SPECIAL ARTICLE T H E PRIMATE M H C W O R K S H O P OEGSTGEEST, T H E N E T H E R L A N D S , 1989
Signal Transduction in B Lymphocytes Chehrazade Brick-Ghannam, Nuala Mooney, and Dominique Charron
ABSTRACT: We have examined the activity and intracellular compartmentalization of protein kinase C (PKC) following activation of human B lymphocytes by anti-human leukocyte antigen (HLA) class I1 antibodies. [2-O-Tetradecanoylpborbol 13-acetate (TPA) treatment increased membrane-associated PKC (between five and nine times greater than the control value) and decreased cytosolic PKC (between 70% and 100% of the control value). In contrash anti-class II antibodies induce an activation of PKC which results either in an increase of cymABBREVIATIONS MHC major histocompasibilitycomplex HLA humanleukocyte antigen HMR half maximalresponse
solic activity or membrane-bound activity without redistribution of cytosolic PKC. The effect of TPA and HLA class 11molecules on total PKC activity was comparable: when TPA induced an increase of total PKC activity so did HLA class II moleculesand when TPA did not, HLA class II molecules did not. Measurement on SDS PAGE of his[one phospharylation confirmed the above results of PKC activity.Taken together, our results suggest that PKC might be implicatedin HLA class [I-induced B lymphocyte activation. H u m a n Immunology 30, 2 0 2 - 2 0 7 (I991)
protein kinase C 12-O-tetradecanoylphorho[ 13-acecate
INTRODUCTION The major histocompatibilky complex (MHC) or human leukocyte antigen (HLA) system consists of a cluster of genes on chromosome 6 in the human which codes for t w o highly polymorphic groups of transmembrane glycoproteins, the class I and the class It antigens. These antigens can be considered as part of the immunnglobulin "superfamily" of cell surface and soluble molecules which share important structural homology. The Ig superfamily includes highly polymorphic molecules which are involved in specific recognition while other members with limited or no polymorphism are adhesion or activation molecules . The HLA system has developed two contrasting properties: a high degree of structural polymorphism resulting in hypervariahle regions and regions of highly conserved sequences. A high degree of interspecies homology also exists supFrom the Laboratoir¢ d'lmmunog~nitique Moltculaire, lmtitut des Carddiers, Paris, France. Addr~s reprint requests to Chehrazade Brick.Ghannam, Laboratoire d'Immuno~nttique l~oliculaire, Institut des Cordeliers, l J, rue de l'Ecole de Mfdecine, 75006 Paris, France. Accepted October I 1, 1990. 202
porting the idea of the evolution of the HLA system from a limited set of primordial molecules. A number of molecules belonging to the immunnglobulin superfamily behave as signal-transducing molecules (Thy-1, lgM, and PDGFr) and the idea that the class I! antigens of the MHC could be involved in signal transductiou has also been examined. Cambier et al.  demonstrated that soluble In-binding ligands induce an increase of cAMP production  and a nuclear translocation of the cytosolic PKC in "the mouse . An augmentation of the proliferative response to pokeweed mitogen stimulation of B cells from healthy donors in the presence of two anti-HLA-DR antibodies was observed . An inhibition of B cell proliferation in response to either SAC or anti-Ig antibodies in the presence of anti-HLA class II antibodies has also been reported . Tanaka et at.  described an inhibition of proliferation and differentiation of B cells from patients with SLE in the presence of anti-HLA class II antibodies leading to the proposal of cellular activation as a direct consequence of recognition of class lI antigens on B cells. H ~ l~unologySO,202-207(t99D © AmericanSociety for Histocompafibilltylindlmmunogenefics,1991
MHC Class II Signaling via PKC Activation
The role of the HLA class II antigens in signal transduction by human resting B lyraphocytes was studied and a proliferative response to immobilized anti-HLA class I! antibodies was described . Surface Ig crosslinking is a well-documented in vitro method of activating B cells and a number of studies suggest that watilgbl-mediated signal transduction involves activation of protein kinase C (PKC) [8, 9]. Our previous studies revealed that MHC class II antigen-mediated signal transduction involved an increased level of intracellular free calcium after cross-linking of the antibodies  and an augmentation of the intracellular free calcium in response to a suboptimal amount of anti-IgM was observed in the presence of an anti-MHC class 1I DR antibody. The two-dimensional PAGE total protein profiles of B lyraphocytes activated via MHC class II proteins also suggested that protein kinase C activation was involved in M H C class II-mediated activation. More supporting evidence for a PKC-mediated pathway came from the observation of increased inositol phosphulipid metabolism following MHC class I1 antigen binding [ 10]. Given the indirect evidence that PKC is involved in MHC class Ii antigen-mediated signal transduction, we have directly investigated PKC activity both in terms of total activity and of transincation. We report an activation of PKC although unlike phorbol esmr-mediated B cell activation, a membrane translocation of PKC was not observed.
MATERIALS AND METHODS
Chemicals. Histone (Ill-s), phosphatidylserine,
1,2diolein, and 12-O-tetradecanoyl phorbol 13-0 acetate (TPA) were from Sigma. (5,32P) ATP (3,000 Ci/mmol) was purchased from Amersham. Protein determination reagents were from Binrad.
Monoclonal antibodies. Purified anti-HLA class II antibody (DI.12: anti-DR ) was prepared from ascitic fluid by ammonium sulphate precipitation followed by DEAE chromatography.
B Cell preparation. Mononuclear cells were obtained from the peripheral blood of healthy adult blood donors by ¢entrifugation on Ficoll Paque. B-cell-enriched pop. ulations were obtained after depletion of raonocytic cells and natural killer cells by L leuciue methyl ester treatment [ 12] and of T cells by two cycles of rosetting with 2 amino-ethyl-isothiouronium bromide hydrobromide treated sheep ¢rythrocytes. The cells obtained by this method contained less than 0.1% monocytes as determined by nonspecific esterase staining  and were
unable to proliferate in the presence of phytohemagglutinin. Low- and high-density populations corresponding to activated and resting B ceils, respectively, were separated on discontinuous Percoll density gradients .
Subcellular fractionation and PKC purification. CeEs were washed twice in phosphare-buffered saline without Ca 2+ and Mgz+. They were suspended at a density of 2 x 107 eells/ml and homogenized in ice-cold buffer A [20 mM Hepes, (pH = 7.5), 300 mM sucrose, 2 mM EDTA, l0 mM EGTA, 2 mM dithiothreitol (DTr), 2 mM phenylmethylsulfonylfluoride, 25 ~g/ml aprotinin, and 10/.~g/ml each of the protease inhibitor leupeptin, soybean trypsin inhibitor and pepstatin]. The homngenate was centrifuged at 900 g for 5 rain to remove cell debris and nuclei and the supernatant was centrifuged at I00,000 g for 1 hr. The supernatant fluid obtained constituted the eytosolic fraction enzyme. The pellet was resuspended and homogenized in buffer A containing 0.1% Triton X100, incubated on ice for 30 rain, and centrifuged at 100,000 g for 45 min. The resulting supernataat constituted the particulate fraction enzyme. Cytosulic and particulate fractions were then applied to a DEAE cellulose DE52 column pre-equilibrated with buffer B [20 mM Hepes (pH 7.5L 2 mM EGTA, 2 mM EDTA, 1 mM DTF). The columns were washed with 5 vol of buffer 13 and PKC was eluted with the same buffer plus 0.12 M NaCL
PKC assay. PKC activity was measured according m the method of Le Peuch et al. [ 15] with few modifications. The standard reaction mixture (final volume 60 tzl) conrained 20 raM Hepes buffer (pH 7.5), 5 mM MgCI2, 0.5 mM CaCI2, I mM D T r , 1 mg/ml lysine~rich histone, 80 lag of phosphatidyl serine/ml, 8/~g of dioleine/ml, 75 /zM (7-32P) ATP (600 cpm/pmol), and 30 /.d of the enzyme solution to be assayed. The reaction was initiated by the addition of('y ~zp) ATP and carried out for 5 rain at 23*(:. The reaction was terminated by pipetring 40 tzl of the reaction mixture onto whatrnan P-81 phosphocenulose paper (2 x 2 cm) . Papers were washed three times with coM water, dried, and assayed for radioactivity in 5 ml of liquiscint (Amersham). Protein was determined with coomassie blue . The enzyme activity was taken as the difference between the activities in the presence and absence of Ca z÷ and phospholipids. ~2p incorporation was linear with time and with protein concentration under the present assay conditions. Qualitative assay of PKC activity. The reaction mixture and the initiation o f the reaction were the same as in PKC assay. The reaction was stopped by addition of
C, Brick-Ghannam et al.
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FIGURE 1 PKC activity in total B-lymphocym population. Cells were incubated for 10, 30, or 60 rain with (A) TPA (20 nM) or (B) D I. 12 (25/zg/ml). Cytosol (@---@)and membrane (0....@) PKC activities were then measured as described in the text. Total PKC activity (~----0) was determined by addition of cytosol and membrane activities. The figure shows a representative experiment.
SDS and boiling the samples for 5 min. After addition of B-mercapthoetbanol, proteins were separated using 10% SDS PAGE gel electrophoresis. Gels were dried under vacumm and autoradiography was performed with Kodak XAR film. RESULTS
Quantitative measurement of PKC activity. Because a large quantity of cells was required (20 x 106 cells/ point) the enzymatic activity was first assayed in the total B-lymphocyte population. Because phorbol esters are known PKC activators [18, 19] that induce translocation of cytosolic enzyme activity to the membrane compartment, TPA was used as a positive control. B cells were treated with 20 mM of TPA for varying lengths of time and assayed for PKC activity recovered from the cytosolic and membrane fractions. Figure 1A shows that exposure to TPA induces the loss of 71% of the cytosolic PKC and an 780% increase in membraneassociated PKC, confirming the ability nf TPA to increase PKC activity and cause its translocation. The ligation of HLA class II antigens resuhs in an increase of total PKC activity which reached a maximum at 30 rain (Fig. 1B). Analysis of the cytosolic and membrane fractions revealed that the increase in PKC activity could be essentially attributed to an increase of the cytosolic activity (579% of the control value). However, a lesser (245% at 10 rain and 179% at 30 rain) but consistent increase in membrane PKC activity was also noted. Resting B cells were separated from activated B lymphocytes on discontinuous pcrcoll density gradient and then tested for their capacity to proliferate in response
to TPA or to D1.12. PKC activity was assayed only in cells that were able to proliferate significantly (data not shown). The translocation capacity of TPA was again observed (Fig. 2A) and was reproducible from one donor to another, When antibody directed against HLA class 1I antigen was tested, no redistribution of cytosoHc PKC was observed (Fig. 2). However, exposure to D1.12 results either in an increase in cymsolic PKC activity without change in membrane activity without important change in the cytosolic fraction (Fig. 2A). The increase of PKC activities was significant as they represent between twofold and eightfold of the control values. Although the pattern of PKC activation was different from one donor to another, the results were reproducible with the same donor. The effect of TPA and D1.12 on total PKC activity was interesting. We observed that the two compounds have a comparable effect: when TPA promoted an increase of total PKC activity, D1.12 did also and when TPA induced translocation of PKC without augmentation of total PKC activity, D I. 12 induced an augmentation of cytosolic or membrane activation without augmentation of total PKC activity (data not shown).
Qualitative measurement of PKC activity. A qualitative assay for measurement of PKC activity was used in order to verify the enzymatic assay. The effect of TPA on the redistribution of cytosolic PKC was first assessed (Fig. 3). B cells were treated with TPA at 8.16 or 32 nM for 30 rain. Historic klnase activity was measured either in the presence or in the absence of phospholipld and Ca z+. This was carried out in order to distinguish PKC (which phosphorylates histone in a phospholipid and Ca2+-dependent fashion) from the other kinases (which can phosphorylate histone in a phospholipid and Ca z+independent fashion). The autoradiograph revealed a
FIGURE 2 PKC Activity in resting B cells. The figure shows two representative experiments using two differents donom (A and B). After 30 rain of treatment cells were tested for cytosolic and membrane PKC activity. The concentrations used are expressed for TPA in naanmolas and for D1.12 in micrograms per milliliter. ¢VTOSOL ©
mNASE C a c n v i r Y [~,.,,u..~M]
MHC Class II Signaling via PKC Activation
FIGURE 3 Cytosol and membrane C kinase phosphor/lation after cell stimulation with TPA. After cell stimulation, cymsol and membrane fractions were prepared, tested for PKC activity in the presence (1-4) or the absence (5-8) of phospholipid and Ca2., separated using SDS-PAGE gel electrophnsesis, and autoradiographed. Cells were treated for 50 rain with control buffer II, 5) or with TPA at 8 nM (2, 6L 16 nM (.5, 7), and 32 aM (4, 8).
major band with molecular mass of approximately 31 kd. In untreated cells, PKC was detected in the cytosolic fraction but not in the membrane fraction. TPA induced a decrease of the cytosolic activity and an increase of the membrane activity in a dose-dependent fashion. The effect of anti-HLA class II antibodies on historic phosphorylation was tested with the same sample used for the quantitative assay (Fig. 2B). Cells were treated with D1.12 at 10, 15, or 25/*g/ml for 30 rain (Fig. 4). The autoradiograph revealed an important augmentation of the histone phusphorylation in the cytosolic compartment. Phosphorylation was maximal with 10 /zg/ml of Dl.12. Only slight phosphorylation was observed in the absence of phosphulipids and Ca z+. Phosphorylation was not detected in the membrane fractions (data not shown) either in the presence or in the absence of phospholipids and calcium.
lymphocytes and to translocate the enzyme to the nuclear compartment [2, 3]. Indirect evident that PKC is involved in MHC class l1 antigen-mediated signal transduction in human B lymphocytes came from our previous studies. An increase of iotracellular free calcium  and inositol phosphoLipid hydrolysis  has been observed following MHC class lI antigen binding. We have undertaken a direct PKC assay in order determine whether or not PKC is implicated in HLA class II-induced human B lymphocyte activation. Because quantities and activities of PKC vary considerably from one cell type to another [21 ], a preliminary study was undertaken to establish the best conditions for measuring human B lymphocyte PKC activity. The following parameters were tested: incubation time enzyme requirement, Ca"" and Mg2+ concentration dependence, and ATP concentration dependence (data not shown). When the rnral B-lymphocyte population was tested the data showed a dramatic increase in cymsolic PKC activity in association with a slight but significant increase in membrane PKC activity. The HLA class ]I molecule induced PKC activation is likely to occur by a mechanism different from that of TPA, which induced a translocation of the enzyme to the membrane. Most of" the knowledge of the physiology of PKC is derived from studies using phorbol esters. Phorbol esters are pharmacological effectors known to bind directly to PKC . it is therefore not surprising that TPA and anti-HLA class li antibodies do not present the same pattern of PKC activation. Furthermore, there are a few
FIGURE 4 Cymsol C klnase phosphorylation after cell stimulation with D1.12. This experiment involved the same donor as in Fig. 2B. Cells were treated for 30 rain with control buffer (I, 5) or with DI.I 2 at 10 t4g/ml (2, 6), 15 ,ug/ml (3, 7), or 25 v-g/ml (4, 8) either in the presence (1-4) or the absence (5-8l of phospholipid and C.az+.
DISCUSSION Since its discovery in 1977 , PKC has been implicated in the activation of many cell types . Specific activity of PKC in human peripheral lymphocytes was found to be 20 times higher than that o f other tissues like liver, kidney, heart, adipose tissue, and skeletal muscle . In B lymphocytes, PKC can be activated by cross-linking o f m l g [8, 9]. In the mouse, anti-HLA class II antibodies have been used to activate PKC in B
|21 ~ 143--[
recent reports suggesting that PKC activation is not necessarily associated with translocation. Warner et el.  have demonstrated that IgE-mediased activation of human basophils is accompanied by an increase in total PKC activity which cart be attributed solely to the increase in membrane-associated PKC activity without any change in cytosolic activity. Another report shows that there is a lack of correlation between translocatioo and biological effects mediated by PKC . The concentration ofphorbol ester, which induces the half maximal expression (HMR) of biological response, was compared with the half maximal translocadon (HMT) in different cell types. An inhibition of alkaline phosphatase expression required an H M R of less than 0.1 mM phorbul, which causes the translocation of approximately 5% of the maximal PKC activity in B lymphocytes. Expression of MHC class II antigens and membrane depolarization of resting B cells required an H M R of 1 to 5 oM, which corresponds to 30% ofrranslocation suggesting that important biological effects can be accompanied with minimal PKC translocation. Our study of resting B cells revealed both quantitatively (Fig. 2) and qualitatively (Figs. 3 and 4) that the ligation of HLA class I1 antigens induced PKC activation. However, in contrast to TPA, a heterogeneity of the response was observed among the donors tested, including an augmentation of cytosolic PKC activation or of membrane PKC activity. The two different types of PKC response observed in different donors could be due to various factors including imperceptible differences in the state of activation of the allegedly "resting" cells, the expression and therefore the binding of the HLA antigens, the availability of PKC substrate(s), the "residual" level of PKC itself, and eventually haplotypic differences. These initial variables are now under investigation. Previous studies in the mouse have found that PKC activation results in a translocation of the enzyme from the cytosol to the nucleus [2, 3]. We could not observe a translocation phenomenon since we have never observed a decrease in cytosolic PKC activity following activation of human B lymphocytes via the class II antigen. Furthermore, we have observed that PKC activation via class II antigen results in an increase of cytosulic and/or particular PKC activity. The difference between the PKC activation pathway in the human and in the mouse following stimulation via class II antigen has yet to be resolved. However, this may result either from intrinsic difference in second messenger signaling systems in the mouse and in the human, or from differences in the antibodies used. Indeed, anti-HLA class II antibodies used in the human recognize monomorphic determinants as opposed to allelic structure in the mouse. Alternatively, the B-cell I~opnlation examined
C. Brick-Ghannam et ai.
may represent a slighdy distinct population or activation stage. The high degree of structural polymorphism of the HLA class I1 system is undoubtedly of importance for the function of antigen presentation. However, the regions of highly conserved sequences may reflect a separate function of the HLA class II molecules which was developed and/or maintained independently of its function in antigen presentation. Evaluation of signal transduction via M H C class II molecules indifferent species will provide further information regarding the evolution of the signal transducing role of the M H C class II molecules. ACKNOWLEDGMENTS The authors wish to thank Mrs. Mutiel Brandel for t/ping the manuscript. We are also grateful to P. Tran. This work was supported by grants from INSERM, ARC, and LNFCC. C. Brick.Ghannam is a recipient of a fellowship from I.NFCC. REFERENCES 1. Williams AF, Barclay AN: The immunnglobulln superfamily domains for cell surface recognition. Ann Rev Immunol 6:381, 1988. 2. Cambier JC, Newell MK, Justemem LB, McGuire JC, Leach KI., Chen, ZZ: la binding llgands and cAMP stimulate nuclear translocation of PKC in B lymphocytes. Nature 327:629, 1987. 3. Chert ZZ, McGuire JC, Learh KL, Cambier J: Transmembrahe signaling through B cell MHC class I1 molecules: Ami-la antibodies induce protein kinase C translocation to the nuclear fraction. J Immunol 138:2345, 1987. 4. Palacios R, Martinez-Maza O, Guy K: Monoclonal antibodies against HLA-DR antigens replace T helper ceils in activation of B lymphocytes. Proc Natl Acad Sci USA 80:3456, 1983. 5. Clement LT, Tedder TF, Gardand GL: Antibodies reactive with class I1 antigens encoded for by the major histocompatibilit/complex inhibit human B cell activation. J Immunol 136:2375, 1986. 6. Tanaka Y, Shivakawa F, Ota T, Suzuki H, Ere S, Yamashita U: Mechanism of spontaneous activation of B cells in patients with systemic lupus erythematosus. J Immunol 140:761, 1988. 7. Mooney N, Gtillot-Courvalin C, Hivroz D, Charron D: A role for MHC class II antigens in B cell activation. J Autoimmunity 2:215, 1989. 8. Chert ZZ, Cnggeshall KM, Cambier JC: Translocation of protein kinase C during membrane immunnglobulin mediated transmembrane signaling in B lymphocytes. J Immunol 136:2300, 1986. 9. Nel AE, Wooten MW, Landreth GE, Goldschmidt-Clermont PJ, Stevenson HC, Miller PJ, Galbraith RM: Tram-
MHC Class II Signalingvia PKC Activation
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the principle of protein dye binding. Anal Biochem 72:248, 1976. 18. CastaguaM, Takai y, Kaibuehi K, Sano K, Kikkawa U, Nishiauka Y: Direct activationof caiciura activated phospholipid dependent protein kinase b~f tumor promoting phorbol esters. J Biol Chetu 257:7847, 1982, 19. NishiankaY: "the role of protein kinase C in cell surface signal mmsduction and tomour promotion. Nature 308:693, 1984. 20. Takal Y, Kishimoto A, Inoue M, Nushizuka Y: Studies on a cyclic nucleotide independent protein kinase and its proenzyme in mammaliantissues. 1. Purificationand characterlzation of an active enzyme from bovine cerebellum. J Biol Cbetu 252:7603, 1977. 21. NishizntmY: Studies and perspectives of protein kinase C. Sciences 223:305, 1986. 22. Og~waY, Yakal Y, Kawahara Y, Kimura S, NishizukaY: A new possible regulatorysystem for protein phusphoryladon in human peripheral lytuphocytes. I. Characterization of a calcium activated phospholipid dependent prorein kinase.J Immunol 127:1369, 1981. 23. Warner JA, MacGlashan DW, Jr: Protein kinase C (PKC) changes in human basophils. IgE mediated activation is accotupanied by an increase in total PKC activity.J Itutuunol 142:1669, 1989 24. Bosca L, Marquez C, Martinez-A C: Lack of correlation between translocationand biologicaleffects mediated by protein kinase C: An sppraisaL Immnnol Today 10:223, 1989.