Immunoregulatory aberrations in patients with polyarticular juvenile rheumatoid arthritis

3LINICAL

IMMUNOLOGY

AND

Immunoregulatory

IMMVNOPATHOLOGY

47,

62-14 (1988)

Aberrations in Patients with Polyarticular Juvenile Rheumatoid Arthritis

GEORGE C. TSOKOS,*+ GIORGIO INGHIRAMI,” STANLEY R. PILLEMER,” ANTIGONE MAVRIDIS,~ AND DANIEL B. MAGILAVY’ *Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Discuses. National Institutes of Health, Bethesda, Maryland 20892: iUniformed Services University qf the Heafth Sciences, Bethesda, Maryland 20892; #Children’s Hospital National Medical Center. Washington, D.C. 20010; and 5Division of Rheumatology. LaRabidu Institute. University qf Chicago. Chicago, Illinois 60637 The presence of hypergammaglobulinemia and various circulating autoantibodies in children with polyarticular juvenile rheumatoid arthritis (JRA) implies an immunoregulatory disorder. We report here experiments planned to elucidate the underlying cellular aberrations in this disease. Twelve children with polyarticular JRA were studied. Percentages of Leu-1, Leu-2, and Leu 3 T cells were comparable to those of normal individuals. Immunofluorescent double staining studies demonstrated elevated numbers of activated (DR+) T cells of both Leu-2 and Leu-3 phenotype. B cells characterized both phenotypically (Leu-12) and functionally (as spontaneous plaque-forming cells. PFC) were elevated. In vitro PFC responses to pokeweed mitogen (PWM) and Epstein-Barr virus (EBV) were diminished. The levels of concanavalin A-induced suppressor cells of the PWM-stimulated PFC responses were comparable to control values. In contrast, the EBV-associated suppressor T cells were significantly impaired in both EBV-seropositive and EBV-seronegative patients. These studies indicate that peripheral blood B-cell activity is abnormal in polyarticular JRA. Defective T-cell responses in vitro suggest that this may be due to disruption of normal regulatory circuits between B and T cells and may contribute to the pathogenesis of this disease. B 1988 Academic Press. Inc.

INTRODUCTION The presence of hypergammaglobulinemia, circulating immune complexes, antinuclear antibodies (ANA), rheumatoid factor, and other autoantibodies in patients with juvenile rheumatoid arthritis (JRA) is suggestive of an autoimmune process (1-15); furthermore, the association with antigens of the major human histocompatibility complex indicates that the disease might have an immunogenetic basis (16-21). The status of the cellular immune system at both the immunoregulatory and the effector cell level have been only partially studied. Limited reports have indicated that the proliferative responses of the peripheral mononuclear cells (MNC) to mitogens are compromised (22-24); regarding the B-cell function, it has been reported that the in vitro plaque-forming cell (PFC) responses in response to pokeweed mitogen (PWM) are deficient (25) while anti-8 antibodies enhance the staphylococcal protein A-induced B-cell colony formation (26). JRA represents a heterogeneous group of diseases which have been categorized on the basis of clinical manifestations into polyarticular, pauciarticular, and sys’ To whom all correspondence and reprint requests should be addressed at National Institutes of -Iealth, Building 10, Room 3N114, Bethesda, MD 20892. 62 OO90-1229/88 $1.50 Copyright 8 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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temic subsets. This feature of this disease(s) should be addressed in any investigation of its pathogenesis. In the present article, we report the studies of the cellular immune response in 12 patients with polyarticular JRA. Increased levels of serum immunoglobulin were associated with increased numbers of circulating B cells, increased numbers of cells spontaneously secreting IgG and IgM, and increased numbers of activated T cells. Peripheral MNC showed a decreased response to PWM in vitro. At the immunoregulatory cell level, concanavalin A (Con A)-induced suppressor cells were at levels comparable to those of normal individuals, while Epstein-Barr virus (EBV) failed to induce suppressor T cells in patients who had circulating anti-EBV antibodies. These results indicate that increased numbers of activated B cells are responsible for the overproduction of autoantibodies; activated T cells and/or deficient suppressor cells are probably responsible for this B-cell overactivity. MATERIALS

AND METHODS

Patient population. Twelve patients who had established diagnosis of polyarticular JRA (27) and were followed at the Rheumatology Clinic at the Children’s Hospital National Medical Center, Washington, D.C., were included in this study. All had a history of involvement of greater than five joints and had no manifestations of systemic disease (i.e., quotidian fever, rash, serositis, lymphadenopathy, or hepatosplenomegaly). None of the patients had rheumatoid nodules. Peripheral blood (5-10 ml) was drawn aseptically in heparinized syringes after obtaining an informed consent from the parent. A summary of the clinical and serological features of the studied patients is shown in Table 1. Eight of the patients were girls. The mean (‘t SEM) age of the patients was 10.1 +- 1.3; the range was 2.2-15 years. An arbitrary scale for the disease activity ranging from 0 (inactive) to 3 (very active) was constructed and each patient was given a rank by two physicians. One patient was on prednisone treatment (5 mg/day); in this patient blood was drawn 24 hr after the last dose of prednisone. Three patients had been on gold treatment (2-5 years) and two more were receiving D-penicillamine at the time of the study (250 mg per day for 1 and 2 years, respectively). All patients were on nonsteroidal anti-inflammatory medications. Normal healthy individuals aged 15 to 30 years old served as controls in this study. MNC isolation. Blood specimens were centrifuged at 400g for 10 min and the plasma was kept frozen (- 20°C) for the serological studies. The cellular sediment was diluted l/4 with phosphate-buffered saline (PBS); MNC were obtained by standard Ficoll-Hypaque (specific gravity 1.070 g/ml) gradient centrifugation. Serological studies. Fluorescent ANA (Hep-2 cells were used as substrate), rheumatoid factors, serum immunoglobulins (IgA, IgG, and IgM), and Clq binding assays for the detection of circulating immune complexes were performed by the Nichols Institute (San Juan Capistrano, CA) using standard techniques. EBV antibody titers were kindly performed by Dr. Werner Henle (Children’s Hospital, Philadelphia, PA) according to standard techniques. Staining of MNC. MNC (2 x 105) were suspended in 100 ~1 of Hanks’ balanced salt solution (HBSS) containing 1% bovine serum albumin and 0.1% NaN, (sorter medium), and 1 p,g of phycoerythrin (PE)-conjugated anti-DR antibody and 1 (*g

64

TSOKOS ET AL. TABLE I CLINICAL AND SEROLOGICAL FEATURES OF PATIENTS WITH POLYARTICULAR JRA Patients with polyarticular

JRA

Feature

RF negative

RF positive

Number Age during study (mean years 2 SEMI (Range) Disease duration (mean years t SEM) Sex (male/female) ratio Activity index (scale O-3) mean t SEM ESR (Westergren, mm/hr) Treatment status (number of patients) D-Penicillamine Gold Prednisone ANA (83% positive)” Serum DNA binding Serum IgG Serum IgA Serum IgM

9 10.1 t 1.3 (2.2-15) 4.0 + 1.2 2/l 2.43 t 0.48 55 -+ 4

3 7.1 2 2.8 (2.5-12.5) 2.7 t 0.9 211 2.3 i 0.3 60.2 I 3.0

2 3 I 317 ” 5.1 0% 1674 2 137h 160 t 19 162 +- 24

0 0 0 38 ? 4.0 0% 1953 _t 455 205 2 32 235 2 75

u Hep-2 cells were used as substrate. All positive sera showed speckled pattern of staining with the exception of sera from five patients with polyarticular JRA in whom homogeneous and speckled patterns were observed simultaneously. Geometric mean was calculated for the positive (titer > 1120) sera. b Normal range for IgG is 639-1349 mg/dl: for IgA, 70-312 mg/dl; and for IgM, 56-352 mgidl.

of fluorescein isothiocyanate (FITC)-conjugated Leu-I , Leu-2. Leu-3, or Leu-4 monoclonal antibody (Becton-Dickinson, Mountain View, CA) were added, incubated at 0°C for 30 min, and washed twice with cold sorter medium before analysis. IgG-PE and IgG-FITC served as controls. When Leu-7 or Leu- 12 were used, only single staining was performed; they were developed by using goat anti-mouse F(ab);-FITC conjugated (0°C for 30 min). Cytojluorometry. Stained cells were anaIyzed by using a FACS IV flow microfluorometer interfaced with a CONSORT-40 computer and equipped with argon laser; 64 by 64 deurent matrices were used to characterize cell-associated fluorescence. Data were collected by using logarithmic amplification of each fluorescent signal. Fractionation of MNC. MNC were fractionated into sheep red blood cell (SRBC) rosette-forming (Et) and non-SRBC rosette-forming (E-) cells by the use of SRBC pretreated with 2-aminoethylisothiouronium bromide hydrobromide (AET; Sigma, St. Louis, MO). SRBC were incubated for 15 min at 37°C with 0.15 M AET (PI-I 9.0) and then washed six times with normal saline and resuspended in PBS containing 10% of fetal calf serum FCS; GIBCO, Grand Island, NY) at a concentration of 4%. Equal volumes of SRBG and MNC (10 X 106 ml) suspensions were incubated for 10 min at room temperature, spun down (2OOg for 5 min), further incubated at 4°C for 30 min, layered over Ficoll-Hypaque cushions. and spun for 25 min at 400g. The interface containing the E- cells and the pellet

IMMUNOREGULATION

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6.5

containing the E+ cells were collected in different tubes. The Et cells were incubated for 10 min at room temperature with 10 ml of ACK lysing buffer (NIH, Media Unit) and then washed two times with RPM1 1640. E- cells were rerosetted on most occasions, and the percentage of E+ cells in final preparations was always less than 1%. E+ and E- cells were also stained (indirect immunofluorescence) with Leu-1 monoclonal antibody to check for purity (Becton-Dickinson). Approximately 95% of the E’ cells stained with this antibody, whereas E- cells contained less than 2% positive cells. In vitro culture techniques. Cultures were carried out in 24-well flat-bottomed plates (Costar, Cambridge, MA); each well contained 2 ml of MNC or E- cells suspension (0.5 x lo6 ml) in RPM1 1640 with 50 pg/m.l of streptomycin, 50 U/ml of penicillin (NIH, Media Unit) and 10% heat-inactivated FCS. PWM (GIBCO) was used at a final concentration 1% of the stock solution. EBV-containing supernatants of a lymphoblastoid cell line (B95-8) infected with EBV were used at a concentration 5% of the final culture volume, which provided optimal responses for PFC. Cultures were harvested and assayed on Day 7 or 14. In some experiments Con A (Sigma) was added at the initiation of the cultures at a concentration IO pg/ml in order to test the generation of suppressor cells. Plaque assay. B-cell responses were estimated by the use of a previously described reverse hemolytic plaque assay slightly modified in our laboratory (28). SeaPlaque agarose (FMC Corp., Rockland, ME) was dissolved (0.8%) in RPM1 1640 containing 2 m&Z Hepes buffer (Microbiological Associates, Walkersville, MD) by heating to boiling, then left to equilibrate at 42°C in a water bath. Agarose, 0.85 ml, was mixed with 0.05 ml of Stuphylococcus protein A, coupled to SRBC by the use of chromic chloride, and with 0.1 ml of MNC to be tested. The mixture was vortexed and poured into 15 x 66-mm petri dishes (Falcon, Oxnard, CA) prelayered with 1.2% SeaKem agarose (FMC Corp.) in PBS. After the gel had solidified, 1 ml of developing antiserum was added. The developing antiserum was the IgG fraction of rabbit anti-human immunoglobulins (IgA + IgG + IgM) purchased from Cappel Laboratories (Cochransville, PA), diluted l/SO in RPM1 1640. The petri dishes were incubated for 2 hr at 37”C, and then the developing antiserum was replaced with 1 ml of guinea pig complement (Cappel Laboratories) diluted l/l0 in Veronal-buffered saline containing 0.5% gelatin (NIH, Media Unit). Statistics. Geometric means are used throughout this paper to express results of PFC assays, and the antilog of the SD of the logs are given. Two-tailed t tests for equal variances were performed using the means of the log-transformed data. RESULTS

Serological Studies (Table 1). Three of the 12 patients with polyarticular type of JRA had circulating IgM rheumatoid factor. The titers were 1:1320, 1:2560, and 1: 10240; disease onset was at 21/2, 10, and 2 years of age, respectively. ANA were present in 83% of patients at titer higher than 1:20. The mean geometric titer among the positive sera was 139 + 1.6. Patients with seropositive (RF) disease had higher titers (317 5 5.1) of ANA than the seronegative group. The pattern of staining was speckled in all positive sera; in five sera a concomitant homogeneous

66

TSOKOS ET AL.

staining was detected. The mean serum IgG was elevated (more in the seropositive group), while serum IgA and IgM levels were not increased. Lymphocyte subpopulations. Lymphocyte subpopulations are readily identified by the use of murine MAb which identify cell surface membrane antigens; certain of these antigens have been associated with specific functional.abilities. Leu-1 and Leu-4 MAb identify all peripheral T lymphocytes while Leu-2 and Leu-3. the suppressor/cytotoxic and the helper/inducer subpopulations, respectively; Leu-7 recognizes a portion of the natural killer cells. The group of 12 patients with polyarticular JRA included in this study had percentages of total T lymphocytes and their subpopulations comparable to those of normal individuals (Fig. 1). In contrast, the percentage of B lymphocytes in the peripheral blood of these patients which are identified by the MAb Leu- 12 was significantly higher (P < 0.05). DR-positive circulating T lymphocytes. In order to identify the presence of DR+ cells among various T-cell subpopulations, we performed double staining studies of peripheral blood MNC using FITC-labeled Leu-1, Leu-4, Leu-2, and

I-

b 0

.

.

t

: .

1

.!I[ 0

11

. . . -

-

i .

0 .

.

i .” :

-

cv z?

. . .

r? 3 -

d ? 3 3 -

FIG. 1. MNC subpopulations in the peripheral blood of patients with polyarticular JRA. Total MNC were incubated with FITC-conjugated mouse monoclonal antibodies for 30 min at 0°C. Mouse myeloma IgG FITC conjugated served as control. Leu-7 and Leu-12 were not directly labeled and they were developed by a goat anti-mouse FITC-labeled antiserum- Solid circles represent the individual entries while horizontal bars and the vertical shaded areas indicate the mean t- SEM. The vertical bar on the right represents the mean k SEM of the normal individuals. Leu-12 + staining cells (B celts) are significantly higher in patients with JRA. Open circles represent the patients with seropositive (RF) disease.

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Leu-3 MAb-and PE-conjugated anti-human DR MAb. The percentages of Leu-l+DR+, Leu-4+DR+, Leu-2+DR+, and Leu-3+DR+ MNC were higher in the group of patients with polyarticular JRA. Due to the wide spread of the obtained measurements only the Leu-2+DR+ (P < 0.05) and the Leu-4+DR+ (P < 0.025) reach statistically significant levels (Fig. 2). These studies indicate the presence of activated T lymphocytes in the peripheral blood of patients with polyarticular JRA. The subgroup of patients with seropositive (RF) disease can not be distinguished from the rest of the group in terms of lymphocyte subpopulations . Spontaneous Zg-secreting lymphocytes. The numbers of Ig-secreting lymphocytes were enumerated in the peripheral blood of patients with JRA using a reverse hemolytic PFC assay. Total Ig-secreting cells were increased significantly (P < 0.001) in patients with JRA. The group of the three patients with seropositive (RF) disease had significantly (P < 0.001) higher numbers of spontaneously Igsecreting cells than the group of seronegative patients (Fig. 3). Mitogen-induced PFC. In order to test the ability of peripheral MNC from patients with JRA to secrete Ig in response to the polyclonal T-cell-dependent B-cell activator, we cultured peripheral MNC with PWM and 6 days later we

> RF positive B RF negativt

FIG. 2. Double staining (T and DR+) of MNC from patients with JRA. MNC were incubated with FITC-conjugated anti-human T-cell murine monoclonal antibodies (Leu-1, Leu-4, Leu-2, and Leu-3) and PE-conjugated anti-human DR antibodies for 30 min at 4”C, washed, and analyzed by the use of the cell sorter. Open circles represent the patients with seropositive (RF) disease.

68

TSOKOS ET AL. 2x103

1

1 23

PV A G POLYARTICULAR JRA

M

PV NORMAL

A

G

M

INDIVIDUALS

FIG. 3. Spontaneous Ig-secreting cells in the peripheral blood of patients with polyarticular JRA. Fresh MNC were mixed with SPA-coated SRBC in agar in petri dishes and incubated with rabbit anti-human polyvalent serum (PV) or rabbit anti-human IgA (A), IgG (G), or LgM (M) sera at 37°C before being developed with normal guinea pig serum (source of complement). Height of bars represents the geometric mean. Numbers at the top of the bars represent the standard deviation.

enumerated the PFC. Despite the fact that patients with polyarticular JR-A have increased numbers of spontaneously PFC in the peripheral blood, they have impaired PFC responses in vitro in response to PWM (Fig. 4). Patients with seropositive (RF) disease also impaired PWM-induced PFC responses. Partial depletion of monocytes in one patient with polyarticular arthritis improved the PWMinduced PFC responses in vitro (data not shown); this indicates that excessive monocytic suppression might be responsible for the observed poor responses. Inhibition of PWM-induced PFC in the presence of Con A. Addition of Con A at the initiation of cultures of human MNC with PWM abrogates the PFC responses. We added 10 pg of Con A at the beginning of cultures of MNC from patients with JRA in order to test its ability to generate suppressive signals to the B cells responding to PWM. Indeed, the presence of Con A inhibited the PFC responses in all patients by more than 85% (Fig. 4) which is comparable to that seen in normal individuals. EBV-associated immunoregulation. Serum antibodies to Epstein-Barr virusspecific antigens are acquired gradually during childhood and the majority of adults are seropositive. All normal individuals included in this study were seropositive; the mean titer of antibodies to virocapsid antigen (VCA) was 108 5 1.24 and the mean titer of antibodies to Epstein-Barr nuclear antigens (EBNA) was 29 5 1.55 (Table 2). Although the titers of antibodies to VCA in the seropositive patients tended to be higher than those in normal individuals (P < 0.05), the mean titer in the whole group of patients with polyarticular JRA was comparable to that of normal individuals. We subsequently tested the ability of EBV to generate PFC responses ifpresent in cultures of MNC in vitro. MNC were cultured with EBV and tested on Days 6 and 13. EBV-induced PFC responses tested on Day 6 of the cultures were lower

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105-J

104v c r

f 8 7 v it 103:

R

2 4

102-5 ::

a 6 u +

1 a 06 +

FIG. 4. PFC responses of peripheral MNC in viro in the presence of medium (0), PWM, or PWM plus Con A. Cells were harvested and assayed on Day 6; PFC are expressed as geometric means. Numbers at the top of the bars are the anti-log of the standard deviation of the logs of the entries.

in patients with JRA (P < 0.001). Individuals seropositive to EBV exhibited good responses on Day 6 (5040 2 1.21) and poor responses on Day 13 (1289 5 153). This late phase suppression is thought to be secondary to the generation of EBVspecific suppressor cells (29,30). Seronegative individuals fail to show this late suppression and instead the 13th day responses are significantly higher. Seropositive patients with polyarticular JRA (n = 9) failed to show the expected late suppression; instead the 13th day responses were 3.4-fold higher than those of the 6th day. In this group were included the three patients with RF-seropositive disease. Seronegative patients with polyarticular type of JRA showed the expected enhancement of the 13th day PFC next day responses (Fig. 5). MNC were subsequently separated into T and non-T subpopulations and syn-

70

TSOKOS ET AL. TABLE

ANTIBODY

RESPONSES

TO EBV

ANTIGENS

Subjects

VCA”

Polyarticular JRA

9112’ (254 ” 1.29)b 711 (108 “, 1.35)

Normal individuals

2

IN PATIENTS

WITH

JRA AND NORMAL EA (R)

EBNA

3112 (20 “, 1) 117 (25 ” 1.34)

9112 (29 “- 1.55) 611 (25 ” 1.34)

EA 0) 1112 (20) 017 .~(20)..~

INDIVIDUALS

LIVCA denotes the virocapsid antigen, EA (D) the diffuse component of the early antigen, EA (R) the restricted component of the early antigen, and EBNA the Epstein-Barr nuclear antigen. b Positive (titer >l/lO)/total number tested and the geometric mean z SEM are given.

geneic or allogeneic mixtures (T:non-T, 3:l) were cultured in the presence of EBV. PFC responses were tested again on Days 6 and 13. Three seropositive patients with polyarticular form of disease were studied and a representative experiment is presented in Table 3. T lymphocytes from patients (seropositive or seronegative) failed to suppress both normal and patient B-cell responses to EBV. In contrast normal T cells suppressed both normal and patient B-cell responses. DISCUSSION

The cause and pathogenesis of JRA is not known; infectious agent, stress, and inheritance have been considered causative factors. In the present study, we

I1 211

0 (1.231

DAY ‘02’

11 521

11.811 0

6

13 EBV Ab POS

EBV Ab NEG

POLYAPTICULAR

NORMAL INDIVIDUALS JRA

FIG. 5. PFC responses to EBV of peripheral MNC from patients with polyarticular JRA; seropositive (EBV Ab pas) or seronegative (EBV Ab neg) in reference to EBV is presented separately. Cells were harvested and assayed on Days 6 (PFC to EBV) and 13 (late suppression phenomenon). 0, Cells were cultured with EBV alone; 0, cells were cultured with~EBV plus Con A (see legend to Fig. 3).

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IN JRA

attempted to dissect the peripheral blood immunoregulatory events which are responsible for the serological manifestations in patients with active polyarticular JRA. The frequencies of rheumatoid factor (25%), ANA (83%), and immune complexes (80%, data not presented) in this group of patients are within the ranges reported by others (4,6,12,13). Up to a quarter of patients with JRA and primarily those with the polyarticular form have 19 S IgM rheumatoid factor (1,7); more than half of the patients though have the so-called “hidden” IgM rheumatoid factor which is identified by separation of the serum immunoglobulins by acid-gel filtration (7,13). The frequency of ANA in the sera of patients with JRA, detected by immunofluorescence, has been reported in variable percentages ranging from less than 10% to more than 80% of the patients. The increased frequency of ANA positivity in our group of patients is apparently due to the usage of Hep-2 cells as substrate. The pattern of the immunofluorescent staining is usually speckled and sometimes homogeneous (4,12). A distinct class of autoantibodies seems to be that of anti-lymphocytic antibodies. These antibodies bind specifically to suppressor T-cell subpopulations or the subpopulations responsible for the induction of suppressor cells (9,11,15,3 1,32). Although the existence of such autoantibodies has been disputed by other investigators who were unable to identify them in sera devoid of immune complexes (33) their presence in the sera of some of the patients might explain some of the observed T-cell abnormalities. In analyzing the peripheral MNC from patients with polyarticular JRA, we found that the mean percentage of B cells was significantly elevated, while the mean percentages of T lymphocytes and their subpopulations were comparable to those of normal individuals. The variance of the group of the patients is quite large indicating that certain patients might have decreased numbers of B cells, while others might have abnormal numbers of T cells or their subsets. The possibility of existence of subgroups of patients with lymphocyte subpopulation aberrations exists and it can be formally be addressed by studying larger numbers of patients. This study provides new evidence on the presence of activated T cells in the peripheral blood of patients with JRA, detected by the expression of DR antigens on their surfaces. Mitogenic or antigenic stimulation of human T lymphocytes TABLE 3 PFC RESPONSES OF T- AND B-CELL

MIXTURES

Cell mixture”

TO EBV Day of harvestb

B

T

6

13

P N P P N N

P N P N

2.500 1250 3ooo 2900 1100 4800

5100 1200 4!900 1250 6400 100

a Cell mixture. at B:T ratio 1:3. b Data from one out of three studied patients with polyarticular

arthritis (all seropositive).

72

TSOKOS

ET

AL.

leads to the expression of DR antigens (34,3.5), transferrin (36), insulin (37), and interleukin 2 receptors (38,39), which are known as activation markers. Activated T cells have been described in patients with rheumatoid arthritis (40,41) and SLE (42-45) and they have been considered as playing a pathogenic role. We have been unable to detect any associations between the numbers of the T cells and/or their subpopulations and any of the lymphocyte functional studies; the small number of patients might be responsible for this. Increased levels of serum Ig has been associated with increased numbers of spontaneously Ig-secreting B cells in several diseases including SLE (28), RA (46), and primary billiary cirrhosis (47). Our group of patients with polyarticular JRA exhibited increased levels of serum Ig, increased numbers of phenotypically (Leu-12+) characterized B cells, and increased numbers of immunoglobulinsecreting cells detected by a reverse hemolytic PFC. Total PFC as well as IgG and IgM PFC were elevated in this group of patients. Spontaneous PFC have been reported increased earlier (25). In order to dissect the status of the immunoregulation among the peripheral MNC in JRA, we examined (a) the MNC PFC responses to PWM, (b) the PFC responses of MNC and B cells of EBV, (c) the ability of Con A to stimulate the production of suppressor cells, and (d) the induction of suppressor T cells in vitro by EBV. PWM is a T-cell-dependent B-cell mitogen; the presence of excessive suppressor T cells or monocytes results in a diminished B-cell response. Patients with RA (46) and SLE (48) have poor PFC responses to PWM in vitro and this is believed to be partially due to excessive monocytic suppression. Patients with JRA have diminished PFC responses to PWM (Fig. 4). Partial depletion of monocytes resulted in significantly improved PFC responses. The PFC responses of MNC or T-cell-depleted cells to EBV were significantly impaired in our group of patients. EBV is a T-cell and monocyte independent B-cell stimulus (29.30) and, hence, it can be postulated that B cells from patients with JRA might have an intrinsic B-cell defect. PFC responses to EBV in vitro have been shown to be normal in patients both with RA (29) and with SLE (30). JRA B cells have been claimed to exhibit specific, not shared with other autoimmune diseases, B-cell abnormalities (26). Con A-induced suppressor cells in vitro from JRA MNC were normal. The presence of Con A in cells cultured along with PWM or EBV markedly suppressed the responses to the latter. Con A is a potent T-cell stimulus and generates normal levels of suppressor T cells in the presence of subtle suppressor cell deficits (49). In contrast, EBV-associated suppressor T cells were deficient in our group of patients. These findings indicate that patients with active polyarticular JRA exhibit cellular abnormalities similar to those of patients with adult onset RA (50) SLE (30) and primary biliary cirrhosis (47) and that JRA is another disease in which EBV-associated immunoregulation is disturbed. Furthermore, we found no differences (a) between the patients who had received immunomodulatory drug treatment (gold, D-penicillamine, and prednisone) and (b) those who were IgM rheumatoid factor positive and those who were not. A major criticism of our study can be the fact that we have used as controls individuals older than the patients; thus our data should be interpreted cautiously.

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B-cell numbers tend to be increased in younger children, while the PWM-induced Ig synthesis is reduced (51,52). T-cell subsets seem also to vary with age (53). The present studies strongly indicate that the presence of circulating autoantibodies in patients with active polyarticular JRA is associated with a number of cellular immunoregulatory abnormalities. Further experimentation will be necessary in order to better characterize these aberrations and identify the pathogenetic links between them. ACKNOWLEDGMENTS The authors are grateful to Dr. Werner Henle of the University of Pennsylvania for performing the anti-EBV serological studies, Dr. Patience White of the George Washington University for allowing us to study some of her patients, Dr. Ian T. Magrath of the National Cancer Institute for valuable discussions and guidance, and Linda Adams for excellent secretarial assistance.

REFERENCES 1. 2. 3. 4.

Cassidy, J. T., and Valkenberg, H. A., Arthritis Rheum. 10, 83, 1967. Torrigiani, G., Ansell, B. M., Chown, E. E. A., Roitt, I. M., Ann. Rheum. Dis. 28, 424, 1969. Bell, C., Talal, N., and Schur, P. H. Arthritis Rheum. 18, 535, 1975. Schaller, J. G., Johnson, E. J., Holborrow, 9. M., Ansell, B. M., and Smiley, W. K., Arthritis Rheum. 20, 1485, 1977. 5. Rossen, R. D.. Brewer, D. A., Person, J. W., Templeton, J. W., and Lipsky, M. D., Arthritis Rheum. 20, 1485, 1977. 6. Petty, R. E., Cassidy. J. T., and Sullivan, D. 9.. Arthritis Rheum. (Suppl.) 20, 260, 1977. 7. Moore, T. L., Dorner, R. W., Weiss, T. D.. Baldassare, A. R., and Zuckner, J., Pediatr. Res. 14, 1135, 1980.

8. Miller, J. J., III, Osborne, C. L., and Hse, Y.-P., J. Rheumatol. 7, 665, 1980. 9. Morimoto, C. E., Reinherz, E. L., Borel, Y., Mantzouranis, E., Steinberg, A. D., and Schlossman, S. R., J. Clin. Invest. 67, 753, 1981. 10. Moore, T. L., Sheridan, P. W., Traycoff, R. B., Zuckner, J., and Domer, R. W.. J. Rheumatol. 7, 395, 1982. Il. Morimoto, C.. Reinherz, E. L., Borel, Y., and Schlossman, S. F., J. Immunol. 130, 157, 1983. 12. Osborn, T. G., Patel, N. J., Moore, T. L., and Zuckner, J., Arthritis Rheum. 27, 1286, 1984. 13. Moore, T. L., Osborn, T. G., Weiss, T. D., Sheridan, P. W., Eisen-Winter, R. K., Miller, A. V., Domer. R. W., and Zuckner, J., Semin. Arthrifis Rheum. 13, 329, 1984. 14. Borel, Y. U.. Morimoto, C., Cairns, L., Mantzouranis, E., Strelkauskas, A. J., and Schlossman. S. F., J. Rheumatol. 11, 56, 1984. 15. Borel, Y., Morimoto, C., Cams, L., Marcus, and Shearer, W. R., J. Rheumntol. 11, 56, 1984. 16. Stastny, P., and Fink, C. W., J. Clin. Invest. 64, 124, 1979. 17. Stastny, P., and Fink, C. W., J. Clin. Invest. 63, 124, 1979. 18. Glass, D., Litvin, D. A., Wallace, K., Chylack. L., Garovoy, M., Carpenter, C. B,, and Schur, P. H., J. Clin. Invest. 66, 426, 1980. 19. Glass, D., and Litvin, D. A., Arthritis Rheum. 23, 296, 1980. 20. Forre, O., Dobloug, J. H., Hoeraal, H. M.. and Thorsby, E., Arthritis Rheum. 26, 35, 1983. 21. Clemens, L. E., Albert, E., and Ansell, B. M., Ann. Rheum. Dis. 42, 431, 1983. 22. HoyeraaI, M. S., Forland, S., and Wisloff, F., Stand. J. Immunol. 4, 801, 1975. 23. Miller, J. J., III, and Olds-Arroyo, L., J. Rheumatol. 8, 716, 1981. 24. Fort-e, O., Egeland, T., Dobloug, J. H., Kvien, T. K., and Natvig, J. B., Stand. J. Immunol. 16, 173, 1982.

25. Oen, K., J. Rheumatol. 12, 728, 1985. 26. Muraguchi, A., Kishimoto, A. T., Kurinati, Y., and Yamanura, Y., J. Zmmunol. 125, 2638, 1980. 27. Brewer, E. J., Bass, J. C., Cassidy, J. T., Duran, 9. X., Fink, C. W., Jacobs, J. E., Markowitz,

74

28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 43. 44. 45.

TSOKOS ET AL. M., Reynold, W. E., Schaller, J., Stillman, J. S., and Wallace, S. T., BUN. Rheum. Dis. 23, 712, 1973. Tsokos, G. C., and Balow, J. E., Clin. Immunol. Immunopathol. 21, 172, 1981. Tosato, G., Steinberg, A. D., and Blaese, M. K., N. Engl. J. Med. 305, 1238, 1981. Tsokos, G. C., Magrath, I. T., and Balow, J. E., J. Immunol. 181, 1797, 1983. Strelkauskas, A. J., Schauf, V., Wilson, B. S., et al.. .I. Immunol. 120, 1278, 1978. Strelkauskas, A. J., Callehy, R. T., McKowell, J., Bore], Y., and Schlossman, S. F.. Proc. Nurl. Acad. Sci. USA 75, 5150, 1978. Froelich, C. J., Bankhurst, A. D., Crown, W. E., Williams, R. C., Jr., Warner, M. L., and Levinson, J. E., Arthritis Rheum. 24, 457, 1981. Reinherz, E. L., Kung, P. C., Pesando, J. M., Ritz, J., Goldlstein, G., and Schlossman, S. F., J. Exp. Med. 150, 1472, 1979. Yu, D. T. Y., McCune, J. M., Fu, S. M., Winchester, R. J., and Kunkel, H. G., J. Exp. Med. 152, 89s 1980. Trowbridge, I. S., and Omary, M. B., Proc. Natl. Acad. Sci. USA 78, 3039, 198i. Helderman, J. H., and Strom, T. B., J. Biol. Chem. 254, 7203, 1979. Uchiyama, T. S., Broder, S., and Waldmann, T. A., J. Zmmunol. 126, 1393, 1981. Robb, R. J., and Greene, W. C., J. Exp. Med. 158, 1332, 1983. Pincus, S. H., Clegg, D. O., and Ward, J. R., Arthritis Rheum. 28, 8, 1985. Haraoui, B., Wilder, R. L.. Malone, D. G., Allen. J. B., Katona, I. M., and Wahl, S. M., J. Immunol. 133, 697, 1984. Yu, D. T. Y., Winchester, R. J., Fu, S. M., Gibofsky, A., Ko, H. S., and Kunkel, H. G.. J. Exp. Med. 151, 91, 1980. Okudaira, K., Seales, R. P., Ceuppens, J. L., Goodwin, J. S., and Willisma, R. C., J. C/in. Invest. 69, 17, 1982. Okudaira, K., Seales, R. P., Ceuppens, J. L., Goodwin, J. S., and Willisma, R. C., J. C/in. Invest. 69, 17, 1982. Inghirami, G., Balow, J. E., and Tsokos, G. C., submitted for publication. Linker-Istraeli, M., Bakke, A. C., Quismorio, F. P., Jr., and Horowitz, D. A., J. C&z. Invest. 75, 762, 1985.

46.

47. 48. 49. 50.

Decker, J. L., Malone, D. G., Haraoui, B., Wahl, S. M., Schrieber, L., Klippel, J. I-I., !Qeinberg. A. D., and Wilder, R. L., Ann. Intern. Med. 101, 310, 1984. James, S. P., Jones, E. A., Hoofnagle, J. H., and Strober, W., J. Clin. Immunol. 5, 254. 1985. Tsokos, G. C., and Balow, J. E., Prog. Allergy 35, 93, 1984. Tsokos, G. C.. and Balow, J. E., J. Clin. Lab. Immunoi. 8, 83, 1982. Kahan, A., Kahan, A.. Amor, B., and Menkes, C. J., Arthritis Rheum. 28, %l, 1985.

Received May 11, 1987; accepted with revision October 29, 1987