these nodes contributed significantly to the enumeration of B and T lymphocytes. The observation of both B and T cells isolated from a lung
MONONUCLEAR
CELL
SUBPOPULATIONS
IN
SARCOIDOSIS
139
FIG. 2. Lymph node from PC, demonstrating reaction oflymphocytes with EAC cells. Note that the EAC cells react only with the surrounding mantle of B lymphocytes and not with the central granuloma. Hematoxylin and eosin stain, x 190.
FIG. 3. Lymph node from PC, reacted with fluroescein-conjugated anti-IgG demonstrating fluorescence only among those mononuclear cells (i.e., lymphocytes) surrounding the central histiocytic core of the sarcoid granuloma. x 190.
140
TANNENBAUM.
PINKCIS
AND
XHL’K
granuloma, a non-lymph node structure, is further confirmation constitute an integral part of the sarcoid granuloma.
that B and T cells
DISCUSSION
Depression of cutaneous delayed type hypersensitivity reactions to a variety of antigens has long been considered a feature of sarcoidosis. In vitro studies reveal impaired lymphoblastic transformation of lymphocytes from sarcoid patients in response to stimulation with phytohemagglutin (PHA) (16,17). In one study. T-lymphocyte function, as assessed by PHA stimulation, was demonstrated to be impaired only in patients with extrathoracic dissemination of sarcoidosis but was normal in patients whose disease was confined to the intrathoracic cavity (16). Furthermore, the impaired PHA response could not be attributed to a decrease in number of T lymphocytes as T-cell quantity was normal (18). Other investigators have demonstrated a decrease in T lymphocytes in both untreated and steroidtreated patients with sarcoidosis (19,21). The observed cutaneous anergy appears not to correlate either with these in vitro observations or with the clinical severity of the disease (16,19). It has been suggested that the immunological defect in sarcoidosis might be related to a serum inhibitor or immunosuppressive factor rather than to dysfunction or depletion of lymphocytes (19,20,22). The presence of hyperglobulinemia and rheumatoid factors in the sera of patients with sarcoidosis is indirect evidence of B-cell hyperfunction. An increase in the percentage and absolute numbers of circulating B cells, especially in patients with disseminated sarcoidosis, has been demonstrated (18,21). In other studies and in patients with sarcoidosis limited to the lung the B-cell quantity was unchanged from control values (18,19). Our studies, however, indicate a normal percentage of B and T cells in the peripheral blood, regardless of the extent of disease activity. A statistically significant diminution in the absolute number of circulating B and T cells was present in our sarcoidosis group due to the relative lymphopenia compared with the control subjects. The in situ observations on cryostat sections of sarcoidosis tissue indicate that the surface membrane of the histiocytes located within the center of the sarcoid granuloma has a receptor for 7s y-globulin. In addition, some of these cells (six of nine specimens) have surface receptors for complement. Thus, these histiocytes resemble more closely the immunological characteristics of lymph node histiocytes in that they appear to have receptors for both complement and cytophilic antibody, unlike splenic histiocytes which have surface receptors for only cytophilic antibody. In only two instances did these histiocytes simultaneously bind both EA-7S and EAC cells avidly. It is not known why the histiocytes in some granulomas bind EAC avidly (two cases), while EAC binding was minimal in four cases and absent in three others (Table 1). The histiocytes did not react with fluorescein-tagged antiimmunoglobulin antisera. The number of histiocytic cells reacting with EA-7S in situ appeared greater than what was observed in the cell suspensions. Similar observations have been noted with splenic and lymph node histiocytes and it has been suggested that histiocytic cells enmeshed in a collagen framework are not readily removed by teasing apart the gland (10). Surrounding the histiocytic cells was a rim of B lymphocytes as recognized by their reactions with EAC and fluorescein-tagged anti-immunoglobulin antisera. A
MONONUCLEAR
CELL
SUBPOPULATIONS
IN
SARCOIDOSIS
141
number of plasma cells was observed among these lymphocytes but not within the central region of the granuloma. Cell suspensions of lymph node and lung revealed a predominance of T cells which was in contradistinction to the immunopathologic observations that suggested predominance of B cells. Underestimation of the T cells from the tissue section observations alone may be due to close intermingling of B and T cells in situ. The EAC reagent cells forming rosettes with B lymphocytes in tissue section may overlap some of the T cells but not react with them, and one can erroneously conclude that greater numbers of B cells actually are present in the tissue. It is evident that additional studies involving characterization of dense collections of lymphocytes are best studied by both tissue section and cell suspension techniques. One of the first lines of host defense is the tissue histiocyte or macrophage. The macrophage can engulf and digest foreign material and, in addition, has an important role in processing new antigens. Perhaps the elusive, etiologic agent of sarcoidosis resides within the center of the sarcoid granuloma. The 7S y-globulin surface receptor on the centrally located histiocyte can then initiate host defense mechanisms against this agent. The mantle of B lymphocytes surrounding these histiocytes closely intermingled with T cells can then mount a conjoint effort to contain this mysterious etiologic agent of sarcoidosis. ACKNOWLEDGMENTS The authors acknowledge the expert technical assistance of Ms. M. Stone and the secretarial assistance of Ms. K. Matheson and would like to thank Dr. J. Andrews and Dr. A. L. Sheffer ofthe New England Deaconess Hospital for allowing us to study their patients.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Raff, M. D., Immunology, 19, 637, 1970. Gajl-Peczalska, K. J., Biggar, W. D., Park, B. H., and Good, R. A., Lance? 1, 1344. 1972. Cooper, M. D., Lawton, A. R., and Bockman, D. E., Lancet 2,791, 1971. Grey, H. M., Rabellino, E., and Pirofsky, G., J. Clin. Invest. 50, 2368, 1971. Katz, D. H., and Benacerraf, B., Advan. Zmmunol. 15, 2, 1972. Talal, N., Sylvester, R. A., Daniels, T. E., Greenspan, J. S., and Williams, R. C., Jr., J. Clin. Invest. 53, 180, 1974. Chusid, T. M., Hardin, J. A., Frank, M. M., and Green, I., J. Immune/. 112, 641, 1974. Tannenbaum, H., Pinkus, G. S., Anderson, L. G., and Schur, P. H., Arthritis Rheum. 18, 305, 1975. Edelson, R. L., Smith, R. W., Frank, M. M., and Green, I., J. Invest. Dermatol. 61, 82, 1973. Jaffe, E. S., Shevach, E. M., Frank, M. M., Berard, C. W., and Green, I., N. Engl. J. Med. 290, 813, 1974. Shevach, E. M., Herberman, R., Frank, M. M., and Green, I., J. Clin. Invest. 51, 1933, 1972. B&ton, M. C., and Schur, P. H., Arthritis Rheum. 14, 87, 1971. Allison, A. C., and Young, M. R., Life Sci. 3, 1407, 1964. Lay, W. H., Mendes, W. F., Bianco, C., and Nussenzweig, V., Nature (London) 230,531, 1971. Ploem, J. S., Leitz-Mih Wiss. Tech. 4: 225, 1969. Kataria, Y. P., Sagone, A. L., LoBuglio, A. F., and Bromberg, P. A., Amer. Rev. Resp. Dis. 108, 767, 1973. Topilsky, M., Siltzbach, L. E., Williams, M., and Glade, P. R., Lancef 1, 117, 1972. Kataria, Y., James, K., Hurtubise, P., LoBuglio, A., and Bromberg, P., Clin. Res. 21,891, 1973 (abstract). Dwyer, J. M., Mangi, R. J., and Kantor, F. S.,J. Allergy Clin. Immunol. 53,69, 1974 (abstract). Mangi, R. J., Dwyer, J. M., and Kantor, F. S., J. Allergy Clin. Immunol. 53,86, 1974 (abstract). Papamichael, M., Holborow, E. J., Keith, H. I., and Currey, H. L. F., Lancer 2, 64, 1972. Belcher, R. W., Carney, J. F., and Nankervis, G. A., C/in. Res. 20, 415, 1972 (abstract).
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
5,
133-141 (1976)
Immunological Characterization of Subpopulations of Mononuclear Cells in Tissue and Peripheral Blood from Patients with Sarcoidosisl HYMAN
TANNENBAUM,~‘~
S. PINKUS,
GERALDINE
AND
PETER
H.
SCHUR
Department of Medicine, Montreal General Hospital, Montreal, Canada, Robert B. Brigham Hospital, Boston,Massachusetts 02120, and the Department of Pathology, Peter Bent Brigham Hospital, Boston, Massachusetts, 02115
Received July 25, 1975 The immunological characteristics of the mononuclear cells comprising sarcoid granulomas were studied in cryostat sections of eight lymph nodes and one lung biopsy. In eight of the nine tissues, the histiocytes in the center of the sarcoid granuloma had surface receptors for 7S y-globulin. Surrounding these histiocytes was a mantle of B lymphocytes as identified by their complement receptors. The percentage of B and T lymphocytes in the circulation was normal in 24 patients with sarcoidosis, but a diminution in the absolute number of B and T cells was present due to the relative lymphopenia observed in the sarcoidosis subjects compared with the control group. It is postulated that the anatomic distribution of the various immunocompetent cells within the sarcoid granuloma may play an important role in the pathogenesis of sarcoidosis.
INTRODUCTION
Human lymphocytes may be classified, based upon animal experiments and studies in patients with immunodeficiency states, into at least two groups, thymicdependent lymphocytes, T cells, and thymic-independent or bone marrow-derived B cells (l-5). T cells are thought to mediate reactions of cellular immunity whereas B cells are primarily concerned with humoral immunity. Different types of mononuclear cells may be recognized in peripheral blood, in cell suspensions and in tissue sections because of differences in cell membrane receptors (6-10). Differentiation between B lymphocytes, T lymphocytes, histiocytes and plasma cells within pathologic lesions has now become possible. Sheep erythrocytes (E) may be reacted with antibody and complement to form complement-coated EAC cells which can recognize B lymphocytes by virtue of the complement receptor on these cells. B lymphocytes also have surface receptors for anti-immunoglobulins and the Fc portion of IgG. Monocytes, histiocytes and macrophages also have complement receptors but differ from B lymphocytes in that they have a receptor for cytophilic antibody which will bind E cells coated with IgG (EA-7s) (9, 10). Recent work has suggested that histiocytes in lymph nodes are similar to blood monocytes in that both cells have a receptor for EAC and EA-7S, * Supported in part by U.S. Public Health Service grants, No. AM 11414, AM 12051, AM 05577, and FR 05669. Presented in part at the Seventh International Conference on Sarcoidosis and other Granulomatous Disorders, New York City. Oct. 6-10, 1975. * Formerly Fellow of the Medical Research Council of Canada. 3 Address reprint requests to: Dr. H. Tannenbaum, Room 7116, The Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec, Canada. 133 Copyright All rights
@ 1976 by Academic Press, Inc. of reproduction in any form reserved.
134
1 .~ivNENR.S~‘M.
I’lYKlS
\.\I)
SC t-1I.K
whereas histiocytes in splenic tissue have only a receptor for EA-7S (10). 7 lymphocytes can be readily identified by formation of nonimmune rosettes with E cells in cell suspensions but have not been detected in tissue sections with this method by most investigators (9, 10). Plasma cells can be recognized by the presence of cytoplasmic granules of immunoglobulin detected by fluorescein tagged anti-immunoglobulin antisera. Immunological characterization of the mononuclear cells within the noncaseating granuloma observed in sarcoidosis may contribute to a better understanding of the pathogenesis of this disease. We have studied tissue sections and cell suspensions of lymph node and lung and peripheral blood lymphocytes from patients with sarcoidosis in an attempt to clarify the nature and the in sits interrelationships of the infiltrating mononuclear cells present within sarcoid granuloma. METHODS
AND MATERIALS
Patient population. Fresh lymph nodes and lung tissues were obtained at the Peter Bent Brigham and/or New England Deaconess Hospitals from eight patients undergoing biopsy procedures as part of their diagnostic evaluation. Nine specimens (four scalene lymph nodes, four hilar lymph nodes and one lung biopsy) diagnostic of sarcoidosis were examined by cryostat section. and cell suspensions were made from four of these tissues (Tables 1 and 2). Peripheral blood lymphocytes were obtained from three of these patients as well as from 21 other patients with sarcoidosis (Table 3). Peripheral blood lymphocytes from 27 normal healthy volunteers comprised the control group (Table 3). In addition, histologically normal lymph nodes and spleen tissue obtained at surgery from patients with other diseases, e.g., cirrhosis, idiopathic thrombocytopenic purpura, and reactive follicular hyperplasia, served as controls for the in sitrr binding characteristics of the various sheep erythrocyte reagents. Preparation of .sheep red blood cell (SRBC’) recrgcnts and rlntiserurn. Complement-coated SRBC were made fresh daily by a modification of the method
Histiocytes” Patient
Biopsy
site
PC AH WH IM RE LL BK JM
Hilar node Lung Hilar node Hilar node Scalene node Scalene node Scalene node Scalene node Hilar node
e Intensity complement; reactive with
of EAC and EA-7S graded on a O-4+ EA-753, sheep erythrocytes coated with fluorescein-conjugated anti-immunoglobulin;
Lymphocytes” --_____ _____~
EAC
EA-7S
I!4
EAC
0 1+ 0 3+ 1+ I+ 3+ I+ 0
2-c 2+
0 0 0 0 0 0 0 0 0
4+ 1+ 4t 4+ 3t 4t 3t 3t 4+
434t 24
40 2+
EA-7S
k
0
3+ ?A 73 -+ 3t 3+ 3t it 3+
0 0 I 4 0 I -0 0 0
scale. EAC, sheep erythrocytes coated with rabbit 7s immunoglobulin; Ig, number of cells graded on O-4+ scale.
MONONUCLEAR
CELL
SUBPOPULATIONS
TABLE PERIPHERAL
BLOOD
STUDIES
AND
IN
135
SARCOIDOSIS
2 CELL
SUSPENSION
EXPERIMENTS
Percentage of Cells Patient
Source
PC
Hilar node Lung Peripheral blood Hilar node Peripheral blood Hilar node Peripheral blood
AH WH
EAC
EA-7S
E
53 14 18
ND ND ND
45 36 69
40 16
4
59 326
ND 11 ND
30
11
56 76
a ND, not done. b Received 60 mg of prednisone per day for 3 days prior to test. TABLE PERIPHERAL
Cells Lymphocytes (No./mm3) B lymphs (%) T lymphs (%) B lymphs (No./mm3) T lymphs (No./mm3)
BI.OOD
LYMPHOCYTE
STUDIES
3 IN PATIENTS
WITH
SARCOIDOSIS
Sarcoidosis (N = 24)
Control (N = 27)
Significance
1521 rt 673
1964 + 588
P < 0.02
16.0 +
16.2 + 66.7 k
NS NS P < 0.01
4.9
61.6 + 11.7 231.7 + 112.6 951.4
2 484.9
3.6 6.5
316.9 + 113.7 1292.3
k 397.8
P < 0.01
described by Shevach et al. (11). A 2% suspension of washed SRBC in Verona1 buffer, pH 7.4, containing 0.1% gelatin, Mg2+ (5 x 10-4M) and Ca2+ (1.5 x lo-“M) salts (GVB2+) was incubated with equal volumes of a l/2000 dilution of a rabbit IgM antibody to SRBC (Cordis Lab, Miami, Fla.) for 10 min at 23°C to form EA-19S cells. Pooled mouse serum as a source of complement previously frozen at -70°C was diluted l/10 with GVB2+ and then incubated with an equal volume of 1% EA-19s cells for 30 min at 37°C forming EAC cells. The cell suspension was then washed three times in tissue culture medium 199 with Hanks’ balanced salt solution (TCM 199) (Microbiological Associates, Bethesda, Md.) and adjusted to a 0.5% suspension. EA-7S cells were formed by incubating SRBC with a l/100 dilution of a rabbit IgG antibody directed against SRBC (Cordis Lab) for 10 min at 23°C. The EA-7S and unsensitized SRBC (E) were washed three times in TCM 199 and adjusted to a final concentration of 0.5%. Monospecific fluorescein conjugated anti-immunoglobulin antisera to IgG, IgM, and IgA were prepared as previously described (12). Isolation of mononuclear cells from peripheral blood and tissues. Twenty milliliters of peripheral blood were collected by venipuncture in plastic syringes containing 1000 U of heparin (Organon, West Orange, N.J.) and 2 ml of 6% dextran (Pharmacia Fine Chemicals, Piscataway, N.J.). The blood was allowed to sediment for 45-60 min at 37°C and the leukocyte-rich plasma was removed and centrifuged.
136
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SCHUR
The residual plasma was discarded and the sedimented cells were then resuspended in 4 ml ofTCM 199 supplemented with 10% heat-inactivated (56°C. 30 mitt) fetal calf serum (FCS) (Gibco, Grand Island, N.Y.). This suspension was layered carefully over 3 ml of a solution composed of 24 parts of 9% Ficoll (Pharmacia Fine Chemicals) and 10 parts of 34% Hypaque (Winthrop Laboratories, New York, N.Y.) and centrifuged at 4008 for 35 min at 23°C. The lymphocyte-rich layer at the interface was aspirated with a Pasteur pipet, washed twice in TCM containing FCS, and centrifuged at 200g for 10 min at 23°C. The final cell suspension consisted of lymphocytes (2 x lOVm1) contaminated with a small number (less than 15%) of monocytes and polymorphonuclear cells. Identification of these later cells in the final suspension could readily be made by use of the special staining techniques outlined below. Cell suspensions were prepared from three lymph nodes and one lung biopsy by placing a portion of the tissue in a petri dish containing TCM with FCS. The lymph node cells were gently teased apart with a small forceps, centrifuged over a Ficoll/Hypaque gradient at 4008 for 35 min, washed twice in TCM with FCS and adjusted to a final concentration of 2 x lo6 cells/ml. EAC and EA-7s rosette formation in ceil suspensions. A 0.4ml portion of a 0.5% suspension of EAC or EA-7S was incubated with an equal volume of mononuclear cells at 2 x lo6 cells/ml at 37°C for 30 min. A drop of Euchrysine 3RX, lo-* g/ml, (Chroma-Gesellschaft, Schmid & Co., Stuttgart-Unterturkheim, West Germany4) in TCM was added to this suspension, and the cells were examined by ultraviolet light. The cytoplasmic lysosomes in monocytes and polymorphonuclear leukocytes take on a red fluorescence with this supravital stain (13). The lymphocyte nucleus appears green and there is a conspicuous absence of any red cytoplasmic fluorescence due to the sparse number of lysosomes. This stain thus readily differentiates monocytes from lymphocytes. The number of EAC or EA-7S rosettes (minimum of three SRBC per mononuclear cell) forming between the mononuclear cells was enumerated by using simultaneous ultraviolet and white light. E rosette formation in cell suspension. Two-tenths of a milliliter of lymphocytes was mixed with an equal volume of 0.5% E cells, centrifuged at 2008 for 5 min at 23°C and then placed on ice for 90 min (14). The cell button was gently resuspended with a Pasteur pipet, and one drop of this suspension was placed on a microscope slide previously coated with 0.1% toluidine blue in 90% methanol; the slides were allowed to evaporate to dryness prior to being used. Differentiation between polymorphonuclear leukocytes and lymphocytes present in the cell suspension could readily be observed by this staining procedure. This staining procedure did not alter the number of E rosettes formed with lymphocytes, and no E rosettes were formed by polymorphonuclear leukocytes. Each E, EAC, and EA-7S test was done in duplicate and a minimum of 400 mononuclear cells was counted. Cell viability was assessed by trypan blue exclusion. Identification of lymphocytes, histiocytes and plasma cells in frozen tissue sections. Tissue specimens were quick frozen in cryostat and embedded in OCT (Ames Co., Elkart, Ind.). Sections 6-microns-thick were cut and stored at -20°C until used. When ready for testing the sections were air dried for 30 min at room tempera’ Distribution
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810 18th Street.
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Washington,
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CELL
SUBPOPULATIONS
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ture, washed three times in cold GVB2+ and then overlaid with the various SRBC reagents for 30 min at 23°C (EA-7s) or at 37°C (EAC and EA-19s). Incubation with E cells was performed for 1 hr at 23°C or 1 hr at 37°C followed by overnight incubation at 4°C. After incubation the slides were gently washed by dipping up and down in a dish containing GVB2+ until there were no SRBC visibly coming off from the tissue section. The tissue was then fixed with 3% glutaraldehyde, stained with hematoxylin and eosin (H & E) and examined by light microscopy. The density of SRBC adherent to a given area of tissue was scored on an arbitrarily defined O-4+ scale. Adjacent cryostat sections were air dried for 30 min, fixed in acetone, washed twice in cold 0.01 M phosphate-buffered saline, pH 7.2 (PBS), and then covered with fluorescein-conjugated anti-immunoglobulin for 30 min at room temperature. The tissue was then washed twice in PBS, mounted in glycerol and observed by means of a Leitz Orthoplan microscope with an Osram HBO 200 mercury-arc lamp and Ploem vertical illuminator (15). Peripheral
RESULTS Blood Lymphocytes
A normal percentage of circulating B and T lymphocytes was observed in the sarcoidosis patients (Table 3). However, the absolute number of circulating B and T cells was significantly diminished in the sarcoidosis group due to the relative lymphopenia of these patients compared with the control subjects. Localization of B Cells, Histiocytes Immunopathology of Normal Lymph
and Plasma Cells in Tissue Sections Node and Spleen Frozen Tissue Sections
In control experiments with histologically normal-appearing human lymph node and spleen tissue, the EAC reagent was observed to adhere to lymphoid follicles, with strongest binding (4+) being present in the germinal centers. This reagent did not react with the paracortex of lymph nodes or with the splenic periarteriolar lymphoid sheath regions, which represent the thymic-dependent zones. The EA-7S reagent reacted with sinusoidal histiocytes of lymph nodes and histiocytes in the splenic red pulp. The EA-19s reagent, as anticipated, always gave negative results as a heterologous IgM receptor and has not been identified on human cells (10). The unsensitized SRBC(E) also did not react with the tissues. Zmmunopathology on Sarcoidosis Frozen Tissue Sections Routine histology. Sections from the nine tissues were stained with hematoxylin
and eosin and each showed multiple noncaseating granulomas comprising between 75 and 95% of the biopsy specimen. Zmmunopathology. 1. Histiocytes. The EA-7S cells reacted with the histiocytic cells located within the center of the noncaseating granuloma (Fig. 1) in eight of nine tissues (Table 1). EAC cells reacted with the central histiocytic cells in six of nine tissues. This reaction was of 3+ intensity in two specimens (WH,LL) and of l+ intensity in the remaining four specimens (PC, IM,RE,BK). The histiocytic cells did not react with either E or EA-19s cells or with fluorescein-conjugated antiimmunoglobulin antisera (Table 1). 2. Lymphocytes. The mantle of lymphocytes observed surrounding the central collection of histiocytes reacted with EAC (Fig. 2) in all specimens (Table 1). These lymphocytes did not react with E or EA-19s cells, and in only two instances was a
FIG:. 1,
Lymph
node
from
PC, demonstrating
reaction
of histiocytes
Nith
EA-7S
cell\.
The
the figure is identical to the left-hand side except that it was taken under conditions microscopy which causes the eosin-stained sheep red Mood cells to appear as white dots. EA-7S cells are confined only to the granuloma and do not react with the lymphocytes upper nght-hand corner of the photograph. Hematoxylin and eo\in stain. 1 140
right
side
of
of dark field Note that the located in the
weak reaction with EA-7S observed (WH,RE). The surface membrane of approximately 50-60% of these cells reacted with fluorescein-conjugated anti-IgG (Fig. 3). The surface membrane of approximately 35% of lymphocytes reacted with anti-IgM and only scattered cells reacted with anti-IgA. Frequent plasma cells were identified by cytoplasmic fluorescence among these lymphocytes but were never observed among the central collection of histiocytes. Cell Suspensions
from Tissue
Involved
with
Sarcoidosis
Cell suspensions were made from four sarcoid tissues (three lymph nodes, one lung biopsy (Table 2). E rosettes formed with 36-59% of the isolated mononuclear cells. EAC rosettes formed with 14-53% and EA-7S rosettes formed with 4-l 1% of the isolated mononuclear cells (Table 2). Many of the mononuclear cells observed in the cell suspension of lung tissue were much larger than lymphocytes, contained numerous lysosomes, and did not react with either EAC or E cells. These cells may have represented lung histiocytes. Testing with EA-7S was not performed on these cells. T lymphocytes in our experiments could not be identified in situ by their reactions with E cells. However, cell suspensions from lymph nodes involved with sarcoidosis indicate that T cells constitute approximately 4S-59% of the isolatable mononuclear cells from these abnormal glands (Table 2). As most of the nodes were involved by the granulomatous process, it is highly unlikely that the residual or normal lymphocytes of these nodes contributed significantly to the enumeration of B and T lymphocytes. The observation of both B and T cells isolated from a lung
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FIG. 2. Lymph node from PC, demonstrating reaction oflymphocytes with EAC cells. Note that the EAC cells react only with the surrounding mantle of B lymphocytes and not with the central granuloma. Hematoxylin and eosin stain, x 190.
FIG. 3. Lymph node from PC, reacted with fluroescein-conjugated anti-IgG demonstrating fluorescence only among those mononuclear cells (i.e., lymphocytes) surrounding the central histiocytic core of the sarcoid granuloma. x 190.
140
TANNENBAUM.
PINKCIS
AND
XHL’K
granuloma, a non-lymph node structure, is further confirmation constitute an integral part of the sarcoid granuloma.
that B and T cells
DISCUSSION
Depression of cutaneous delayed type hypersensitivity reactions to a variety of antigens has long been considered a feature of sarcoidosis. In vitro studies reveal impaired lymphoblastic transformation of lymphocytes from sarcoid patients in response to stimulation with phytohemagglutin (PHA) (16,17). In one study. T-lymphocyte function, as assessed by PHA stimulation, was demonstrated to be impaired only in patients with extrathoracic dissemination of sarcoidosis but was normal in patients whose disease was confined to the intrathoracic cavity (16). Furthermore, the impaired PHA response could not be attributed to a decrease in number of T lymphocytes as T-cell quantity was normal (18). Other investigators have demonstrated a decrease in T lymphocytes in both untreated and steroidtreated patients with sarcoidosis (19,21). The observed cutaneous anergy appears not to correlate either with these in vitro observations or with the clinical severity of the disease (16,19). It has been suggested that the immunological defect in sarcoidosis might be related to a serum inhibitor or immunosuppressive factor rather than to dysfunction or depletion of lymphocytes (19,20,22). The presence of hyperglobulinemia and rheumatoid factors in the sera of patients with sarcoidosis is indirect evidence of B-cell hyperfunction. An increase in the percentage and absolute numbers of circulating B cells, especially in patients with disseminated sarcoidosis, has been demonstrated (18,21). In other studies and in patients with sarcoidosis limited to the lung the B-cell quantity was unchanged from control values (18,19). Our studies, however, indicate a normal percentage of B and T cells in the peripheral blood, regardless of the extent of disease activity. A statistically significant diminution in the absolute number of circulating B and T cells was present in our sarcoidosis group due to the relative lymphopenia compared with the control subjects. The in situ observations on cryostat sections of sarcoidosis tissue indicate that the surface membrane of the histiocytes located within the center of the sarcoid granuloma has a receptor for 7s y-globulin. In addition, some of these cells (six of nine specimens) have surface receptors for complement. Thus, these histiocytes resemble more closely the immunological characteristics of lymph node histiocytes in that they appear to have receptors for both complement and cytophilic antibody, unlike splenic histiocytes which have surface receptors for only cytophilic antibody. In only two instances did these histiocytes simultaneously bind both EA-7S and EAC cells avidly. It is not known why the histiocytes in some granulomas bind EAC avidly (two cases), while EAC binding was minimal in four cases and absent in three others (Table 1). The histiocytes did not react with fluorescein-tagged antiimmunoglobulin antisera. The number of histiocytic cells reacting with EA-7S in situ appeared greater than what was observed in the cell suspensions. Similar observations have been noted with splenic and lymph node histiocytes and it has been suggested that histiocytic cells enmeshed in a collagen framework are not readily removed by teasing apart the gland (10). Surrounding the histiocytic cells was a rim of B lymphocytes as recognized by their reactions with EAC and fluorescein-tagged anti-immunoglobulin antisera. A
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number of plasma cells was observed among these lymphocytes but not within the central region of the granuloma. Cell suspensions of lymph node and lung revealed a predominance of T cells which was in contradistinction to the immunopathologic observations that suggested predominance of B cells. Underestimation of the T cells from the tissue section observations alone may be due to close intermingling of B and T cells in situ. The EAC reagent cells forming rosettes with B lymphocytes in tissue section may overlap some of the T cells but not react with them, and one can erroneously conclude that greater numbers of B cells actually are present in the tissue. It is evident that additional studies involving characterization of dense collections of lymphocytes are best studied by both tissue section and cell suspension techniques. One of the first lines of host defense is the tissue histiocyte or macrophage. The macrophage can engulf and digest foreign material and, in addition, has an important role in processing new antigens. Perhaps the elusive, etiologic agent of sarcoidosis resides within the center of the sarcoid granuloma. The 7S y-globulin surface receptor on the centrally located histiocyte can then initiate host defense mechanisms against this agent. The mantle of B lymphocytes surrounding these histiocytes closely intermingled with T cells can then mount a conjoint effort to contain this mysterious etiologic agent of sarcoidosis. ACKNOWLEDGMENTS The authors acknowledge the expert technical assistance of Ms. M. Stone and the secretarial assistance of Ms. K. Matheson and would like to thank Dr. J. Andrews and Dr. A. L. Sheffer ofthe New England Deaconess Hospital for allowing us to study their patients.
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