A defect in the protein kinase C system in T cells from patients with systemic lupus erythematosus

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

60,

220-231 (1991)

A Defect in the Protein Kinase C System in T Cells from Patients with Systemic Lupus Erythematosus YOSHIFUMI TADA, KOHEI NAGASAWA, YASUO YAMAUCHI. HIROSHI TSUKAMOTO, AND YOSHIYUKI NIHO First

Department

of Internal

Medicine,

Faculty

of Medicine,

Kyushu

University,

Fukuoka,

Japan

To determine whether there is an intrinsic defect in T cells from patients with systemic lupus erythematosus (SLE), we studied signal transduction systems, assaying the total protein kinase C (PKC) levels and the phorbol myristate acetate (PMA)-induced activation of PKC in PHA-treated T cells. T cells from SLE patients showed a decrease in proliferation in response to PMA, but not to PHA, thereby suggesting the existence of an intrinsic abnormality in the PKC-mediated activation pathway. Total PKC activity in the T cells from SLE patients was significantly decreased. Although stimulation with PMA induced a translocation of PKC from the cytosol to the particulate fraction, translocated PKC activity after 2 IL&I PMA treatment was decreased in the SLE T cells. Furthermore, PMA-induced phosphorylation of t?O-kDa substrates was also decreased in SLE T cells. These results suggest that there is a reduced PKC activity and an impaired PKC activation in response to PMA in the SLE T cells, a finding which may explain, if partially, the defect in T cell activation in patients with SLE. o 1~1 Academic PRESS, IX

INTRODUCTION In patients with systemic lupus erythematosus (SLE), a typical autoimmune disease, there are abnormalities in T cells, B cells, monocytes, and serum factors. In the T cells, there is an impaired proliferation and interleukin 2 (IL-2) secretion in response to mitogens and antigens (l-6). As the proliferative response of T cells, as measured by tritiated thymidine uptake or IL-2 secretion, sometimes reflects in part the function of accessory cells as well, and, in some cases, different culture conditions or concentrations of stimulants will yield different results. Such being the case, it is important to determine whether these T cells have intrinsic defects, and if so, it should be clarified which step, from the receptorligand binding to the expression of function, is responsible for the defect in the T cells from SLE patients. Protein kinase C (PKC) is an important transmembrane signaling system in T cell activation (7-11). Mitogenic stimulants such as phytohemagglutinin (PHA) or anti-CD3 antibodies induce phosphatidylinositol hydrolysis in T cells (12-14). Of the hydrolytic products, inositol 1,4,5trisphosphate (IP,) mobilizes free calcium from intracellular stores, and diacylglycerol (DG) activates PKC (15, 16). Phorbol myristate acetate (PMA) binds to PKC intracellularly and directly activates PKC resulting in proliferation of T cells (9, 17-20). We evaluated total PKC levels and activation of PKC in an attempt to clarify whether there is an intrinsic defect in signal transduction systems in T cells from patients with SLE. We obtained evidence that the total PKC level is decreased and that the activation of PKC in response to PMA is impaired in T cells from 220 0090-1229/91 $1.50 Cotwinht

B 1991 by Academic

Press, Inc.

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patients with SLE. This may explain, to some extent, the defect in T cell activation in patients with SLE. PATIENTS

AND METHODS

Patients. Nineteen patients (4 men and 15 women) who fulfilled revised criteria of the American Rheumatism Association for SLE (21) were studied. Thirteen were considered to have active SLE, according to the lupus activity criteria count (LACC) proposed by Urowitz et al. (22). Six patients were not treated with corticosteroids and 13 were prescribed 5-40 mg/day of prednisolone. No patient was treated with immunosuppressive agents. All subjects were studied at least 18 hr after the last prescription of corticosteroids. The normal control population consisted of 17 age-matched healthy individuals (7 men and 10 women). Not all of the assays described below were performed in all the patients. Cell preparation. Peripheral blood mononuclear cells were separated from heparinized venous blood by Ficoll-Conray density gradient centrifugation. The cell suspension was incubated in plastic petri dishes at 37°C for 1 hr and the nonadherent cells were recovered. T cells were prepared by the neuraminidase-treated sheep red blood cell rosetting technique. The resultant T cell population was >92% reactive with the anti-CD3 monoclonal antibody. Cell proliferation assay. Proliferation was measured by [3H]thymidine uptake. T cells (1 x 10’) were cultured in 0.2 ml RPM1 1640 medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% fetal calf serum (Sera-Lab, Ltd., Sussex, England), 100 U/ml penicillin, and 100 pg/ml streptomycin in flat-bottomed microtiter culture plates (Costar 35%) at 37°C in 5% CO*. Cells were stimulated with PMA (Sigma, St. Louis, MO) for 72 hr, and 0.5 &i/well of [3H]thymidine (Amersham, Buckinghamshire, England) was pulsed 10 hr before termination. Cells were harvested onto glass fiber filters and the radioactivity incorporated was measured with a scintillation counter. Cell activation. T cells were suspended at 1 x 106/ml in complete media and stimulated with 0.1% PHA (Difco, Detroit, MI) for 3 days. Alternatively, T cells were stimulated with PHA for 4 days, washed, and incubated with 50 U/ml recombinant IL-2 (provided by Shionogi & Co., Osaka, Japan) for another 2 days and then washed and incubated without stimulants for 24 hr. The cell population was >95% viable by the trypan blue dye exclusion test. PKC preparation. PKC was prepared from unstimulated or PMA-stimulated cells. PMA stimulation was as follows. Activated T cells were suspended at 1 x lO’/ml in complete media and were stimulated with the indicated concentration of PMA in a water bath at 37°C. Cells removed at indicated time intervals were washed twice with phosphate-buffered saline (PBS). Unstimulated or PMAstimulated cells were resuspended in 3 ml buffer A (20 mM Tris, pH 7.5, 2 miV EDTA, 0.5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride (PMSF)) containing 50 mM 2-mercaptoethanol and sonicated with a Branson Model Sonilier for 45 set at 20 W. The homogenates were centrifuged for 60 min at 100,OOOg; the supematant served as the cytosol fraction. The pellets were sonicated for 90 set at 20 W, treated with 0.5% Nonidet-P40 for 1 hr on ice to extract the PKC, and centrifuged for 45 min at 100,OOOg. The supematant served as the particulate fraction. PKC

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from the cytosol and the particulate fraction was partially purified by applying each sample to a 1S-ml DEAE-Sepharose column equilibrated with buffer A. The column was washed with 5 ml buffer A and 0.5 ml 0.15 M NaCI, and the eluate by 2 ml of 0.15 M NaCl was collected. All of the PKC activity was present in this fraction. PKC assay. PKC activity was determined as described (23). The reaction mixture, in a final volume of 250 pl, consisted of 80 pg/ml phosphatidyl serine (PS)(Sigma), 20 mM Tris, pH 7.5, 0.1 mM CaCl,, 5 mM magnesium acetate, 2.5 nM y-32P-ATP (100-200 cpm/pmol) (Amersham, Buckinghamshire, England), 50 p,g histone HI (Sigma), and 80 l.~l of sample in the absence or presence of 1 &ml PMA. Incubation was carried out for 5 min at 30°C. The reaction was terminated by the addition of 25% trichloroacetate. The acid-precipitated materials were collected on a membrane filter and 32P incorporated into histone HI was counted. PKC activity was determined by subtracting background phosphorylation counts obtained in the absence of PMA and PS and was expressed in nanomoles per minute per milligram protein. The PKC assay was done in duplicate and data were obtained from a single sample. In each experimental procedure, the patient’s samples were always tested together with at least one sample from a normal control. To minimize interexperimental variance in the PKC assay, a frozen control PKC sample from a human T lymphoblastic cell line was always assayed together with all samples. Phosphoprotein analysis. Freshly isolated T cells were suspended at 2.5 x 106/ml and incubated for 2 hr at 37°C in phosphate-free RPM1 1640 containing 5% dialyzed FCS and carrier-free 32P (0 .2 mCi/ml)(Amersham). Labeled cells were stimulated with PMA for 20 min. Cell lysis was performed with 1% Triton X-100 in buffer B (10 mM Tris/HCl, pH 7.4,2 mM EGTA, 50 mM NaF, 1 mM PMSF), and the cytoplasmic lysate was boiled for 10 min. Precipitated proteins were removed for 10 min at 10,OOOg and soluble proteins were precipitated overnight at -20°C in acetone (90%, v/v). Pellets were desiccated and phosphoproteins were analyzed by SDS-PAGE on a 9% gel under reducing conditions (24). Gels were stained and autoradiography was performed with Kodak XAR film. Statistical analysis. Student’s t test was used to determine the statistical significance . RESULTS Proliferative response of T cells to PMA. Figure 1 shows the dose-response proliferation curve of T cells to PMA after 72 hr culture, from a representative patient with SLE and from a normal control. Maximal proliferation was observed at 4-16 nM PMA. The proliferation of T cells from a SLE patient was apparently lower than that from a normal control, regardless of the concentration of PMA. Table 1 shows a comparison of T cell proliferation in response to 2 nM, 8 nM PMA, and 0.1% PHA. There was a significant decrease in the response of T cells from the active SLE patients to both concentrations of PMA, whereas the T cell response to PHA was not significant. Since PMA directly binds to and activates PKC, thereby leading to T cell proliferation, this result suggests that a possible

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PKC IN SLETCELLS

- ,,

1

2

4

8

16

80 P M A (nM)

FIG. 1. T cell proliferative response to various concentrations of PMA in a representative patient with SLE (A) and a normal control (0). T cells were stimulated with PMA for 72 hr and [3H]thymidine incorporation was measured. Standard errors of triplicate measurements were less than 5% of the means.

abnormality in the PKC-mediated activation pathway may exist in T cells from SLE patients. This prompted us to evaluate the PKC activity in T cells from SLE patients. Total PKC levels in T cells. As it is most difftcult to obtain sufftcient numbers of T cells from SLE patients, T cells from the peripheral blood were activated with PHA for 3 days and the PKC activity was examined. Most of the PKC activity was located in the cytosol fraction (>95%) in T cells both from SLE patients and from normal controls (data not shown); we then evaluated the cytosol PKC activity, as the total activity. Figure 2 shows the kinetics of the cytosol PKC activity in T cells from two control subjects, activated with PHA. PKC activity in the cultured T cells increased as the T cells were activated and proliferated. However, the PKC activity did not change or was gradually decreased when it was expressed as specific activity “per protein content.” We expressed PKC activity in this manner throughout the following experiments. As shown in Figure 3, the cytosol-specific PKC activity in the PHA-treated T cells from SLE patients (seven active and four inactive) was significantly decreased as compared to that from normal controls TABLE 1 T CELL PROLIFERATIVERESPONSETO PMA AND PHA IN SLE PATIENTSANDNORMALCONTROLS [‘H]Thymidine

Medium 2nhfPMA 8nMPMA 0.1% PHA * P < 0.05 versus control.

incorporation

(cpm X lo-*)

Control (n = 6)

SLE (n = 6)

2*1 134 f 40 140 + 46 769 + 236

I”0 71 k 19* 75-c40* 629 5 152

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ET AL.

day FIG. 2. Kinetics of the PKC activity in activated T cells. T cells from two normal controls were stimulated with 0.1% PHA for the time periods indicated, and PKC activity in activated T cells was measured. PKC activity was expressed in two ways: per cell count at the start of culture (0) and per protein content (A). Results are expressed as the ratio of PKC activity in nonstimulated T cells. Standard error of PKC activity (cpm) averaged less than 10% of the means.

(0.392 2 0.108 vs 0.533 -+ 0.145, P < 0.01). This means that SLE T cells had less specific PKC activity than control T cells, because SLE T cells showed a comparable response to PHA as control T cells did. We next investigated an activation of PKC, in response to PMA. Trunslocation of PKC in response to PMA. PMA and other mitogenic stimuli for T cells, such as anti-CD3 antibodies, induce translocation of PKC from the cytosol to the particulate fraction. This process is considered to be a manifestation of PKC activation and translocated PKC to the particulate fraction is thought to be “activated PKC” (9,25). Figure 4 shows the translocation of PKC after 10 min stimulation of PMA in normal T cells. PKC activity in cytosol and particulate fractions dose dependently changed reciprocally each other. This translocation was found in SLE T cells as well. It is noteworthy that the translocation of PKC was dose dependent on PMA up to 16 nM, a finding that was similar in PHAactivated and freshly isolated T cells (data not shown), but as shown in Fig. 1, T cell proliferation reached the plateau at 4 nM. Therefore, activation of a half of total PKC seems to induce a maximum proliferation of T cells. We compared the translocation of PKC in activated T cells from SLE patients and from normal controls. Here, T cells from six active and two inactive SLE patients were stimulated with PHA and IL-2 for 6 days, rested 1 day without stimulants, and then used for the translocation study. These T cells were stimulated with 2 nM PMA, the same dose as used in proliferation assay, for 10 min. The PKC activity in the cytosol and particulate fractions was simultaneously measured and the extent of translocation of PKC from the cytosol to the particulate was compared. Figure 5 shows that the particulate PKC activity in PMA-stimulated T cells from SLE patients was significantly lower than in cells from normal controls (0.162 + 0.048 vs 0.220 +- 0.038, P < 0.05). The results from the same experiments are summa-

PKC

IN

SLE

225

T CELLS PcO.01

I

normal control

1

SLE

FIG. 3. PKC activity in PHA-treated T cells (Day 3) from SLE patients and normal controls.

rized in Table 2. In the case of both controls and SLE patients, 53 and 57% of cytosol PKC remained in the cytosol fraction, respectively. However, the rate of PKC recovered in the particulate fraction after PMA treatment was decreased in T cells from SLE patients compared with normal controls (20% vs 2%, P < 0.05). Thus, in the SLE T cells, the rate of PKC which disappeared from the cytosol but which was not recovered in the particulate fraction is higher than that in normal T cells. PMA-induced phosphorylation of the cytosol proteins in T cells. To examine directly the activation of PKC, peripheral blood T cells were labeled with 32Pand PMA-induced phosphorylation was analyzed by SDS-PAGE. A representative experiment is shown in Fig. 6. A Coomassie brilliant blue staining revealed no significant differences in the pattern and intensities of bands of cytoplasmic proteins between T cells from SLE patients and normal controls (data not shown). The 80-kDa phosphoprotein is often used as a substrate of PKC (27,29). We also found the 80-kDa phosphorylation in response to PMA in a dose-dependent manner up to 16 ti in normal T cells (lanes 14). T cells from a SLE patient (active,

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ET AL.

0 P M A (nM)

FIG. 4. Translocation of PKC induced by PMA in PHA-treated T cells. Activated T cells from a normal control were stimulated with various concentrations of PMA for 10 min, and PKC activity in the cytosol (0) and the particulate fraction (A) was measured. These data are representative of two experiments.

steroid-free) showed a decreased phosphorylation of the 80-kDa protein in response to 16 nM PMA (lane 6), which was even less than that with 4 ti PMA in control T cells. Apparently, a reduced 80-kDa phosphorylation was found in three of four active SLE patients tested. These results indicate that PMA-induced phosphorylation was defective in peripheral blood T cells from SLE patients. DISCUSSION Here we first demonstrated the impairment of SLE T cells in the PKC system by assaying the enzyme activity directly and by showing the PKC-mediated protein phosphorylation. In T cells, stimulation with mitogenic lectins or the antiCD3 antibody leads to hydrolysis of phosphatidylinositol, the products of which activate PKC in the presence of calcium and phospholipids (12-15). PMA binds to and directly activates PKC, bypassing phosphatidylinositol hydrolysis, and induces cell activation (17, 18). We found that SLE T cells had a deficient proliferation in response to PMA (Fig. 1, Table 1). Concerning the steps of activation mediated by PKC, several possibilities are considered: (i) the total PKC level is decreased in SLE T cells, (ii) PKC is less activated by PMA in SLE T cells, or (iii) PKC in SLE T cells has a reduced kinase activity. The result in Fig. 3 suggests the first possibility since the total specific PKC activity is reduced in T cells from SLE patients. We examined the second possibility, using 7-day PHA-treated T cells, and obtained the data suggesting the impaired PMA-induced translocation of PKC in SLE T cells as shown in Fig. 5 and Table 2. PKC translocation to the cell membrane has been shown to correlate with actual PKC activation (9, 25). All the PKC activity that disappeared from the cytosol fraction could not be completely recovered in the particulate fraction. Some portions of PKC might be either degraded and inactivated with PMA or translocated to the nuclei or cytoplasmic organella. It is of

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PKC IN SLE T CELLS

pco.05 I

I

0

normal control

SLE

FIG. 5. PKC activity in the particulate fraction in PHA-treated T cells stimulated with PMA. PHA-treated T cells (Day 7) from SLE patients and normal controls were stimulated with 2 nM PMA for 10 mitt, and PKC activity in the particulate fraction was measured.

interest that the PKC activity which disappeared from the cytosol but was not recovered in the particulate is greater in SLE T cells than in normal T cells (Table I). Shoji et al. (26) reported that a subclone of an acute myeloid leukemia cell line that is resistant to the PMA-induced differentiation showed PKC translocation from the cytosol to the perinuclear region. A similar “ineffective” PKC translocation might be increased in SLE T cells. The third possibility still remains to be clarified. The number of PKC molecules in T cells or the cofactor requirement of PKC could not be investigated because of the limited samples from SLE patients. We investigated the in situ protein phosphorylation of endogenous substrates in

~. Subject Control SLE

TABLE 2 SUBCELLULARREDISTRIBUTIONOFCYTOSOL PKC AFTER PMA TREATMENT INPHA-ACTIVATEDTCELLS .- ____ Stimulation Particulate (%)” Cytosol (%)” ____ 100 2nMPMA(n = 6) 53 t 10 29 + 6 100 2n&fPMA(n = 6) 57 rt 10 20 k 5*

0 PKC activity in the cytosol and the particulate fraction after stimulation with PMA was expressed as a percentage of the cytosol PKC activity, before stimulation, in each subject. * P < 0.05 versus control.

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123456

-8OkDa

FIG. 6. PMA-induced phosphorylation of the SO-kDa protein. “P-labeled T cells from a normal control (lanes 1 to 4) and a SLE patient (lanes 5 and 6) were treated with medium alone (lanes I and 5), 1 n&f PMA (lane 2), 4 nM PMA (lane 3). or 16 n&I PMA (lanes 4 and 6). Cells were lysed and boiled for 10 min. Heat-stable proteins were analyzed by SDS-PAGE on a 9% gel and autoradiography was performed. The numbers on the left of the gel refer to M, markers (X 10e3).

T cells in response to PMA to ascertain the defective PKC activation. Some PKC-specific substrates have been reported (7, 27), for example, the y chain of the CD3 complex (11, 28) or 80- and 19-kDa proteins (29). In our experiment, the dose-dependent phosphorylation of the 80-kDa protein was identified after PMA stimulation, and we found that phosphorylation of the 80-kDa protein was reduced in SLE T cells, suggesting that the PKC-induced phosphorylation in SLE T cells was defective. The mechanism of this phosphorylation reduction may be the reduced kinase activity of PKC. Alternatively, a small amount of substrate protein might be the case, but this is less probable since a Coomassie brilliant blue staining of cytoplasmic protein revealed a similar pattern in SLE and normal T cells. Our data, which suggest a decreased PKC activity in SLE T cells, were obtained by using PHA-stimulated T cells instead of fresh or resting T cells. This was because it was difficult to obtain sufficient numbers of T cells (>l x 10’ cells) from active SLE patients to produce reliable results. However, the results from PHA-stimulated T cells seem to be applicable to in vivo T cells since there was no difference in PHA responsiveness between SLE and normal T cells, and PKCmediated phosphorylation in fresh T cells was decreased compared with that in fresh normal T cells. T cells from SLE patients have an impaired responsiveness to the various kinds of stimuli (l-6). However, studies on intracellular events in SLE T cells have been considerably limited. Murakawa and Sakane (30) reported that decreased IL-2 production by T cells from patients with inactive SLE in response to PHA was correctable if PMA was added; therefore, the signal activating PKC might be impaired with PHA stimulation. Huang et al. (31) reported that SLE T cells showed a decreased IL-2 production in response to PHA plus PMA, a finding which suggests that PKC activation by PMA is (and other signals induced by PHA might be) defective. These observations imply a defective phosphatidylinositol hydrolysis and PKC activation and may explain some of the impairment of SLE

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T cells. On the other hand, in lupus-prone MRL fprllpr mice, lymph node T cells showed a markedly deficient PHA-induced phosphatidylinositol hydrolysis, whereas the PKC level and PMA-induced translocation were intact (32). Kammer et al. has reported a defect in the CAMP-protein kinase A pathway and a defective CAMP-dependent phosphorylation in SLE T cells (33-35). PKC abnormality in SLE T cells needs to be elucidated on several points. Recently, two types of PKC isoforms were identified in T cells, the activities of which showed different dependencies on phospholipid and free calcium (36). Lack of or a specific abnormality of one type might be the event related to PKC abnormality in SLE T cells. Alternatively, a specific T cell subpopulation in SLE patients might be highly defective and might reflect the low PKC activity in whole T cells. ACKNOWLEDGMENTS The authors thank Y. Taniguchi and T. Shigeto for technical assistance and M. Ohara for critical comments.

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34. Kammer, G. M., Birch, R. E., and Polmar, S. H., Impaired immunoregulation in systemic lupus erythematosus: Defective adenosine-induced suppressor T lymphocyte generation. J. Zmmunol. 130, 1706-1712, 1983. 35. Hasler, P., Schultz, L. A., and Kammer, G. M., Defective CAMP-dependent phosphorylation of intact T lymphocytes in active systemic lupus erythematosus. Proc. Nutl. Acad. Sci. USA 87, 1978-1982, 1990. 36. Beyers, A. D., Hanekom, C., Rheeder, A., Strachan, A. F., Wooten, M. W., and Nel, A. E., Characterization of protein kinase C and its isoforms in human T lymphocytes. J. Immunol. 141, 3463-3470, 1988. Received September 5, 1990; accepted with revision March 27, 1991