B-cell Signaling: Protein Kinase CδδPuts the Brakes on

Dispatch

Diane Mathis and George L. King

The phenotype of mice lacking the delta isoform ofprotein kinase C reveals that this isoform curtailssignaling events after engagement of the antigen-specific receptor on B cells. The result is a state ofnon-responsiveness, termed anergy, that representsone form of immunological self-tolerance.

Protein kinase C (PKC) is one of the cell’s most impor-tant signal-transducing molecules, responding to adiversity of extracellular cues to regulate a number ofcentral processes, including proliferation, metabolism,migration and death [1]. It is also one of the morecomplex signal transducers, constituting a family ofserine/threonine kinases that can phosphorylate amultitude of cellular substrates. The family currentlyincludes 12 isoforms, which are structurally relatedand have been grouped into 3 classes according towhether or not they require diacylglycerol (DAG)and/or Ca2+ for activation. The various isoforms havedifferent cell-type distributions, and show differentialcompartmentalizations within cells upon activation.More confounding, a single isoform can have distinct,even opposing, functions in different cell types. It hasbeen a major challenge to penetrate this complexity inorder to attribute to specific isoforms specific func-tions in specific cell types. Fortunately, some keyadvances have come in the past few years thanks tothe development of transgenic mouse lines over-expressing a particular PKC isoform, knockout mouselines lacking a single isoform, and molecule-specificinhibitors. A good example is provided by two recentpapers that reported an important role for PKCδδ in tol-erance induction in B lymphocytes [2,3].

PKC activation appears to influence multiple facetsof immune system function, from T and B lymphocytedifferentiation to various forms of leukocyte activation.Mice overexpressing PKCαα in T lymphocytes exhib-ited decreased T-cell proliferative responses, a biasedpattern of T-cell cytokine secretion and reduced pro-duction of antibodies by B cells [4]. Mice lackingPKCββ showed reduced B-cell proliferative responsesand impaired humoral immunity [5]. Since these twomolecules belong to the ‘classical’ class of isoforms,which requires both DAG and Ca2+ for activation,investigations into PKC activities focused for sometime on immune system functions that depend onboth of these intracellular mediators. However, recentreports [2,3,6] of T- and B-cell abnormalities in mice

lacking PKCθθ and PKCδδ, respectively, have changedthis emphasis because these kinases are both of the‘novel’ class of isoforms, whose activation requiresDAG but not Ca2+. Several observations have piquedinterest in examining the role of PKCδδ in the immunesystem. For example, this isoform has the unusualproperty of being tyrosine phosphorylated [7], and thisphosphorylation event takes place within a minute ofengagement of the surface immunoglobulin (Ig) recep-tors on B cells [8,9]. In addition, this isoform can havea potent negative influence on cell behavior, inhibitingproliferation and enhancing death [7].

When PKCδδ-deficient mice were screened forimmune system manifestations, the most overt abnor-malities were splenomegaly and lymphadenopathy,both attributed to an increase in the number of B cellsof the conventional, B2, type. This augmentation wasnot observed in the bone marrow, indicating that it isa post-maturation phenomenon. Abnormal B-cellaccumulation was also revealed by the unusually highnumbers of germinal centers in the spleen and lymphnodes in the absence of intentional antigenic stimula-tion. Interestingly, smooth muscle cells from theseknockout mice were previously reported to showaberrant homeostasis, but the mechanism appearedto differ from that for B cells: a normal proliferativecapacity [3] and decreased propensity for cell death[2,3] for the former; increased proliferation and normaldeath for the latter [10]. The B-cell abnormalities inPKCδδ-deficient mice had pathological consequences.Concentrations of serum IgG1 and IgA were increased,and auto-antibodies recognizing a variety of auto-anti-gens could be detected after 6 months of age. Igswere aberrantly deposited in the kidneys, and B-cellinfiltrates were found in perivascular regions of multi-ple organs. Clearly, then, B-cell tolerance to self-anti-gens was somehow compromised.

To explore the mechanisms underlying the loss ofB-cell tolerance in mice lacking PKCδδ, Mecklen-brauker et al. [2] introduced the knockout mutationinto a well-studied transgenic mouse system focusedon Igs reactive to hen egg lysozyme (HEL). Single-transgenic mice (IgHEL) that carry pre-rearranged Iggenes encoding the heavy and light chains of an anti-body that recognizes HEL develop a B-lymphocyterepertoire highly skewed for HEL-reactive cells, andproduce large amounts of anti-HEL antibodies uponantigenic stimulation [11]. Double-transgenic mice(IgHEL/mHEL) that also harbor a second transgeneencoding a membrane-bound form of HEL as a self-antigen lack mature HEL-responsive B cells becausethese cells are deleted during maturation in the bonemarrow [12]. If, instead, the second transgene (sHELin IgHEL/sHEL mice) encodes a soluble form of HELas the self-antigen, significant numbers of matureHEL-reactive B cells are generated, but these do notproduce anti-HEL antibodies because they have beenrendered non-responsive, or anergic [11]. This has

Current Biology, Vol. 12, R554–R556, August 20, 2002, ©2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)01052-7

Sections on Immunology and Immunogenetics, and VascularCell Biology and Complications, Joslin Diabetes Center andDepartment of Medicine, Brigham and Women’s Hospital,Harvard Medical School, One Joslin Place, Boston,Massachusetts 02215, USA. E-mail: cbdm@joslin.harvard.edu

proven a very powerful system for probing the signal-ing pathways implicated in anergy induction [13–17].The current picture, schematized in Figure 1, is thatthe anergic B lymphocytes from IgHEL/sHEL mice arein an activated state compared with antigen-naïve B cells from IgHEL mice, but that the activation isincomplete in comparison with that of antigen-stimu-lated B cells from IgHEL animals. The aborted activa-tion involves low calcium oscillations, muted proteintyrosine phosphorylation, and mobilization of the tran-scription factor NFAT and the MAP kinase ERK; but itstops short of a typical biphasic calcium response, astandard pattern of protein tyrosine phosphorylation,and additional mobilization of the NF-κκB transcriptionfactor and the MAP kinase JNK [13,15,16]. The ulti-mate result is a negative response: lack of prolifera-tion, reduction in survival, blockade of differentiation,and a failure to express T-cell co-stimulatory mol-ecules [13,14], reflecting induction of genes whichencode negative regulators of signaling and transcrip-tion but not genes promoting proliferation [17]. Wheredoes PKCδδ come in?

Breeding of the PKCδδ knockout mutation into double-transgenic IgHEL/mHEL mice had no overt effect: asusual, potentially auto-reactive HEL-responsive B cellswere deleted in the bone marrow. However, introduc-tion of the mutation into IgHEL/sHEL animals showeda striking influence: HEL-reactive B cells emerged intothe periphery as usual, but they were no longer toler-ant to HEL, no longer anergic. These animals had sig-nificant titers of anti-HEL antibodies in the serum, andtheir B cells resembled those of non-transgenic or

IgHEL mice in many features of their behavior, culmi-nating in a normal response to HEL. Thus, chronicexposure of PKCδδ-deficient B cells to self-antigenstimulation failed to induce tolerance, via anergy, as itdoes for wild-type B cells; in marked contrast, acuteexposure to the same antigen was perfectly capableof eliciting immunity.

The search is now on for the molecular basis of theB-lymphocyte abnormalities exhibited by PKCδδ-defi-cient mice. One group [3] observed an augmentation ofinterleukin-6 production upon engagement of surfaceIg (and the costimulatory molecule CD40), attributed toan increase in the transcription factor NF-IL6. Thisseems an attractive explanation for at least some ofthe B-cell aberrancies because mice lacking PKCδδ andtransgenic mice overexpressing IL-6 exhibit a numberof similarities. The other group [2] focused on the tran-scription factor NF-κκB, no doubt prompted by a previ-ous report [15] that this factor is not activated inanergic B cells, neither ex vivo nor after cognateantigen stimulation in vitro. This lack of activity is dueto reduced nuclear translocation of NF-κκB, a reflectionof inefficient degradation of an inhibitor that seques-ters it in the cytoplasm, IκκB. Indeed, the non-anergic Bcells from IgHEL/sHEL mice lacking PKCδδ showedenhanced NF-κκB activation, greater than that of anergicB cells from double-transgenic animals expressingPKCδδ, both with and without exogenous HEL stimula-tion, and even greater than that of non-anergic B cellsfrom IgHEL animals, whether or not they expressedPKCδδ. Rather surprisingly, degradation of IκκB was not diminished in parallel with enhanced activation of

Current BiologyR555

Figure 1. Different signals emanatingfrom the B cell antigen receptor.

Left, events downstream of an activationstimulus (e.g. a foreign antigen). Right,events downstream of a tolerogenic stim-ulus (such as a soluble self-antigen). Theupper curve represents the plasma mem-brane, and the inner represents thenuclear membrane. Ca2+ depicts thecalcium signal, with the size of the trianglereflecting the strength and duration of thissignal. ERK, extracellular signal regulatedkinase; JNK, c-Jun N-terminal kinase;NFAT, nuclear factor of activated T cells;NFκκB, nuclear factor κκB. ERK NFAT NF-κB JNK

Ca2+

Selfantigen

Immunity

Activation signal (acute)

Tolerance (anergy)

Positive programProliferationMigration into folliclesDifferentiationAntibody productionCostimulatory capacityIncreased survival

Negative programBlock in: Migration Differentiation Antibody production CostimulationDecreased survival

ERK

Nucleus

Cellmembrane

NFAT NF-κB JNK

Current Biology

Immuno-globulin

Ca2+

Foreignantigen

Tolerance signal (chronic)

DispatchR556

NF-κκB, suggesting that there might be an alternativeroute to boost activation. The differences in NF-κκBbehavior in the presence and absence of PKCδδ were aproperty of the anergic state, as they were not observedwith B cells from either non-transgenic or IgHEL mice.

These new findings pointing to a ‘braking’ role forPKCδδ during anergy induction in B cells are of signifi-cant interest from a number of perspectives. Firstly,they pose again the question, in a perfectly matchedsetting, of how the same signal transducer can havedivergent effects in two cell types: increased prolifer-ation [3], normal death [2,3] in B cells; and normal pro-liferation, increased death in smooth muscle cells [10].The answer must await painstaking dissection of thesequence of events downstream of PKCδδ activation inthe two cell types. Secondly, these studies raise theissue of what transducer might have an analogousfunction in T cells, which also undergo anergy induc-tion when confronted with a self-antigen under theappropriate circumstances. In fact, there are similari-ties in the activation state of anergic T and B cells,notably normal NFAT [15,18] but defective NF-κκB[15,19] activation. No T-cell abnormalities wereobserved in the PKCδδ-deficient mice. PKCθθ has anumber of features in common with PKCδδ,, such as notrequiring Ca2+ for activation and perhaps being moredependent on phosphatidylinositol 3-kinase than DAGas an upstream regulator [8,20]. The results on PKCθθknockout mice argue for this isoform having a positiverole in the T-cell response, however, being essentialfor activation of mature T cells through the antigenreceptor [6]. It is imperative now to look elsewhere.Finally, one cannot help but evoke the potential ofPKCδδ-specific modulators to prevent or reverse B-cell-mediated autoimmune diseases, in particular sys-temic lupus erythematosus.

References1. Liu, W.S. and Heckman, C.A. (1998). The sevenfold way of PKC reg-

ulation. Cell. Signal. 10, 529–542.2. Mecklenbrauker, I., Saijo, K., Zheng, N.Y., Leitges, M. and

Tarakhovsky, A. (2002). Protein kinase Cdelta controls self-antigen-induced B-cell tolerance. Nature 416, 860–865.

3. Miyamoto, A., Nakayama, K., Imaki, H., Hirose, S., Jiang, Y., Abe,M., Tsukiyama, T., Nagahama, H., Ohno, S., Hatakeyama, S. et al.(2002). Increased proliferation of B cells and auto-immunity in micelacking protein kinase Cdelta. Nature 416, 865–869.

4. Ohkusu, K., Du, J., Isobe, K.I., Yi, H., Akhand, A.A., Kato, M., Suzuki,H., Hidaka, H. and Nakashima, I. (1997). Protein kinase C alpha-mediated chronic signal transduction for immunosenescence. J.Immunol. 159, 2082–2084.

5. Leitges, M., Schmedt, C., Guinamard, R., Davoust, J., Schaal, S.,Stabel, S. and Tarakhovsky, A. (1996). Immunodeficiency in proteinkinase c beta-deficient mice. Science 273, 788–791.

6. Sun, Z., Arendt, C.W., Ellmeier, W., Schaeffer, E.M., Sunshine, M.J.,Gandhi, L., Annes, J., Petrzilka, D., Kupfer, A., Schwartzberg, P.L.et al. (2000). PKC-theta is required for TCR-induced NF-kappaBactivation in mature but not immature T lymphocytes. Nature 404,402–407.

7. Gschwendt, M. (1999). Protein kinase C delta. Eur. J. Biochem. 259,555–564.

8. Popoff, I.J. and Deans, J.P. (1999). Activation and tyrosine phos-phorylation of protein kinase C delta in response to B cell antigenreceptor stimulation. Mol. Immunol. 36, 1005–1016.

9. Barbazuk, S.M. and Gold, M.R. (1999). Protein kinase C-delta is atarget of B-cell antigen receptor signaling. Immunol. Lett. 69,259–267.

10. Leitges, M., Mayr, M., Braun, U., Mayr, U., Li, C., Pfister, G., Ghaf-fari-Tabrizi, N., Baier, G., Hu, Y. and Xu, Q. (2001). Exacerbated veingraft arteriosclerosis in protein kinase Cdelta-null mice. J. Clin.Invest. 108, 1505–1512.

11. Goodnow, C.C., Crosbie, J., Adelstein, S., Lavoie, T.B., Smith-Gill,S.J., Brink, R.A., Pritchard-Briscoe, H., Wotherspoon, J.S., Loblay,R.H., Raphael, K. et al. (1988). Altered immunoglobulin expressionand functional silencing of self-reactive B lymphocytes in trans-genic mice. Nature 334, 676–682.

12. Hartley, S.B., Crosbie, J., Brink, R., Kantor, A.B., Basten, A. andGoodnow, C.C. (1991). Elimination from peripheral lymphoid tissuesof self-reactive B lymphocytes recognizing membrane-bound anti-gens. Nature 353, 765–769.

13. Cooke, M.P., Heath, A.W., Shokat, K.M., Zeng, Y., Finkelman, F.D.,Linsley, P.S., Howard, M. and Goodnow, C.C. (1994). Immunoglob-ulin signal transduction guides the specificity of B cell-T cell inter-actions and is blocked in tolerant self-reactive B cells. J. Exp. Med.179, 425–438.

14. Cyster, J.G. and Goodnow, C.C. (1995). Antigen-induced exclusionfrom follicles and anergy are separate and complementaryprocesses that influence peripheral B cell fate. Immunity 3,691–701.

15. Healy, J.I., Dolmetsch, R.E., Timmerman, L.A., Cyster, J.G., Thomas,M.L., Crabtree, G.R., Lewis, R.S. and Goodnow, C.C. (1997). Differ-ent nuclear signals are activated by the B cell receptor during pos-itive versus negative signaling. Immunity 6, 419–428.

16. Dolmetsch, R.E., Lewis, R.S., Goodnow, C.C. and Healy, J.I. (1997).Differential activation of transcription factors induced by Ca2+

response amplitude and duration. Nature 386, 855–858.17. Glynne, R., Akkaraju, S., Healy, J.I., Rayner, J., Goodnow, C.C. and

Mack, D.H. (2000). How self-tolerance and the immunosuppressivedrug FK506 prevent B-cell mitogenesis. Nature 403, 672–676.

18. Li, W., Whaley, C.D., Mondino, A. and Mueller, D.L. (1996). Blockedsignal transduction to the ERK and JNK protein kinases in anergicCD4+ T cells. Science 271, 1272–1276.

19. Sundstedt, A., Sigvardsson, M., Leanderson, T., Hedlund, G.,Kalland, T. and Dohlsten, M. (1996). In vivo anergized CD4+ T cellsexpress perturbed AP-1 and NF-kappa B transcription factors. ProcNatl. Acad. Sci U.S.A. 93, 979–984.

20. Villalba, M., Bi, K., Hu, J., Altman, Y., Bushway, P., Reits, E., Neefjes,J., Baier, G., Abraham, R.T. and Altman, A. (2002). Translocation ofPKC[theta] in T cells is mediated by a nonconventional, PI3-K- andVav-dependent pathway, but does not absolutely require phospho-lipase C. J. Cell Biol. 157, 253–263.