CLINICAL
IMMUNOLOGY
AND
Lymphocyte
IMMUNOPATHOLOGY
14, 130-
136 (1979)
Subpopulations in Minimal Nephrotic Syndrome
Change
HOWARD 0. KERPEN, J. GANESH BHAT,’ ROBERT KANTOR. BERNARD GAUTHIER, KANTI R. RAI, ROBERT G. SCHACHT, AND DAVID S. BALDWIN
Received
January
24. 1979
Minimal change nephrotic syndrome (MCNS) was characterized by a relative lymphocytopenia, a normal T-cell count, and a discordance between B cells enumerated by the presence of surface immunoglobulins (Bsig) and by C3B receptors (Beact. By either technique of B-cell count, the total number of T and B lymphocytes in MCNS exceeded that of controls. suggesting the presence of “double-marked” cells or decreased null cell population. This alteration in surface receptor characteristics of lymphocytes which was observed both in relapse and in remission. documents an abnormality, in MCNS. possibly of cell-mediated immunity, which may be relevant to its pathogenesis.
INTRODUCTION
Minimal change nephrotic syndrome (MCNS) is a glomerular disease of unknown etiology and pathogenesis. Although usually controlled by corticosteroids, patients frequently relapse, requiring long-term use of steroids or cytotoxic drugs ( 1, 2) with potential for adverse side effects. Various lines of evidence suggest lymphocyte involvement in the pathogenesis of MCNS. The most significant evidence is the consistent induction of remission of the nephrotic syndrome following immunosuppressive agents (corticosteroids and cytotoxic drugs). This response, despite the absence of demonstrable immune deposits or complement in the glomerulus, may imply an alteration in lymphocyte function in MCNS. Additional evidence includes remissions induced by measles (3), where cell-mediated immunity (CMI) is known to be depressed. Depressed serum IgG, IgA, and elevated serum IgM levels are also seen in patients with MCNS (4). Other immune-related phenomena include increased serum IgE (5). increased susceptibility to infection (6), occurrence of lymphocytotoxic antibodies (7), circulating blastogenesis-inhibiting factor in the serum (S), and the possible production of a vascular permeability enhancing factor by lymphocytes (9). Also. CM1 directed against the kidney may be inferred by the demonstration in migra. tion inhibition experiments (10) that human lymphocytes from patients with MCNS are or have been sensitized to fetal kidney antigens. In addition, nephrotic syndrome with glomerular pathology indistinguishable from MCNS occurs in ’ Present address: Institute for Geriatric 410 Lakeville Road.
Department of Medicine, Long Island Jewish Hillside Medical Care, New Hyde Park. N.Y. To whom requests for reprints New Hyde Park. N.Y. 11040. 130
0090-1229/79/090130-07$01.00/O Copyright Q 1979 by Academic Press. Inc. All rights of reproducfmn in any form reserved.
Center and Jewish may be addressed:
LYMPHOCYTE
SUBPOPULATIONS
IN
MCNS
131
some cases of Hodgkin’s lymphoma. The activity and remission of the glomerular lesion parallels the status of the disease (11 - 13). It has been hypothesized that MCNS is caused by a functional abnormality of lymphocytes arising either de ~OVO or due to specific antigenic stimulus. Abnormal production of a lymphokine with specific effects on vascular permeability, particularly of the glomerular basement membrane, could be a pathogenetic mechanism. In a disease which is characterized by a remarkably consistent response to steroids and cytotoxic drugs, some altered parameter of cellular and/or humoral immunity should be demonstrable which correlates with disease activity and remission. We have studied the major subpopulations of peripheral blood lymphocytes, with respect to numbers and cell surface marker characteristics, in 16 patients with MCNS, 14 healthy age- and sex-matched controls, and 3 patients with proteinuria due to causes other than MCNS. We have found a population of lymphocytes in MCNS that bear receptors for both T and B cells, i.e., doublemarked cells. The possible significance of these findings are discussed below. MATERIALS
AND METHODS
Observations were made in three groups of individuals. Sixteen patients who were known to us from the onset of nephrotic syndrome were studied. The diagnosis of MCNS was made initially on the basis of typical features of the syndrome: massive proteinuria, unremarkable urinary sediment, absence of azotemia or hypertension, and response to either corticosteroids or cyclophosphamide. In three patients, the diagnosis was confirmed by percutaneous renal biopsy. The ages of the entire group ranged from 3 to 26 years. At the time of study, six patients were in relapse as indicated by the presence of nephrotic syndrome and ten were in clinical remission (Table 1). Seven of the ten patients without proteinuria were receiving no medications at the time of study, although they had been treated either with prednisone or cyclophosphamide in the past. Fourteen healthy children, eight boys and six girls, varying in age from 5 to 13 years (mean age 10 years), with no evidence of proteinuria, formed the control group. In addition, three male patients and one female with proteinuria not due to MCNS were also studied. Their ages ranged from 8 to 16 years. Three of these had focal sclerosis on renal biopsy and the fourth had orthostatic proteinuria. Collection of blood and separation of mononuclear cells. Blood (20 ml) was collected in sterile disposable syringes with 10 units heparin/ml. Lymphocytes were isolated using density gradient centrifugation (14). The blood was diluted with an equal volume of calcium and magnesium-free Hanks’ balanced salt solution (HBSS) (GIBCO). Of this diluted peripheral blood, 4 ml was layered over 3 ml of Ficoll-Hypaque and centrifuged at 400g for 20 min at room temperature. The mononuclear cells at the plasma- Ficoll- Hypaque interface were aspirated aseptically and washed three times with HBSS. The cells were resuspended to a final concentration of 2 x lo6 mononuclear cells/ml. The resultant cell suspension usually contained greater than 95% lymphocytes, of which at least 90% were viable when tested with trypan blue dye exclusion technique. Contamination with monocytes was determined by latex particle phagocytosis. Aliquots of this total mononuclear cell population were used for T- and B-cell determinations. Cell surface marker studies. T cells were determined by a modification of the technique of Wybran and Fudenberg (15) using sheep red blood cell (SRBC)
132
KERPEN ET AL. TABLE CLINICAL
No.
Age
Sex
1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16
3 12 11 26 12 13 12 11 4 7 5 8 I5 20 II 5
F F M M M M M M M M M F M M M M
Proteinuria
FEA-KJRES
Serum albumin
+ i + + + + -
I
OF 16 PATIENTS
Serum cholesterol
WITH
MCNS” Therapy at time of study P P
i i 1 I
1
N N N N N N N N N N
N N N N N N N N N N
Past therapy -P. 1977 CY. 1972 .-
CY. P CY P -.
-
P. P, P. P. P. CY. CY. P.
t’ 1972 1976 1972 1970 1972 IV77 1977 1977
o P. prednisone; CY. cyclophosphamide
rosettes. 0.5 ml of a 2 x 10Yml lymphocyte suspension was mixed together with 0.5 ml of a 0.5% SRBC suspension and incubated to 37°C for 5 min, centrifuged at 5Og for 5 min and then incubated at 4°C overnight. The lymphocyte - SRBC pellet was gently resuspended by inversion of the tube and an aliquot removed and counted in a hemocytometer. At least 200 lymphocytes were counted in each duplicate preparation, and only those cells showing three or more adherent SRBC were considered as positive rosettes. Results were expressed as a percentage of rosetted mononuclear cells. B cells were identified by two techniques: (a) Surface immunoglobulin (sIg)bearing cells were determined using the technique of Preud’homme and Seligmann (16). Aliquots of the viable cells isolated above were incubated at 4°C for 30 min with FITC-labeled polyvalent anti-immunoglobulin serum (Meloy Labs). Cell suspensions were washed three times with 5% bovine serum albumin in phosphatebuffered saline. Duplicate wet mount slides were prepared and sealed with nail enamel. A minimum of 200 lymphocytes per slide were counted, using a vertical illuminator-equipped Leitz Orthoplan fluorescence microscope. Results were expressed as a percentage of tagged cells of the total lymphocyte population. (b) Complement receptor lymphocytes were determined using the technique of Bianco et al. (17). Sheep erythrocytes (E) were sensitized with rabbit anti-sheep erythrocyte IgM (A) and then treated with a 1: 10 dilution of mouse complement (C). Then 0.5 ml of a 2 x 10’Vml suspension of lymphocytes and 0.5 ml of a 0.5 suspension of sensitized sheep cells (EAC) were mixed together and centrifuged at 50g for 5 min. This mixture was incubated at 37°C for 30 min, gently resuspended and counted in a hemocytometer. At least 200 lymphocytes were counted, and only those lymphocytes showing three or more adherent EAC cells were scored as positive. Results were expressed as a percentage of EAC-positive cells.
LYMPHOCYTE
SUBPOPULATIONS
133
IN MCNS
TABLE 2 LYMPHOCYTE SUBPOPULATION IN MCNS” MCNS Mean
Controls (SEM)
Mean
(SEM)
P NS
Total WBC count
7486
(853)
6548
(514)
Percentage lymphocytes Absolute lymphocyte count
35.8 2583
(3.31 (333)
48.6 3135
(5.5) (399)
Percentage T cells Absolute percentage cells
71.3 1813
W) (222)
67.5 2090
(2.2) (255)
NS NS
Percentage B cells (sIg) Absolute B cells (slg)
21.5 565
(99)
16.2 523
( 1.0) (71)
NS NS
Percentage B cells (EAC) Absolute B cells (EAC)
33.4 908
14.8 442
11.3)
(1873
(6%
IMMUNOLOGY
AND
Lymphocyte
IMMUNOPATHOLOGY
14, 130-
136 (1979)
Subpopulations in Minimal Nephrotic Syndrome
Change
HOWARD 0. KERPEN, J. GANESH BHAT,’ ROBERT KANTOR. BERNARD GAUTHIER, KANTI R. RAI, ROBERT G. SCHACHT, AND DAVID S. BALDWIN
Received
January
24. 1979
Minimal change nephrotic syndrome (MCNS) was characterized by a relative lymphocytopenia, a normal T-cell count, and a discordance between B cells enumerated by the presence of surface immunoglobulins (Bsig) and by C3B receptors (Beact. By either technique of B-cell count, the total number of T and B lymphocytes in MCNS exceeded that of controls. suggesting the presence of “double-marked” cells or decreased null cell population. This alteration in surface receptor characteristics of lymphocytes which was observed both in relapse and in remission. documents an abnormality, in MCNS. possibly of cell-mediated immunity, which may be relevant to its pathogenesis.
INTRODUCTION
Minimal change nephrotic syndrome (MCNS) is a glomerular disease of unknown etiology and pathogenesis. Although usually controlled by corticosteroids, patients frequently relapse, requiring long-term use of steroids or cytotoxic drugs ( 1, 2) with potential for adverse side effects. Various lines of evidence suggest lymphocyte involvement in the pathogenesis of MCNS. The most significant evidence is the consistent induction of remission of the nephrotic syndrome following immunosuppressive agents (corticosteroids and cytotoxic drugs). This response, despite the absence of demonstrable immune deposits or complement in the glomerulus, may imply an alteration in lymphocyte function in MCNS. Additional evidence includes remissions induced by measles (3), where cell-mediated immunity (CMI) is known to be depressed. Depressed serum IgG, IgA, and elevated serum IgM levels are also seen in patients with MCNS (4). Other immune-related phenomena include increased serum IgE (5). increased susceptibility to infection (6), occurrence of lymphocytotoxic antibodies (7), circulating blastogenesis-inhibiting factor in the serum (S), and the possible production of a vascular permeability enhancing factor by lymphocytes (9). Also. CM1 directed against the kidney may be inferred by the demonstration in migra. tion inhibition experiments (10) that human lymphocytes from patients with MCNS are or have been sensitized to fetal kidney antigens. In addition, nephrotic syndrome with glomerular pathology indistinguishable from MCNS occurs in ’ Present address: Institute for Geriatric 410 Lakeville Road.
Department of Medicine, Long Island Jewish Hillside Medical Care, New Hyde Park. N.Y. To whom requests for reprints New Hyde Park. N.Y. 11040. 130
0090-1229/79/090130-07$01.00/O Copyright Q 1979 by Academic Press. Inc. All rights of reproducfmn in any form reserved.
Center and Jewish may be addressed:
LYMPHOCYTE
SUBPOPULATIONS
IN
MCNS
131
some cases of Hodgkin’s lymphoma. The activity and remission of the glomerular lesion parallels the status of the disease (11 - 13). It has been hypothesized that MCNS is caused by a functional abnormality of lymphocytes arising either de ~OVO or due to specific antigenic stimulus. Abnormal production of a lymphokine with specific effects on vascular permeability, particularly of the glomerular basement membrane, could be a pathogenetic mechanism. In a disease which is characterized by a remarkably consistent response to steroids and cytotoxic drugs, some altered parameter of cellular and/or humoral immunity should be demonstrable which correlates with disease activity and remission. We have studied the major subpopulations of peripheral blood lymphocytes, with respect to numbers and cell surface marker characteristics, in 16 patients with MCNS, 14 healthy age- and sex-matched controls, and 3 patients with proteinuria due to causes other than MCNS. We have found a population of lymphocytes in MCNS that bear receptors for both T and B cells, i.e., doublemarked cells. The possible significance of these findings are discussed below. MATERIALS
AND METHODS
Observations were made in three groups of individuals. Sixteen patients who were known to us from the onset of nephrotic syndrome were studied. The diagnosis of MCNS was made initially on the basis of typical features of the syndrome: massive proteinuria, unremarkable urinary sediment, absence of azotemia or hypertension, and response to either corticosteroids or cyclophosphamide. In three patients, the diagnosis was confirmed by percutaneous renal biopsy. The ages of the entire group ranged from 3 to 26 years. At the time of study, six patients were in relapse as indicated by the presence of nephrotic syndrome and ten were in clinical remission (Table 1). Seven of the ten patients without proteinuria were receiving no medications at the time of study, although they had been treated either with prednisone or cyclophosphamide in the past. Fourteen healthy children, eight boys and six girls, varying in age from 5 to 13 years (mean age 10 years), with no evidence of proteinuria, formed the control group. In addition, three male patients and one female with proteinuria not due to MCNS were also studied. Their ages ranged from 8 to 16 years. Three of these had focal sclerosis on renal biopsy and the fourth had orthostatic proteinuria. Collection of blood and separation of mononuclear cells. Blood (20 ml) was collected in sterile disposable syringes with 10 units heparin/ml. Lymphocytes were isolated using density gradient centrifugation (14). The blood was diluted with an equal volume of calcium and magnesium-free Hanks’ balanced salt solution (HBSS) (GIBCO). Of this diluted peripheral blood, 4 ml was layered over 3 ml of Ficoll-Hypaque and centrifuged at 400g for 20 min at room temperature. The mononuclear cells at the plasma- Ficoll- Hypaque interface were aspirated aseptically and washed three times with HBSS. The cells were resuspended to a final concentration of 2 x lo6 mononuclear cells/ml. The resultant cell suspension usually contained greater than 95% lymphocytes, of which at least 90% were viable when tested with trypan blue dye exclusion technique. Contamination with monocytes was determined by latex particle phagocytosis. Aliquots of this total mononuclear cell population were used for T- and B-cell determinations. Cell surface marker studies. T cells were determined by a modification of the technique of Wybran and Fudenberg (15) using sheep red blood cell (SRBC)
132
KERPEN ET AL. TABLE CLINICAL
No.
Age
Sex
1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16
3 12 11 26 12 13 12 11 4 7 5 8 I5 20 II 5
F F M M M M M M M M M F M M M M
Proteinuria
FEA-KJRES
Serum albumin
+ i + + + + -
I
OF 16 PATIENTS
Serum cholesterol
WITH
MCNS” Therapy at time of study P P
i i 1 I
1
N N N N N N N N N N
N N N N N N N N N N
Past therapy -P. 1977 CY. 1972 .-
CY. P CY P -.
-
P. P, P. P. P. CY. CY. P.
t’ 1972 1976 1972 1970 1972 IV77 1977 1977
o P. prednisone; CY. cyclophosphamide
rosettes. 0.5 ml of a 2 x 10Yml lymphocyte suspension was mixed together with 0.5 ml of a 0.5% SRBC suspension and incubated to 37°C for 5 min, centrifuged at 5Og for 5 min and then incubated at 4°C overnight. The lymphocyte - SRBC pellet was gently resuspended by inversion of the tube and an aliquot removed and counted in a hemocytometer. At least 200 lymphocytes were counted in each duplicate preparation, and only those cells showing three or more adherent SRBC were considered as positive rosettes. Results were expressed as a percentage of rosetted mononuclear cells. B cells were identified by two techniques: (a) Surface immunoglobulin (sIg)bearing cells were determined using the technique of Preud’homme and Seligmann (16). Aliquots of the viable cells isolated above were incubated at 4°C for 30 min with FITC-labeled polyvalent anti-immunoglobulin serum (Meloy Labs). Cell suspensions were washed three times with 5% bovine serum albumin in phosphatebuffered saline. Duplicate wet mount slides were prepared and sealed with nail enamel. A minimum of 200 lymphocytes per slide were counted, using a vertical illuminator-equipped Leitz Orthoplan fluorescence microscope. Results were expressed as a percentage of tagged cells of the total lymphocyte population. (b) Complement receptor lymphocytes were determined using the technique of Bianco et al. (17). Sheep erythrocytes (E) were sensitized with rabbit anti-sheep erythrocyte IgM (A) and then treated with a 1: 10 dilution of mouse complement (C). Then 0.5 ml of a 2 x 10’Vml suspension of lymphocytes and 0.5 ml of a 0.5 suspension of sensitized sheep cells (EAC) were mixed together and centrifuged at 50g for 5 min. This mixture was incubated at 37°C for 30 min, gently resuspended and counted in a hemocytometer. At least 200 lymphocytes were counted, and only those lymphocytes showing three or more adherent EAC cells were scored as positive. Results were expressed as a percentage of EAC-positive cells.
LYMPHOCYTE
SUBPOPULATIONS
133
IN MCNS
TABLE 2 LYMPHOCYTE SUBPOPULATION IN MCNS” MCNS Mean
Controls (SEM)
Mean
(SEM)
P NS
Total WBC count
7486
(853)
6548
(514)
Percentage lymphocytes Absolute lymphocyte count
35.8 2583
(3.31 (333)
48.6 3135
(5.5) (399)
Percentage T cells Absolute percentage cells
71.3 1813
W) (222)
67.5 2090
(2.2) (255)
NS NS
Percentage B cells (sIg) Absolute B cells (slg)
21.5 565
(99)
16.2 523
( 1.0) (71)
NS NS
Percentage B cells (EAC) Absolute B cells (EAC)
33.4 908
14.8 442
11.3)
(1873
(6%
