Interleukin-2 receptor expression in peripheral blood lymphocytes from systemic lupus erythematosus patients: Relationship to clinical activity

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY 47, 354--362 (1988)

Interleukin-2 Receptor Expression in Peripheral Blood Lymphocytes from Systemic Lupus Erythematosus Patients: Relationship to Clinical Activity 1 DELBERT R. W I G F A L L , REBECCA S. SAKAI, D A N I E L J. W A L L A C E , AND STANLEY C . JORDAN

Division of Pediatric Nephrology, Renal Immunology Laboratory, and Division of Rheumatology, Department of Internal Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048 Deficient intedeukin-2 (IL-2) production and other T-cell dysfunctions have been demonstrated in active systemic lupus erythematosus (SLE). The generation of IL-2 receptors is known to be important to the growth and differentiation of T and B lymphocytes. This study investigated IL-2 receptor expression in peripheral blood lymphocytes (PBL) from patients with active and inactive SLE. PBL from 27 SLE patients, diagnosed by revised ARA criteria, were assayed for IL-2 receptor expression, IL-2 and immunoglobulin (Ig) production. PBL from SLE patients with active disease spontaneously expressed increased numbers of IL-2 receptors compared to those with inactive disease (P < 0.01) and normal donors (P < 0.01). There was no significant increase in IL-2 receptors expression in PBL from active SLE patients in response to mitogenic stimulation with PHA compared to inactive SLE patients and normal donors. There was negligible IL-2 production in response to mitogenic stimulation and increased spontaneous IgG production by PBL from active SLE patients compared to normal donors (P < 0.001). Purified B cells isolated from active SLE patients showed significant spontaneous IL-2 receptor expression when compared to spontaneous IL-2 receptor expression by normal B cells (P = 0.005). Therefore, in addition to derangements in Ig and IL-2 production, the level of spontaneous expression of IL-2 receptors may represent a cellular indicator of disease activity, and hence, may he a useful parameter in monitoring disease activity in SLE patients. The significance of the increased IL-2 receptor expression on B cells of active SLE patients is unknown, but may represent a marker of polyclonal activation of these cells. © 1988AcademicPress, Inc.

INTRODUCTION

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by hypersecretion of antibodies including a number of autoantibodies commonly associated with polyclonal lymphocyte activation and altered immunoregulation (1). Defective production of interleukin-2 (IL-2) is thought to further contribute to the observed alterations by resultant attenuation of normal T lymphocyte proliferation and differentiation (2). IL-2 is produced as a result of activation of helper-T cells (3). IL-2, in turn, upregulates the expression of IL-2 receptors (IL-2R) in the stimulated cells (4). The recent demonstration (5, 6) that B cells express IL-2R of similar density and t This work was supported by the Rebecca Sakai Memorial Fund and Grant DK38831 from the National Institute of Health. Dr. Jordan is the recipient of a Research Career Development Award (DK01783-05) from the NIH. This work was presented in part at the Annual Meeting of the Society of Pediatric Research, 1987. 354 0090-1229/88 $1.50 Copyright© 1988by AcademicPress, Inc. All rightsof reproductionin any form reserved.

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affinity as those of T cells has stimulated further interest in the IL-2, IL-2R system, especially in SLE. In this study, we investigated IL-2R expression, IL-2 production, and spontaneous immunoglobulin production in PBL from patients with active and inactive SLE and determined how these parameters correlate with the state of disease activity. MATERIALS AND METHODS

Patient Population The study group included 27 SLE patients with a mean age of 38 -+ 14 years (range 9-69 years). Diagnosis was made according to the revised American Rheumatism Association (ARA) criteria (7). Using the Lupus Activity Criteria Count as designed by Urowitz et al. (8), 13 patients were categorized as active and 14 patients inactive. Using this methodology, a patient was considered to have active disease if two or more of the following manifestations were present: arthritis, abnormal hematologic or serologic findings such as decreased C3 or CHso, and positive anti-DNA, rash or ulcers, serositis, neurologic disease, vasculitis, or hematuria. Inactive disease was defined as absence of clinical symptoms described above. Nine normal donors served as controls. Lymphocyte Isolation Lymphocytes were isolated from heparinized venous blood samples by FicollHypaque gradient centrifugation using established techniques (9). In specific experiments, the cell preps were further enriched for B lymphocytes using an antibody-mediated complement lysis procedure. Briefly, using a B-cell isolation reagent, Lympho-Kwik (One Lambda, Inc., Los Angeles, CA), the whole PBL from nine active patients and nine normal controls were pelleted in separate Fisher tubes, then incubated for 1 hr with the first reagent which contains antibodies to monocytes, granulocytes, red blood cells, and platelets. The treated cells were repeUeted and exposed to complement, centrifuged, and washed. The resultant B-enriched cells were resuspended to appropriate concentration. Using indirect immunofluorescent techniques to examine the preparations (10), the B-cell purity was determined to be >95%. Cell Cultures The isolated cells (PBM) were washed twice and then cultured in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% AB normal human sera (NHS) (Sigma, St. Louis, MO). The cells were cultured for 3 days with or without PHA for IL-2 production or IL-2R expression. Additional cultures were also prepared with or without PWM in media supplemented with FBS for 12 days to determine immunoglobulin production. All cultures were incubated in a 5% CO2, 37°C humidified atmosphere. In other experiments, purified B cells were maintained in RPMI 1640 with 10% AB NHS prior to performance of the IL-2R assay. The determination of spontaneous IL-2R expression was performed on the day B cells were isolated.

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IL-2 Receptor Assay At the end of the culture period, the cells were pelleted, washed, and assayed for IL-2R presence by enzyme immunoassay (EIA) using murine monoclonal anti-IL-2R antibody (anti-Tac antibody) kindly provided by Dr. T. A. Waldman (NIH), and affinity purified sheep-anti-mouse-IgG conjugated with HRP (BectonDickinson, Mountain View, CA). The cells were first treated with a 1:500 dilution of anti-Tac in I%BSA-PBS (100 p~l/106 cells). After a 45-min incubation at 4°C, the cells were washed and then incubated with sheep-anti-mouse IgG. The cells were then washed, resuspended at 10 6 cells/ml, and aliquoted (50 p~l/well) into a 96-well tray (Titertek, Flow Labs, McLean, VA). o-Phenylenediamine (OPD, New England Nuclear, Boston, MA) was added, and after 15 min at room temperature, the reaction was quenched with 4.25 M sulfuric acid and the absorbance (optical density, O D ) a t 492 nm was measured.

IgG Determination IgG determinations on spontaneous and PWM-stimulated culture supernatants were performed by standard EIA techniques (11). Specifically, 96-well plastic microwell trays (Nunc, Denmark) were coated with F(ab')2 goat anti-human IgG for 16 hr at 4°C, then washed and blocked with 0.1% PBS-Tween. Afterward 50 p~l of test sample was aliquoted in quadruplicates, then incubated for 60 min. Following washing, 50 ~1 of Protein-A (Sigma) diluted in PBS (1:5000) was added to the wells, with subsequent incubation. Finally, OPD was added, and the reaction quenched after 10 min with 4.25 M sulfuric acid. A standard curve in nanograms per milliliter was generated using serial dilutions of stock IgG (CalbiochemBehring Corp., La Jolla, CA). Test values were compared to the standard curve.

IL-2 Assay IL-2 levels were determined using the hexoseaminidase assay (12) employing the IL-2-dependent CTLL-2 cell line. The system is dependent on the interaction of the ubiquitous lysosomal enzyme, N-acetyl-[3-D-hexoseaminidase, found in the live intact cell, with the substrate para-nitrophenyl-N-acetyl-~-D-glucoseaminide (Sigma). The assay entailed a three day culture of the CTLL-2 cells in complete RPMI supplemented with 2% heat inactivated FBS (GIBCO), sodium pyruvate, nonessential amino acids, and 2fl-mercaptoethanol. The cells were suspended and aliquoted at 4 x 103 cells per well (100 p~l) in a 96-well flat bottomed tray (Falcon, Becton-Dickinson). Test samples appropriately diluted in complete media were added in equal volume (100 p~l), with subsequent incubation in a 5% CO2, 37°C humidified incubator for 72 hr. At the end of the culture period, the trays were washed with PBS, and the substrate was added. After a 4-hr incubation, the color reaction was developed and enyzme activity blocked by addition of glycine buffer. Absorbances were measured at 405 nm with a standard curve (in units/ml) created by comparison of samples to a serial dilution of recombinant IL-2 (AmGen, Thousand Oaks, CA).

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Statistical Methods Differences between active and inactive SLE patients or controls were analyzed using the nonparametric Wilcoxon rank sum test. Paired data were analyzed using the Wilcoxon signed rank test. The results were expressed as means -+ SEM.

RESU LTS Figure ] depicts the spontaneous and stimulated [gG production for active and inactive SLE patients and normal donors. As shown, there was no significant spontaneous IgG production by PBL from normal controls and inactive SLE patients, while those from active SLE patients produced significant levels of IgG (P < 0.005). Following mitogen stimulation, there were significant increases in IgG production in the normal and inactive groups (P < 0.001 and 0.01, respectively), but no significant increases in IgG production by the active group reflecting the hyperactivity of those cells in the spontaneous state. Figure 2 depicts IL-2 production under the various conditions described. There was no spontaneous IL-2 production in any of the study groups. After PHA stimulation, the PBL from active SLE patients failed to elaborate significant levels of IL-2 as compared to the normal group (P < 0.001) or the inactive SLE group (P < 0.001). IL-2R expression in the three populations (spontaneous and stimulated) is depicted in Fig. 3. There was low spontaneous IL-2R expression in the normal and inactive groups. However, the spontaneous IL-2R expression in the PBL of active SLE patients was significantly higher than either the normal or inactive groups (P < 0.01). On the other hand, there were significant increases in the IL-2R expression upon PHA stimulation in the normal (P < 0.05) and the inactive PBL (P < 0.02), but no significant change in the PBL from active SLE patients (Fig. 4), suggesting that these were already maximally stimulated in the spontaneous state.

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FIG. 1. IgG production in ng/ml by PBL from active (A) and inactive (I) SLE patients and normal donors (N) under spontaneous (S) and mitogen-stimulated (PWM) conditions. There was significant spontaneous IgG production by active SLE cells compared to normal or inactive cells (P < 0.005).

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FIG. 2. IL-2 production in U/ml by PBL from active (A) and inactive (I) SLE patients and normal donors under spontaneous (S) and mitogen-stimulated (PHA) conditions. There is significantly higher stimulated IL-2 production in the normal group compared to inactive or active (P < 0.001).

There was a 126 --_ 75% increase in IL-2R expression above resting in the normal group and a 66 -+ 78% increase observed in the inactive group (P = NS). However, in the active group, there was only an 11 --_ 16% increase in receptor expression, significantly less than that observed in the normal PBL (P < 0.001) or in the inactive PBL (P < 0.001). Since B-cell hyperfunction and T-cell dysfunction are characteristic of active SLE (13) and since IL-2R appear on activated T and B lymphocytes, we undertook a study to determine whether the B cells in active SLE patients showed

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FIG. 3. IL-2 receptor expression under spontaneous (S) and mitogen-stimulated (PHA) conditions in the normal (N), inactive SLE (I), and active SLE (A) groups. Significantly higher spontaneous IL-2 receptor expression is observed in the active group compared to inactive and normal (P < 0.01). There are significant increases upon mitogen stimulation in the receptor expression in the normal (P = 0.03) and inactive (P = 0.01) group, but not in the active group (P = NS).

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IL-2 RECEPTORS IN SLE p~.03

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FIG. 4. Representative data pairs in active (A) and inactive (I) SLE patients and normal donors (N). There is no significant change in IL-2 receptor expression upon mitogen stimulation.

increased IL-2R expression. In these experiments, purified B-cell preparations were isolated from nine normal individuals and from nine patients with active SLE. As expected, there was a low IL-2R expression in normal nonactivated cells (OD at 492 mm = 0.12 -+ 0.07). The active SLE patients tested had a significantly higher B-cell IL-2R expression (OD at 492 mm = 0.29 -+ 0.12, p = 0.005) when compared to normal unstimulated controls. This data indicates that there is an increased B-cell IL-2R expression in active SLE patients which is partially responsible for the increased spontaneous IL-2R expression seen in active SLE patients. DISCUSSION As previously noted by Kunkel in 1980, one of the most reproducible abnormalities of cellular immune function in SLE is the marked B-cell hyperreactivity (14). There is increased spontaneous IgG production by PBL from active SLE patients as compared to inactive or normals which cannot be enhanced by PWM. Blaese et al. (26) also reported that patients with active SLE have increased IgG and IgA secreting cells in their peripheral blood. These investigators also reported that the parameter correlating most with SLE disease activity was the frequency of IgG secreting cells in the peripheral blood. Steinberg et al. (27) and others (28, 29) have shown that there is an increased stem cell and B-cell hyperactivity in patients with SLE. These investigators (27) have also shown that mice and humans with SLE have increased expression of c - m y c gene attributed to hyperac-

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tivity of B cells. These data suggest abnormal regulation of oncogene expression in SLE resulting in dysfunctional lymphoid maturation and activation. In our study group, we also noted defective IL-2 production in the active and inactive SLE patients in response to mitogen stimulation. Huang has postulated that the IL-2 hyposecretion may be simply a manifestation of cellular exhaustion (15). Of particular interest is the increase in IL-2R in active SLE patients, compared to inactive patients and normal controls, a phenomenon observed in whole PBL as well as in B-enriched preparations. Ishida et al. (24) have recently shown that high affinity IL-2Rs are decreased in PHA-activated T-cell blast from active SLE patients. These authors also found no differences in IL-2R expression on the PHA blast when active SLE patients were compared to inactive and normal controls. Our data would tend to support this conclusion since no significant differences were noted in IL-2R expression in PHA-stimulated populations (Fig. 3). However, these investigators did not assess the spontaneous IL-2R expression or B-cell IL-2R expression in their patients. More recently, Balderas et al. (25) found high levels of IL-2R in the serum of lupus mice whose disease was characterized primarily by B-cell proliferation. These authors speculate that IL-2R determination is a good marker of lymphoid cell activation and may be useful in following patients with autoimmune diseases. It is now accepted that activated T cells and certain populations of activated B cells display receptors for IL-2. It has been demonstrated that B cells exposed to IL-2 manifest increased numbers of receptors. Furthermore, B cells activated by mitogen can be induced to proliferate and synthesize Ig by addition of recombinant IL-2--a response that is abrogated by the addition of anti-Tac (16). However, the significance of IL-2 receptor expression and of IL-2 itself on B-cell function remains controversial (17). It appears that the spontaneous expression of IL-2R in our patients reflects cellular activation in the active disease state regardless of manifestation. This activation does not appear to be secondary to increased IL-2 production as patients with SLE have defective IL-2 production especially in the active state. This is unrelated to immunotherapy, since no patients were excluded from the study by virtue of their current therapy. Our assay system does not distinguish high from low affinity receptors, but other investigations have reported an association between increased low affinity, soluble IL-2Rs, and diseases characterized by increased T-cell activation, such as rheumatoid arthritis, allograft rejection, and SLE (18-20). Ishida et al. (24) have shown that PHA-activated T-cell blast from active SLE patients are poorly responsive to recombinant IL-2 in vitro, suggesting a paucity of high affinity IL-2R on these cells. Despite this, the Tac antigen may represent a potential target for future immunotherapeutic maneuvers. The immunoregulatory effects of monoclonal antibody directed against IL-2R have been well described in animal models where they have been shown to prevent or reverse allograft rejection (21, 22) and to suppress murine lupus nephritis (23). Since increased immunoglobulin production and IL-2R expression by B cells were demonstrated in active SLE patients in this study, attempts to modulate or eliminate

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IL-2 receptor bearing cells with monoclonal antibody may prove a more precise and efficacious therapy for active SLE. SUMMARY

In summary, we have demonstrated cellular markers of disease activity which include IL-2 hyposecretion, increased spontaneous IgG production, and increased IL-2 receptor expression in active SLE PBL. While the functional significance of these findings is still unclear, our data suggest that the presence of IL-2 receptors in unstimulated B cells of active SLE patients may be used as a marker of disease activity. ACKNOWLEDGMENT This manuscript is dedicated to the memory and talent of Rebecca S. Sakai whose work and support was invaluable in its preparation.

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23. Kelley, V. E., and Strom, T., In, "Proceedings, The American Society of Nephrology, December 1986," pp. 167A (abstract). 24. Ishida, H., Kumagai, S., Umehara, H., Sano, H., Tagaya, Y., Yodoi, J., and Imura, H., J. lmmunol. 139, 1070, 1987. 25. Balderas, R. S., Josimovic-Alasevic, O., Diamantstein, T., Dixon, F., and Theofilopoulos, A., J. lmmunol. 139, 1496, 1987. 26. Blaese, R. M., Grayson, J., and Steinberg, A. D., Amer. J. Med. 69, 345, 1980. 27. Steinberg, A. D., Klinman, D., Kastner, D., Seldin, M., Gause, W. C., Scribner, C. L., Britten, J. L., Siegal, J. N., and Mountz, J. D., J. Rheumatol. 14(Suppl. 13), 166, 1987. 28. Glinski, W., Gershwin, M. E., Budman, D. R., et al., Clin. Exp. Immunol. 6, 228, 1976. 29. Budman, D. R., Merchant, E. B., Steinberg, A. D., et al., Arthritis Rheum. 20, 829, 1977. Received October 28, 1987; accepted with revision February 8, 1988