ARTHRITIS & RHEUMATISMVol. 46, No. 5, May 2002, pp 1139–1154DOI 10.1002/art.10192© 2002, American College of Rheumatology

REVIEW

Abnormal T Cell Signal Transduction inSystemic Lupus Erythematosus

Gary M. Kammer,1 Andras Perl,2 Bruce C. Richardson,3 and George C. Tsokos4

Introduction

Systemic lupus erythematosus (SLE) is an acuteand chronic autoimmune disease of indeterminate etiol-ogy characterized by protean clinical symptoms andsigns. Although this syndrome can occur in children orthe elderly (1), SLE predominantly affects women intheir childbearing years. Current information suggeststhat a complex interaction between environmentalagents and disease susceptibility genes may predisposeto SLE. Although many of these genes have not yet beenidentified, it is currently believed that the expression ofsuch genes may predispose to aberrant cellular andhumoral immune responses. Altered immune responsesto autoantigens then result in pathologic autoantibodyproduction, formation and deposition of antigen–antibody complexes, and propagation of a chronic in-flammatory process that destroys organ parenchyma andresults in end-stage organ failure, such as end-stagerenal disease (2).

At the cellular level, T lymphocytes mediate thisaltered immune response in SLE. Indeed, comparedwith normal controls in whom a balance of T cellfunctions exists (Figure 1A), diverse T cell dysfunctionsresult in an imbalance in the functions of T cell subsetsin SLE (3–5) (Table 1 and Figure 1B). A pan–T celldysfunction appears to exist that is characterized byexaggerated CD4 and diminished CD8 T cell activities.Moreover, natural killer (NK) cell functions are alsodefective (6). Due to the loss of effective CD8 T and NKcell feedback on B cells (7), forbidden B cell clonesproduce autoantibodies directed against an array ofintra- and extracellular autoantigens.

Inquiries into the mechanism(s) causing these Tcell dysfunctions have led to the identification of severalnew defects of signal transduction and have begun toreveal their molecular basis. It is timely, therefore, toreview our progress during the 6 years since this subjectwas previously reviewed (8) to assess how abnormalsignaling events contribute to our understanding of Tcell dysfunctions in SLE. Second, we address the impactthese signaling defects are likely to have on regulationand expression of IL2, a representative T cell–specificgene. Third, we summarize new signaling abnormalitiesthat contribute to T cell apoptosis and necrosis, im-paired protein translation, and altered DNA methyl-ation. Fourth, we highlight initial genetic approaches inSLE T cells designed to bypass and to reconstitute asignaling defect and its effect on interleukin-2 (IL-2)production. Finally, we identify likely directions of fu-ture research that are anticipated to yield new informa-tion and novel insights into T cell effector dysfunctionsin SLE.

Aberrant T cell signal transduction in SLE

Several defects of the signaling cascade in SLE Tcells have been discovered over the last decade (8–10).

Supported by grants RO1-AR39501, RO1-AI46526, RO1-AI42269, RO1-DK49221, RO1-AI48079, RO1-AG014783, RO1-AR42525, and RO1-AI42753 from the USPHS, by a Merit grant fromthe Department of Veterans Affairs, by the Lupus Foundation ofAmerica, and by grant MO1 RR07122 from the General ClinicalResearch Center, Wake Forest University School of Medicine.

1Gary M. Kammer, MD: Wake Forest University School ofMedicine, Winston-Salem, North Carolina; 2Andras Perl, MD, PhD:State University of New York Health Science Center College ofMedicine, Syracuse; 3Bruce C. Richardson, MD, PhD: University ofMichigan Medical Center, Ann Arbor; 4George C. Tsokos, MD:Uniformed Services University of the Health Sciences, Bethesda,Maryland, and Walter Reed Army Institute of Research, Silver Spring,Maryland.

Address correspondence and reprint requests to Gary M.Kammer, MD, Section on Rheumatology and Clinical Immunology,Wake Forest University School of Medicine, Medical Center Boule-vard, Winston-Salem, NC 27157. E-mail: gmkammer@wfubmc.edu.

Submitted for publication August 3, 2001; accepted in revisedform November 26, 2001.

1139

Figure 2 shows the topography of these defects, whichcan be roughly divided into 3 regions: proximal, middle,and distal. In addition to these discrete defects ofbiochemical signaling pathways in SLE T cells, theexistence of mitochondrial hyperpolarization and in-creased reactive oxygen species has just recently beenidentified (11). This discovery may explain in part why ahigher proportion of SLE T cells undergoes apoptosis.

Table 2 summarizes the signaling defects in SLE T cellsidentified to date.

Signaling defects in SLE T cells

Proximal pathway defects: diminished T cell re-ceptor (TCR) �-chain expression in SLE T cells. Underphysiologic conditions, binding of major histocompati-bility complex (MHC)–peptide complexes on the surfaceof antigen-presenting cells (APCs) to the TCR crosslinksit and induces polarization and noncovalent associationof CD4 coreceptors with CD3 (12). Efficient signalinitiation requires phosphorylation of tyrosyl residueswithin immune receptor tyrosine-activated motifs(ITAMs) in the cytoplasmic domains of the �, �, �, and� chains. Each chain of the � homodimer possesses 3ITAMs, whereas the other CD3 chains each contain 1ITAM. Fyn kinase phosphorylates the tyrosyl residues ofthe ITAMs, and subsequently, the tyrosine kinase, ZAP-70, docks to Src homology 2 domains of the � chains.Following contact between CD4 and class II MHC, lckkinase then phosphorylates ZAP-70 on tyrosyl residues,activating the kinase (13).

In contrast to normal and rheumatic diseasecontrol T cells, � protein is deficient in the T cells of amajority of SLE patients (14,15). Although multiplemolecular mechanisms may underlie �-chain deficiency,reduced or absent �-chain transcripts implicate de-creased gene transcription (14). In cells that expressed �-chain transcripts, cloning and genomic DNA sequencingrevealed widely divergent mutations and silent polymor-phisms in the coding region. Compared with normalcontrols, the frequency of polymorphisms/mutations wassignificantly higher in SLE (16,17). In addition, �-chaintranscripts displayed an increased frequency of alterna-tively spliced forms in one or more of the 8 exons (16).Although the proximal promoter region of the �-chaingene did not display any structural defects that wouldaccount for the decreased transcription of the gene, thenontranslated downstream region of the �-chain genewas shorter, which may account for decreased messenger

Figure 1. Relationships between T cell subsets in conditions of healthand systemic lupus erythematosus (SLE). A, In conditions of health,there is a balance between the two principal T cell subsets, CD4 Thelper cells (Th) and CD8 T suppressor/cytotoxic cells (Ts/c). Presen-tation of antigen (Ag) by antigen-presenting cells (APCs), such asmacrophages or dendritic cells, results in appropriate expression of cellsurface adhesion molecules and production of cytokines that regulateB cell maturation to plasma cells and production of immunoglobulins(antibodies). B, In SLE, there is an imbalance in T cell subsetfunctions, resulting in enhanced CD4 Th and diminished CD8 Ts/c

regulation of B cell production of immunoglobulins. Reduced CD8 Ts/c

function may contribute to hypergammaglobulinemia due to impairedregulation of forbidden B cell clones. There is also skewed cytokineproduction and altered adhesion molecule expression on immune cells.AutoAg � autoantigen; autoabs � autoantibodies.

Table 1. Diverse T lymphocyte dysfunctions in systemic lupuserythematosus*

Exaggerated activity of CD4 T helper cellsDiminished function of CD8 T suppressor/cytotoxic cellsReduced antibody-dependent cytotoxicityImbalanced Th1 and Th2 cytokine productionReduced proliferative responses to antigens/alloantigens/mitogens

* For review, see refs. 2, 4, 5, and 8.

1140 KAMMER ET AL

RNA (mRNA) stability (17). Taken together, thesemutations are expected to contribute to critical func-tional abnormalities of the � chain. How these mutationsaffect �-chain transcription and translation remains to beestablished.

TCR � chain has also been reported to be de-creased in human immunodeficiency virus–infected Tcells, in tumor-infiltrating T cells (9), and in in vitro–generated effector T cells (18). However, there appearto be some fundamental differences between T cellsunder these circumstances and SLE T cells. For exam-ple, the TCR � transcript is present in T cells under eachof these circumstances, whereas it is often reduced orabsent in SLE. Second, TCR-initiated signals stimulateeffector T cells to produce interferon-� (IFN�), whereasidentical signals result in significantly reduced amountsof IFN� production by SLE T cells (9,18).

Although diminished � chain expression does not

readily explain the markedly higher peak intracellularcalcium concentrations ([Ca��]i) (see below), it canaccount for both the decreased activation-induced celldeath (AICD) and the diminished NK cell activity. Bothfunctions require �-chain expression (9).

Two other chains belong to the �-chain group ofproteins and can replace the missing � chain. Theso-called � chain is similar to the � chain, except thatexon 8 has been replaced by exon 9. Preliminary evi-dence suggests that the � chain expression is alsodecreased. The � chain of the Fc� receptor type I(Fc�RI) has a single ITAM in its intracytoplasmic regionand can signal for the TCR. This chain appears to beoverexpressed in both CD4 and CD8 T cells in SLEpatients compared with controls. Interestingly, the �chain associates with the CD3-� chain as well as with theprotein kinase, syk. Crosslinking of CD3-� leads tophosphorylation of both Fc�RI and syk, suggesting that

Figure 2. Overview of T cell signaling abnormalities identified to date. 1, Deficient CD45 tyrosyl phosphataseactivity; 2, reduced/absent T cell receptor (TCR) � homodimer expression; 3, slightly increased inositoltrisphosphate (IP3) concentrations; 4, increased and prolonged intracellular calcium concentrations followingactivation via the TCR–CD3 complex; 5, reduced protein kinase C (PKC)–dependent protein phosphorylation;6 and 7, deficient type I PKA (PKA-I) and type II PKA (PKA-II) phosphotransferase activities; 8, reducedmitogen-activated protein kinase kinase (MEK)–catalyzed extracellular signal–regulated kinase (ERK) phos-phorylation; 9, increased PKR-catalyzed phosphorylation of eukaryotic initiation factor 2�; 10, diminishedactivity of DNA methyltransferase 1 (Dnmt1). PI-3K � phosphatidylinositol 3-kinase; AC � adenylyl cyclase;PLC-� � phospholipase C-�; MEKK � MEK kinase; JNK � c-Jun N-terminal kinase; JNKK � JNK kinase.

ABNORMAL T CELL SIGNAL TRANSDUCTION IN SLE 1141

the Fc�RI is functional. Indeed, it may take over func-tions assigned to � chain (19).

Proximal pathway defects: increased and pro-longed [Ca��]i in SLE T cells. Initiation of a signal fromthe TCR–CD3 complex results in the rapid ingress ofextracellular Ca�� via membrane Ca�� channels, aswell as the release of intracellular stores of Ca�� fromthe endoplasmic reticulum. Together, this results in arise of the second messenger Ca��, which is required forfunctional activation of Ca��-dependent enzymes, suchas calcineurin phosphatase or calcium/calmodulin ki-nases.

An enhanced and prolonged rise in [Ca��]ifollowing activation via the TCR has been identified inprimary SLE T cells, T cell lines, and antigen-specific Tcell clones (9). This altered [Ca��]i is unrelated toglobal disease activity or any specific organ involvement.However, in its role as a second messenger, Ca��

mediates calcineurin-catalyzed dephosphorylation of thetranscription factor, nuclear factor of activated T cells(NF-AT), resulting in its nuclear translocation. Interest-ingly, genes whose transcription is regulated by NF-AT,such as CD154, Fas ligand, and c-myc, are overexpressedin SLE T cells (20–23). Impaired regulation of [Ca��]imight account for the altered expression of these mole-cules in SLE.

Although the precise mechanisms underlying thisaberrant Ca�� response have not yet been identified,

the interrelationships between this second messengerand the adenylyl cyclase (AC)/cAMP/protein kinase A(PKA) pathway (24) raise the possibility that deficientPKA activity (8,10) (see below) in SLE T cells maycontribute in part to impaired Ca�� homeostasis. Underphysiologic conditions, ligand binding to a G protein–associated receptor in different cell types activates bothAC and phospholipase C (PLC) isozymes (25). More-over, ligand binding to the TCR induces tyrosyl phos-phorylation of PLC-�1 and its activation (26). Theresulting production of inositol 1,4,5-trisphosphate (IP3)induces a rapid rise in [Ca��]i that is down-regulated byPKA-dependent phosphorylation of PLC-�1 and itsinactivation (27–29). Inhibition of PKA-catalyzed pro-tein phosphorylation blocks its inhibitory effect on IP3-induced Ca�� release (30). In SLE, it is conceivable thatthe heightened and prolonged [Ca��]i observed follow-ing T cell activation may in part be a result of deficientPKA-catalyzed phosphorylation of PLC-�1 and inabilityto inactivate PLC-�1 enzymatic activity.

Middle pathway defects: defective cAMP-dependent protein phosphorylation due to deficientactivities of type I and type II isozymes of PKA (PKA-Iand PKA-II). Abnormal signal transduction in SLE Tcells was first identified by abnormal cAMP metabolismand markedly impaired cAMP-dependent protein kinaseprotein phosphorylation (31,32). This abnormal signal-ing pathway was associated with deficient CD8 suppres-sor T cell function (31–33). Later studies also implicatedan abnormal cAMP-dependent signaling pathway inaltered cytoskeletal regulation of CD3, CD4, and CD8receptor mobility within the plane of the plasma mem-brane (33,34). These findings then led to the identifica-tion of a profound deficiency of cAMP-activatable PKAactivity in SLE T cells (35). This was the first identifica-tion of a deficient protein kinase activity in this auto-immune disease.

More recently, we demonstrated that this pro-found reduction of total PKA activity reflects a defi-ciency of the PKA-I and PKA-II isozymes (35,36).Under physiologic conditions, PKA-I is composed of twoholoenzymes: RI�2C2 (also written as PKA-I�) andRI�2C2 (PKA-I�). (RI� and RI� are the � and �isoforms of the type I regulatory [RI] subunit.) PKA-IIalso comprises two holoenzymes: RII�2C2 (also writtenas PKA-II�) and RII�2C2 (PKA-II�).

Among SLE patients, the expected prevalencesof deficient PKA-I and PKA-II activities are �80% and�40%, respectively; furthermore, low enzyme activity ispersistent over time and independent of clinical diseaseactivity (36,37). Although the mean PKA-I activity is

Table 2. Currently identified signaling abnormalities in systemiclupus erythematosus T cells*

Pathway segment, defect Reference

ProximalAltered CD45 tyrosyl phosphatase activity 116Reduced/absent TCR �-chain homodimer 2, 14–17Enhanced tyrosyl phosphorylation and lck

tyrosyl kinase activity14, 114

Heightened [Ca��]i 2, 9Middle

Deficient PKA-I and PKA-II activities 2, 31, 32, 35–41Reduced PKC-catalyzed phosphorylation 113Altered mitochondrial hyperpolarization,

reactive oxygen intermediates, and T cellapoptosis

11

DistalDiminished MAPKK-catalyzed ERK

phosphorylation84

Increased PKR-dependent phosphorylationof eIF2�

105

Reduced Dnmt1 activity 84

* TCR � T cell receptor; [Ca��]i � intracellular calcium concentra-tion; PKA-I � type I isozyme of protein kinase A; MAPKK �mitogen-activated protein kinase kinase; ERK � extracellular signal–regulated kinase; eIF2� � eukaryotic initiation factor 2�; Dnmt1 �DNA methyltransferase 1.

1142 KAMMER ET AL

20–25% of that of normal T cells, the activity can be�10% of that of controls in some subjects (38). Dimin-ished PKA-I activity is the product of reduced RIsubunit protein content (RI� � RI�) (39). On average,SLE T cells possess only 35% and 70% of the normalamounts of RI� and RI� proteins, respectively. Indeed,T cells from �25% of SLE patients have no detectableRI� protein. Thus, deficient T cell PKA-I activity re-flects reduction of both holoenzymes: RI�2C2 � RI�2C2(39). In contrast, deficient PKA-II activity is the result ofan independent disorder: spontaneous activation ofRII�2C2 holoenzyme and subsequent nuclear transloca-tion of the RII� subunit from the cytoplasm and itsnuclear retention. This results in a deficiency of theRII�2C2 holoenzyme in the cytoplasm. No significantchange in RII�2C2 holoenzyme expression or activityhas been identified (36).

Our current evidence suggests that there may bedefects of both transcription and translation in SLE Tcells. At the level of transcription, investigators in ourgroup have demonstrated that steady-state amounts ofRI� mRNA and RI� mRNA are reduced by aboutone-fifth and one-half, respectively, of the physiologiccontent in normal T cells (39). At the level of translation,there is a reduction of both RI� and RI� translation.Notably, T cells that fail to express RI� protein exhibit aprofound block of RI� translation. However, if SLE Tcell–derived RI� complementary DNAs (cDNA) span-ning the coding region of the protein are transientlytransfected into autologous SLE T cells, the cells canproduce the protein. This leads to a significant rise incAMP-activatable PKA activity and a partial reconstitu-tion of IL-2 production in response to activation via theCD3 and CD28 cell surface receptors (40). These resultssuggest that signaling via the PKA-I isozyme mediatesIL-2 production and raise the question of whetherdeficient PKA-I activity might contribute to the inabilityof TCR-activated SLE T cells to secrete IL-2 in anamount comparable to that of normal T cells in vitro (2).

One mechanism that contributes to deficient RI�transcript is the occurrence of transcript (mRNA) mu-tations. RNA editing is the co- or posttranscriptionalmodification of RNA, which results in the insertion,deletion, or substitution of nucleotides. RNA editing cantherefore modify the information encoded within thecorresponding genomic sequence and can frequentlyalter the function of the affected RNAs. In SLE T cells,we have found that heterogeneous transcript mutations,including deletions, transitions, and transversions, areclustered adjacent to GAGAG motifs and CT repeats,regions that may be “hot spots” for transcript editing

and/or molecular misreading. In contrast, no genomicmutations were identified. These results suggest theoccurrence of mRNA editing and/or defective functionof RNA polymerase. Such mutant RI� transcripts maybe pathophysiologically significant, for they might en-code diverse, aberrant RI� proteins, including trun-cated, dominant-negative subunits, resulting in deficientPKA-I activity. This is the first identification of tran-script mutations affecting any gene in an autoimmunedisease (41).

At this time, the overall impact of transcriptediting in the immunopathogenesis of SLE remains tobe established. However, because transcript editing ofRI� leading to abnormal protein structure was the initialdiscovery of such a mechanism in SLE T cells, we predictthat other genes could also be undergoing such editing.If this proves to be true, transcript editing may becomerecognized as an important mechanism resulting inmutated proteins in SLE cells. One mechanism by whichthis might occur is the conversion of adenosine toinosine and cytidine to uridine in mRNA by the editinggene, adenosine deaminases that act on RNA (ADAR).Evidence from recent investigations by our group sug-gests that ADAR transcripts are significantly up-regulated in SLE T cells (42). Because ADAR-mediatedadenosine deamination tends to occur at certain “hotspots” (43), mutations could occur indiscriminately inother genes possessing such motifs, resulting in mutatedproteins.

To date, we do not know how deficient PKA-Iand PKA-II activities affect other components of thesignaling cascade, such as [Ca��]i and PLC-� activity.Of course, to understand the impact of defective signal-ing on T cell effector functions, such as helper orcytotoxic activities, it is first necessary to learn howdeficient PKA activity modifies the functions of othersignaling components. Our current studies are directedtoward this end.

As we discussed above, current evidence suggeststhat SLE is a polygenic disease (2). Indeed, recentgenome-wide searches in families with multiple affectedmembers have revealed chromosomal regions in thegenome associated with SLE (44–46). A recent reviewdiscusses the criteria for inclusion of genes as putativesusceptibility loci for SLE and enumerates candidatesidentified to date (10). Based on these criteria, the RI�gene may be yet another susceptibility locus for SLE. Ina followup sibpair family analysis of potential suscepti-bility loci, the p22 region of chromosome 7 was found tohave a significantly increased logarithm of odds score of2.87 (47). Because the RI� gene resides within this

ABNORMAL T CELL SIGNAL TRANSDUCTION IN SLE 1143

interval (48), this screen may be detecting the RI� gene.Confirmation of RI� as a susceptibility locus for SLEwill have to await completion of our ongoing familystudies.

Middle pathway defects: altered mitochondrialhyperpolarization, reactive oxygen intermediates(ROIs), and T cell apoptosis. Programmed cell death(PCD), or apoptosis, is a physiologic mechanism for elim-ination of autoreactive lymphocytes during development(49). Current evidence suggests that the regulation of PCDis impaired in both human and murine SLE and couldcontribute to the immunopathogenesis of the disease (2).In lpr and gld mice, defects in PCD signaling through theFas pathway appear to predispose to autoimmunity (50).While mutations of the Fas receptor (FasR) or Fas ligand(FasL) have been associated with a lupus-like autoimmunesyndrome in mice with the lpr or gld genetic background(50), Fas-mediated signaling appears to be intact in humanSLE (51). However, SLE T cells demonstrate defectiveAICD, possibly related to decreased intracellular synthesisof tumor necrosis factor (TNF), which would perpetuatethe longevity of autoreactive cells (52). In contrast, in-creased spontaneous apoptosis of peripheral blood lym-phocytes (PBL) has also been observed in SLE (53). Thus,paradoxically, SLE T cells exhibit both defective AICD andenhanced spontaneous apoptosis.

The signaling pathways and their relationships tothe mitochondrion remain incompletely understood.Figure 3 presents a schematic diagram that depicts theserelationships and highlights several key molecules thatregulate viability versus apoptosis. Disruption of themitochondrial transmembrane potential (��m) has beenproposed as the point of no return in apoptotic signaling(54–56). Recent data indicate that elevation of ��m (i.e.,mitochondrial hyperpolarization) occurs in the earlyphase of Fas-, p53-, and H2O2-induced apoptosis. In-deed, elevation of ��m precedes caspase activation (57),phosphatidylserine externalization, and disruption of��m in Fas- and H2O2-induced apoptosis (54,57,58).

Recently, investigators in our group identifieddeviations in key mitochondrial checkpoints that areassociated with abnormal apoptosis of SLE T cells (11).The mitochondrial transmembrane potential and mito-chondrial ROI production were elevated, whereas glu-tathione (GSH) levels were diminished, compared withhealthy controls or controls with rheumatoid arthritis(RA). Low levels of GSH are consistent with increasedROI production due to utilization of reducing equiva-lents. Ongoing oxidative stress in vivo may lead to theskewed expression of the transcription factors activatorprotein 1 (AP-1) and nuclear factor B (NF-B) (59,60),

and, further downstream, to the skewed expression ofIL-2, TNF, and IL-10 (61). Expression of TCR � chain issensitive to H2O2 (62), and thus, increased ROI levelsmay explain, at least in part, low TCR �-chain expressionin SLE T cells (14,15). Of note, increased spontaneousapoptosis of lymphocytes has been linked to increasedIL-10 production, release of FasL, and overexpression ofthe FasR in SLE (63). Elevated nitric oxide (NO)production may also contribute to increased spontaneousapoptosis (64). Indeed, increased ROI levels confersensitivity to H2O2-, NO-, TNF-, and Fas-induced celldeath (65). Therefore, elevated baseline ROI levels andelevated ��m may play key roles in enhanced spontane-ous death of SLE PBL.

The Bcl-2 family of proteins comprises 19 mem-bers that mediate cell viability and apoptosis. Onemechanism by which Bcl-2 proteins control these pro-cesses is via regulation of the ��m (66). In SLE T cells,however, it remains a controversial subject whether ornot the expression of Bcl-2 is physiologic or heightened(67). While dimerization or overexpression of proapop-totic Bcl-2 members, Bax or Bak, promotes disruption of��m and increased ROI production, overexpression ofantiapoptotic Bcl-2 or Bcl-xL helps maintain ��m anddiminishes ROI production (68). While Bcl-2 overex-pression may help sustain ��m and diminish ROI pro-duction, low Bcl-2 levels would favor disruption of ��mand increase ROI production. Therefore, neither in-creased nor decreased Bcl-2 activity would explain theelevation of baseline ��m and ROI production resultingin increased spontaneous cell death of SLE T cells. Ofnote is that phosphorylation of BAD, another Bcl-2family member, by mitochondria-anchored PKA resultsin sequestration of BAD in the cytosol and is antiapop-totic (69). Therefore, diminished PKA activity in SLE Tcells (2,8) may contribute to translocation of BAD intomitochondria, thus increasing susceptibility to apoptosis.

ROIs can modify signaling initiated via the CD3/CD28 receptors. In response to treatment with exoge-nous H2O2, a precursor of ROIs, SLE T cells failed toundergo apoptosis, and cell death preferentially oc-curred via necrosis (11). Endogenous H2O2 is generatedby superoxide dismutase from the ROIs, O2

� or OH�, inmitochondria (70). In turn, H2O2 is scavenged by cata-lase and GSH peroxidase. While H2O2 is freely diffus-ible, it has no unpaired electrons and, by itself, is not anROI (70). Induction of apoptosis by H2O2 requires itsmitochondrial transformation into an ROI (e.g., OH�)(70,71). As investigators in our group have demon-strated (58), H2O2 triggers a rapid increase of ��m andROI production that is followed by apoptosis of PBL in

1144 KAMMER ET AL

Figure 3. Relationship of signaling pathways, mitochondrial transmembrane potential (��m), and redox control of T cell activation and apoptosis.Crosslinking of Fas/tumor necrosis factor death receptors (FasR and TNFR) by their ligands, FasL and TNF, respectively, recruits an adaptorprotein with a Fas-associated death domain (FADD). Caspase 8 is recruited to the death-inducing signaling complex through its death effectordomain, similar to that of FADD. Upon recruitment of caspase 8, its oligomerization causes self-cleavage and activation of downstream effectorcaspases. Caspase 3–activated DNase (CAD) results in host cellular DNA fragmentation. Through DD-containing proteins, TNFR also interactswith TNFR-associated factor (TRAF), which in turn, activates inhibitor of nuclear factor B kinase (IKK) via the mitogen-activated protein kinasekinase kinase/apoptosis signal–regulating kinase 1 (MAPKKK/ASK1) cascade. ASK1 is maintained in its inactive form when bound to thioredoxin(TRX). Reactive oxygen species (ROS) dissociate TRX from ASK1 and promote multimerization required for kinase activity. Subsequently, IKKactivation leads to phosphorylation (P)–mediated degradation of inhibitor of nuclear factor B (IB) and translocation of nuclear factor B (NF-B)into the nucleus. Antigen binding–initiated signaling through CD3/CD28 activates phosphatidylinositol 3-kinase (PI3K) and protein tyrosine kinases(PTKs). This results in Ca2� fluxing into mitochondria, followed by increased production of ROS and NF-B activation. Mitochondrial membraneintegrity is maintained by a balance of membrane-stabilizing Bcl-2 and Bcl-xL, pore-inducing Bax and BAD, and the metabolic capacity to synthesizereducing equivalents, NADPH, glutathione (GSH), and TRX. Controlled increase of ROS levels activates NF-B and promotes cell growth. ExcessROS production and disruption of ��m lead to activation-induced cell death executed by activation of caspase 3 (digesting vitally important proteinspoly[ADP-ribose] polymerase [PARP], 70K U1 RNP, lamin, and actin) and CAD (causing nuclear DNA fragmentation). The ��m is controlled byintracellular GSH/NADH/NADPH levels, integrity of the permeability transition pore complex (PTPC; consisting largely of adenine nucleotidetranslocator [ANT; inner membrane] and voltage-dependent anion channel [VDAC; outer membrane]), and translocation and dimerization of pro-and antiapoptotic Bcl-2 family members in the intermembrane space. Phosphorylation of BAD by mitochondria-anchored PKA results inantiapoptotic sequestration of BAD into the cytosol. AP-1 � activator protein 1; GSSG � oxidized form of glutathione; Sp1 � specificity protein1 (see Figure 2 for other definitions).

ABNORMAL T CELL SIGNAL TRANSDUCTION IN SLE 1145

healthy subjects. Moreover, T cell activation via CD3/CD28 receptors induces mitochondrial hyperpolariza-tion and ROI production in normal PBL (11). In con-trast, H2O2 and CD3/CD28 costimulation fail to elevate��m, increase ROI production, and induce apoptosis inSLE T cells (11). Both CD3/CD28-induced H2O2 pro-duction and H2O2-induced apoptosis require mitochon-drial ROI production. Therefore, SLE T cells show thefollowing deviations of key mitochondrial checkpoints ofT cell activation and apoptosis: 1) deficient elevation of��m, 2) diminished CD3/CD28-induced ROI and H2O2production, and 3) reduced H2O2-induced apoptosis.

Mitochondrial hyperpolarization and increasedROI levels correlate with diminished GSH levels, sug-gesting increased utilization of reducing equivalents inSLE T cells. It is currently unclear whether synthesis ofGSH or its regeneration from its oxidized form is alteredin SLE PBL. GSH is also required for IL-2–dependent Tcell proliferation as well as for CD2- and CD3-mediatedT cell activation (72,73). Thus, low GSH content couldalso hinder CD3-induced H2O2 production. However,GSH deficiency predisposes cells to ROI-induced celldeath (58,65). Therefore, diminished H2O2-induced ap-optosis of cells with low basal GSH levels indicates asevere dysfunction of redox signaling in SLE.

The ��m (negative inside and positive outside) isdependent upon the electron transport chain transfer-ring electrons from NADH to molecular oxygen andproton transport mediated by the F0F1–ATPase complex(71). During oxidative phosphorylation, the F0F1–ATPase converts ADP to ATP, utilizing the energystored in the electrochemical gradient. Alternatively,using the energy of ATP hydrolysis, F0F1–ATPase canpump protons out of the mitochondrial matrix into theintermembrane space, causing ��m hyperpolarization.

Thus, mitochondrial hyperpolarization might oc-cur in one of two ways. First, deficiency of cellular ADPcould cause diminished utilization of the electrochemi-cal gradient, ATP depletion, and hyperpolarization.However, ADP levels were not diminished, but insteadwere slightly elevated in SLE PBL (11). This suggestedthat ATP depletion and mitochondrial hyperpolariza-tion were not due to a lack of ADP in SLE. Second,inhibition of the enzymatic activity of F0F1–ATPasecould decrease utilization of the electrochemical gradi-ent and cause mitochondrial hyperpolarization, ATPdepletion, and ADP accumulation. Since blocking ofF0F1–ATPase by oligomycin led to mitochondrial hyper-polarization and elevated ROI production, preventedH2O2- or CD3/CD28-induced elevation of ��m in nor-mal PBL, and sensitized cells to H2O2-induced necrosis,

a similar mechanism may also be operational in SLE.Indeed, we observed an association between diminishedapoptosis and increased necrosis upon H2O2 treatmentof SLE PBL. Moreover, the rate of H2O2-inducednecrosis correlated inversely with ATP levels.

These findings are consistent with a requirementof ATP for apoptosis and a predisposition to ATPdepletion for necrosis (74). Stimulation of T cells viaCD3/CD28 receptors caused a transient elevation of��m and ATP depletion and sensitized cells to H2O2-induced necrosis. Thus, repetitive T cell activation or asignaling abnormality mimicking T cell activation in vivocould be responsible for prolonged mitochondrial hyper-polarization and ATP depletion in SLE. In turn, ATPdepletion may play a major role in defective processingof subsequent activation and cell death signals.

Distal pathway defects: deficient Ras/mitogen-activated protein kinase (MAPK) activity. Althoughseveral signaling defects have now been identified in theproximal and middle pathways of SLE T cells (seeabove), little is known about distal signaling via theMAPK pathways. The MAPK signaling system com-prises 3 principal pathways: extracellular signal–regulated kinases (ERK-1/ERK-2), c-Jun N-terminalkinase (JNK), and p38 MAPK. Our recent investigationspoint to defective signaling via the ERK pathway inactive SLE T cells and implicate defective ERK pathwaysignaling in the immunopathogenesis of this disease. Theidentification of this signaling defect came from studieson the regulation of DNA methylation in SLE T cells.

One of the biochemical abnormalities that char-acterizes SLE T cells is a global decrease in genomicdeoxymethylcytosine (dmC) content (75). Because meth-ylation of deoxycytosine in regulatory sequences cansuppress transcription of the associated gene (76), ab-normal hypomethylation could contribute to overexpres-sion of some genes in SLE T cells. Importantly, T cellstreated with DNA methylation inhibitors that producehypomethylation, including procainamide and hydral-azine, become autoreactive as a result of lymphocytefunction–associated antigen 1 (LFA-1; CD11a) overex-pression, leading to a lupus-like disease in murinemodels (2,77–79). These experiments provide directevidence that DNA hypomethylation can contribute todisease pathogenesis.

The mechanism by which LFA-1 causes T cellautoreactivity has been studied in some detail. In 1994and again in 1996, our group reported that causingLFA-1 overexpression indirectly by treatment with DNAmethylation inhibitors, or directly by transfection, low-ered the threshold for T cell activation, allowing the cells

1146 KAMMER ET AL

to respond to self–class II MHC molecules presentinginappropriate antigens (80,81). LFA-1 overexpressionappears to cause the autoreactivity at least in part byoverstabilizing the normally low-affinity interaction be-tween the TCR and class II MHC molecules lacking therelevant antigenic peptide, allowing the signaling appa-ratus to assemble and transmit its signal (82). LFA-1–transfected T cells also cause a lupus-like disease closelyresembling that caused by T cells treated with DNAmethylation inhibitors (81). As such, T cells that over-express LFA-1 resemble the hyperactivatable T cellsmore recently proposed to contribute to autoimmunityin MRL mice transgenic for the AND TCR (83).

In mature T cells, DNA methylation is a postsyn-thetic event mediated by the enzyme DNA methyltrans-ferase 1 (Dnmt1) (76). Dnmt1 mRNA content andactivity are decreased by approximately one-half in SLET cells, which may account for the overall decrease indmC content (2,84). Because Dnmt1 levels increasefollowing T cell stimulation (85), it is possible that thesignaling defects in the proximal and middle pathwaysmight be associated with abnormal signaling in the distalpathways as well, contributing to decreased Dnmt1levels. Moreover, Dnmt1 levels are also regulated bysignals that traverse the JNK and ERK pathways, pos-sibly acting through AP-1 sites in an intronic promoter(86,87).

To further understand the mechanisms underly-ing deficient Dnmt1 activity, T lymphocytes from sub-jects with active and inactive SLE, subjects with RA, andhealthy controls were stimulated with phorbol myristateacetate/ionomycin (PMA/IO), and phosphorylation ofERK-1/ERK-2 and JNK was analyzed. Compared withcontrol and RA cells, MAPK kinase (MEK)–catalyzedERK-1/ERK-2 phosphorylation in SLE peripheralblood mononuclear cells (PBMCs) and SLE CD4 T cellswas significantly less, despite normal levels of the ERKproteins. This decrease in ERK phosphorylation wasdirectly proportional to disease activity (84). The func-tional significance of decreased signaling was tested bytreating normal T cells as well as the leukemic Jurkat Tcell line with PD98059, a selective MEK inhibitor. TheMEK inhibitor decreased Dnmt1 mRNA content andDnmt1 enzymatic activity to levels comparable withthose in SLE T cells and caused globally hypomethylatedDNA (84).

These observations support the hypothesis thatdecreased signaling through the ERK pathway contrib-utes to the DNA hypomethylation that is characteristicof SLE T cells, and possibly to the immunopathogenesisof SLE via mechanisms analogous to other DNA meth-

ylation inhibitors (2,77). The precise mechanism(s) caus-ing this defect in SLE T cells is currently unknown.However, the lupus-inducing drug, hydralazine, inhibitsERK pathway signaling, with identical effects on T cellDNA methyltransferase expression and overall dmCcontent (Deng C, et al: unpublished observations).

The observation that ERK pathway signaling isimpaired in SLE T cells is supported by additionalobservations. For example, other investigators usingelectrophoretic mobility shift assays (EMSAs) havedemonstrated that SLE T cells express reduced levels ofAP-1 transcription factor (88), the activation of which isalso regulated by this pathway (89). It should also benoted that inhibiting the T cell ERK pathway withPD98059 decreases production of TNF� and IFN� (90);expression of both cytokines is markedly reduced inphytohemagglutinin-stimulated SLE lymphocytes (2).

Gene transcription factor abnormalities in SLE Tcells

Abnormalities of two transcription factors havebeen recently identified in SLE T cells: 1) reduced/absent p65-RelA subunit of NF-B and 2) increasedphosphorylated cAMP response element modulator (p-CREM) binding to the IL2 promoter. To date, SLE isthe only autoimmune disorder in which these defectshave been identified.

Deficient p65-RelA subunit of NF-�B in SLE Tcells. Owing to the central role of the transcriptionfactor NF-B in proinflammatory responses in generaland in IL-2 production in particular (91), we consideredthe possibility that reduced IL-2 production by SLE Tcells (92–94) might be a product of altered NF-Bactivity. Utilizing EMSAs to quantify NF-B activity innuclear extracts from SLE T cells, we demonstrated thatits binding activity was either significantly depressed orundetectable compared with that from control cells (95).Moreover, in SLE T cells with no NF-B bindingactivity, its binding activity was persistently absent overtime, independent of disease activity or therapy.Antibody-induced supershift analyses revealed that, inSLE T cells with undetectable NF-B activity, p65/p50NF-B heterodimeric complexes were undetectable inthe cytosol. Instead, only p50 homodimeric complexeswere identified. Because p65/p50 heterodimeric com-plexes translocate to the nucleus upon T cell activationand are transcriptionally active, the absence of theseheterodimeric complexes could contribute to impairedproinflammatory responses, particularly IL-2 pro-duction.

ABNORMAL T CELL SIGNAL TRANSDUCTION IN SLE 1147

As discussed above, AP-1 is also reduced in thePBMCs of SLE patients (88). This may be the result ofseveral contributing factors, including decreased ERKpathway signaling and deficient c-Fos protein (a compo-nent of the heterodimeric AP-1 transcription factor)(refs. 84 and 88, and Mishra N, Kammer GM: unpub-lished observations). Thus, it seems likely that reducedexpression of both transcription factors may contributeto diminished IL-2 production by TCR-activated SLE Tcells (see below).

Based on our current understanding of NF-B(91), it seems likely that the profound deficiency ofNF-B will have wide and pathophysiologically signifi-cant effects in this disease. Current studies are aimed atestablishing the prevalence of deficient NF-B activity inSLE, its precise role in low IL-2 production by T cells,and the molecular mechanisms underlying the absenceof the p65-RelA subunit.

Association of increased p-CREM binding to the–180 site of the IL2 promoter with reduced IL-2 produc-tion by SLE T cells. SLE disease activity is oftenassociated with anergy to recall antigens by skin testing.This bedside observation led to the discovery that SLE Tcells stimulated in vitro with antigens or mitogens pro-liferate significantly less than T cells from healthy con-trols (2). Once it was recognized that splenocytes frommice bearing the lpr gene produce low amounts of IL-2in vitro in response to mitogens (96,97), it was soonappreciated that activated human SLE T cells alsosecrete significantly less IL-2 in vitro compared withhealthy control cells (92-94). Since then, it has beenconjectured that anergy to skin testing may reflect T cellanergy due in part to impaired IL-2 production.

Two mechanisms that are likely to contribute todeficient IL-2 production by SLE T cells are the absenceof the p65/p50 NF-B heterodimer and reduced AP-1activity (88,95). A third potential mechanism for lowIL-2 production by antigen/mitogen-stimulated SLE Tcells is inhibition of IL2 enhancer/promoter transcrip-tional activation. CREM is a transcriptional repressorthat binds to cAMP response elements and down-regulates the expression of genes having this binding site(98). Of special interest, however, is the recent demon-stration that p-CREM binds to the –180 region of theIL2 enhancer/promoter and contributes to T cell anergy(99). This raised the possibility that p-CREM binding tothis response element could impede IL-2 production bystimulated SLE T cells, contributing to T cell anergy.

Although activation of normal T cells in vitro viathe TCR produces high levels of IL2, similar activationof SLE T cells generally produces significantly less

cytokine (2). Following activation of normal T cells,phosphorylated cAMP response element binding pro-tein (p-CREB) binding to the –180 site of the IL2promoter is increased 10-fold compared with only min-imal p-CREM binding. Compared with activated normalT cells, activated SLE T cells bound both p-CREM andp-CREB in a 2:1 ratio. Thus, p-CREM predominantlybinds to the –180 site of the IL2 enhancer/promoterduring activation. This skewed binding pattern may hinderefficient and full transcriptional initiation by p-CREBbinding, leading to underproduction of IL-2 (92).

To initiate transcription under physiologic condi-tions, p-CREB binds to two nuclear coactivators, CREBbinding protein (CBP) and p300, to form heteromericcomplexes that interact with the basal transcriptionmachinery (100,101). In contrast to normal T cells, SLET cells revealed abundant p-CREM/CBP/p300 com-plexes. Such heteromeric complexes would be expectedto hinder transcriptional initiation of the IL2 enhancer/promoter. Indeed, transient transfections of SLE T cellswith a luciferase reporter construct driven by two tan-dem –180 site motifs resulted in only a 1.1-fold increasein luciferase activity compared with a 6.8-fold increase inluciferase activity in normal T cells activated by PMA/IO. Moreover, SLE T cells exhibiting increased amountsof p-CREM binding produced the lowest amounts ofIL-2 in vitro. In normal T cells, PKC is one of severalserine/threonine kinases that can phosphorylate CREB.However, we still do not know which kinase is involvedin the phosphorylation of CREM in SLE T cells.

In sum, these experiments suggest that preferen-tial phosphorylation of CREM in SLE T cells resultspredominantly in p-CREM binding to the –180 site ofthe IL2 enhancer/promoter. Therefore, the formation ofp-CREM/CBP/p300 heteromeric complexes hinders IL2transcriptional activation and contributes to very lowproduction of IL-2.

Impaired protein translation

As discussed above, T cells from some subjectswith SLE fail to translate RI� transcript into protein(40). PKR is a serine/threonine protein kinase thatregulates protein translation (102). As an inactive ho-loenzyme, the molecule exists in its dephosphorylatedform. However, binding of double-stranded RNA toPKR results in a conformational change, causing auto-phosphorylation and homodimer formation (102,103).Once activated, PKR can phosphorylate several sub-

1148 KAMMER ET AL

strates, including those regulating protein synthesis. Theinitiation of protein translation is regulated at two steps:1) eukaryotic initiation factor 4e (eIF4e)–mediatedbinding of the 40S ribosomal subunit to the 5� end of themRNA and 2) eIF2�-mediated binding of the initiatingmethionine–transfer RNA (met-tRNA) to the 40S ribo-somal subunit (103). Activated PKR phosphorylateseIF2�, which tightly binds eIF2�, thereby impairingformation of a complex consisting of eIF2, met-tRNA,and the small ribosomal subunit, inhibiting proteinsynthesis and subsequent cellular proliferation (104).Interestingly, mice heterozygous for a PKR-null muta-tion exhibit decreased signaling through the p38 MAPKpathway (102).

We pointed out above that TCR-mediated acti-vation of SLE T cells is markedly impaired and that anincreased proportion of T cells undergoes increasedapoptosis (11,53). Because both can be a product ofimpaired protein synthesis (103), we investigated theregulation of protein translation in SLE T cells. Stimu-lation of normal T cells with PMA/IO increased proteintranslation �90-fold. In contrast, similar treatment of Tcells from subjects with active SLE increased proteinsynthesis only 5-fold, whereas T cells from people withinactive SLE increased protein synthesis only �20-fold.A comparison of PKR levels revealed that stimulation ofSLE T cells caused more rapid and larger increases inPKR compared with control cells, resulting in increasedeIF2� phosphorylation. Further, overexpressing PKR inT cells by transfection suppressed protein synthesis.Comparison of PKR mRNA levels in stimulated SLEand control T cells demonstrated no differences, indi-cating that increased PKR activity in SLE T cells reflectsposttranscriptional regulatory differences (105). Theprecise mechanism underlying PKR overexpression inSLE T cells is currently unknown.

Relationship between defective signaling and T celleffector dysfunctions

Current evidence in human and murine SLEsuggests that defective signaling in SLE contributes to Tcell effector dysfunctions and, ultimately, to the immu-nopathogenesis of SLE. In human SLE, impairedcAMP-dependent protein phosphorylation was initiallylinked to deficient T cell suppressor activity and aber-rant mobility of molecules in the plane of the plasmamembrane (i.e., capping) (31,33,34,106). Of particularinterest was that both defective capping and deficientPKA activity in T cells persisted over time, were inde-pendent of disease activity, and were not affected by

therapy. Based on these findings, investigators in ourgroup proposed that abnormal T cell effector functionswere an outcome of a disorder(s) intrinsic to the cell(3,34).

Similarly, overexpression of cell surface receptorsis likely to augment cell–cell interactions between T cellsand B cells or APCs. Altered Ca�� homeostasis as wellas deficient cbl- and ERK-dependent phosphorylationcontribute to heightened and prolonged CD154 expres-sion (10,84,107). Moreover, as discussed above, overex-pression of LFA-1 (CD11a) results in autoreactivity andpromotes enhanced T cell interactions with endothe-lium, B cells, and APCs bearing CD54 (intercellularadhesion molecule 1) (80).

An inherent limitation of such experiments inhuman SLE is the difficulty of performing gain-of-function studies to establish whether correction of asignaling defect(s) is sufficient to reconstitute physio-logic T cell effector functions. As discussed above, manysignaling abnormalities have now been identified in SLET cells (Figure 2). This makes it unlikely that a singledefect is solely responsible for the diverse T cell dysfunc-tions identified in SLE. Notwithstanding, our group’srecent demonstration that transient transfection of anRI� cDNA into SLE T cells bypasses the block in RI�translation and increases RI� protein, cAMP-activatablePKA activity, and IL-2 production (40) gives credence tothe idea that there is a direct relationship betweendefective signaling and abnormal effector functions inSLE.

This concept is strengthened by the recent recog-nition that a single, inactivating point mutation of mem-brane CD45 phosphatase in murine T cells produces anSLE-like disease (108). A knockin mutation of CD45glutamate 613 to arginine (E613R) caused a lymphopro-liferative disorder characterized by polyclonal T and Bcell activation, heightened IL10 and IFN� transcriptcontent, anti-dsDNA autoantibody, and a severe glo-merulonephritis leading to premature death. This is thefirst example of a single point mutation in a hematopoi-etic cell protein that results in SLE in either humans ormice. Although there are differences as well as similar-ities in T cell effector dysfunctions between this modeland idiopathic human SLE, it serves to demonstrate thatan intrinsic signaling defect can result in lupus-likeautoimmunity.

In this regard, it is also conceivable that theinherent hyperreactivity of T cells to low-affinity anti-gens recently identified in a lupus-prone mouse model(83) may be related to as-yet-unrecognized signalingabnormalities, as observed in human SLE T cells. How-

ABNORMAL T CELL SIGNAL TRANSDUCTION IN SLE 1149

ever, the presence of intrinsic abnormalities of SLE Tcells does not exclude effects on cell functions byextrinsic factors. The most likely candidates are cyto-kines. For instance, circulating IL-10 levels are signifi-cantly increased in both human and murine SLE andhave been associated with increased B cell hyperactivity,polyclonal hypergammaglobulinemia, and enhanced Tcell apoptosis (63,109–111). Other extrinsic factors, suchas binding of immune complexes or autoantibody to Fc�receptors, as well as putative repetitive T cell stimulationby low-affinity self-antigens presented by APCs to TCR(83,112), are likely to exert a modulating effect on T cellsignaling and effector functions. However, this effect hasbeen difficult to quantify in human SLE. In our currentview, it seems likely that both intrinsic signaling defectsand extrinsic factors impact on the T cell to yield theobserved T cell effector dysfunctions.

Future directions of research

Within the next 5 years, we anticipate that addi-tional signaling defects will be discovered in SLE T cells.Based on the widely diverse abnormalities identified todate, it seems likely that these will include abnormalitiesof protein phosphorylation/dephosphorylation, DNAmethylation, histone acetylation/deacetylation, mito-chondrial function, transcription factors, transcriptionalregulation, and translation.

Although defects of protein kinases, such asPKA, have already been well identified in SLE T cells,others remain to be further defined and detailed. Forexample, reports of diminished PKC-, lck-, and cbl-catalyzed phosphorylation suggest abnormal kinase ac-tivities, but further detailed analyses are needed toclarify the precise defects (107,113,114). In particular,considering the pivotal role of the PKC isozymes, suchas PKC (115), in T cell signaling and the previousrecognition that defects of these isozymes are likely toexist (8), it is indeed surprising that so little is knownabout this pivotal kinase. Moreover, additional efforts toconfirm and extend our understanding of reduced CD45phosphatase activity (116,117) and its functional signif-icance should also be undertaken. This is particularlyappropriate now that a single point mutation of CD45 inthe mouse has been linked to autoimmunity (108).

The identification of deficient kinase activitiesraises the question of whether defective signaling leadsto abnormal T cell effector functions, such as helper andcytotoxic activities. Moreover, defective signaling result-ing in abnormal regulation of DNA methylation is likelyto modify gene expression. Future experiments should

address these pertinent issues by establishing a firmcause-and-effect relationship between deficient activitiesof specific protein kinases, skewed gene expression, andabnormal effector functions.

Essentially nothing is yet known about the impactsignaling defects might have on mitochondrial functions.Several questions might be posed. First, how do signal-ing pathways, such as cAMP/PKA and PKC, regulate��m? What is the role of phosphorylation of Bcl-2family proteins, such as BAD, in this process? Second,how do mitochondrial hyperpolarization and heightenedROI products contribute to accelerated apoptosis inSLE? Third, how do reduced levels of GSH and ATP(11) predispose cells to apoptosis or necrosis? Finally,the mitochondrion possesses enormous capacity to se-quester Ca�� (71). In fact, ��m elevation and inhibitionof the respiratory chain can limit mitochondrial Ca��

uptake (118), thus facilitating sustained Ca�� responses.Is mitochondrial hyperpolarization linked to heightenedCa�� signaling following a TCR signal in SLE T cells?

Transcription factors are the pivotal link betweensignaling pathways and genes. Their aberrant expressionor regulation could modify gene expression. The discov-eries that AP-1 and NF-B are deficient and that CREMis preferentially phosphorylated and binds to the IL2promoter provide the first clues as to why their targetgenes, such as IL2 (101), are dysregulated. Becausemany transcription factors depend upon phosphoryla-tion to modify their functions, it seems likely thatdefective protein kinase–catalyzed phosphorylation willresult in impaired transcription factor binding to regu-latory elements and gene dysregulation. Future experi-ments should explore this potential mechanism.

Alternatively, disorders of transcriptional initia-tion, transcript stability, and/or transcript-specific trans-lational repression may be discovered that could alsoalter gene expression. Although no report has yet in-voked translational silencing as a mechanism for alteredgene expression in autoimmunity, it seems plausible thatinhibitory factors binding to the 3�-untranslated regionsof transcripts could be an as-yet-undiscovered mecha-nism. Indeed, it has already been recognized that in-creased IL-6 production in SLE is linked to enhancedIL6 mRNA stability and a skewed distribution of 3�AT-rich minisatellite alleles (119).

Within the past few years, great strides have beenmade in understanding how acetylation and deacetyla-tion of histones by histone acetyltransferase and histonedeacetylases modify gene expression (120–122). Under-standing how acetylation/deacetylation modifies geneexpression may be directly relevant to lupus immuno-

1150 KAMMER ET AL

pathogenesis. In SLE, T helper cells exhibit increasedand prolonged expression of cell surface CD40 ligand(CD154) and spontaneously overproduce IL-10, butunderproduce IFN� (2). Recently, investigators in ourgroup demonstrated that the imbalance of these geneproducts may reflect skewed expression of CD154, IL10,and IFN� genes due to diminished histone acetylationand/or increased deacetylation (123). If this is so, thenstrategies to reverse hypoacetylation and to restore abalance between the two processes may have therapeuticsignificance in SLE. Our current evidence that thehistone deacetylase inhibitor, trichostatin A, correctsthis aberrant gene expression may lead to new avenuesof research in gene regulation in SLE.

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