Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4⁺ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of Calmodulin, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of Calmodulin in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti–TNF-α therapy in vivo, RA PBMC Calmodulin transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced Calmodulin transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

Rheumatoid arthritis (RA) is a chronic disorder of the joints in which the synovium becomes hyperplastic, secondary to proliferation of the lining layer and infiltration by inflammatory cells (1). Its pathogenesis is unknown but linkage to the class II region of the MHC, the presence of circulating autoantibodies, and response to some T-cell targeted therapies, suggest an autoimmune contribution (2). Additionally, RA T cells have an activated phenotype, characterized by upregulated expression of HLA-DR (3). On the other hand, these cells demonstrate features of anergy in vitro and do not proliferate when stimulated with mitogens or recall antigens (4). They are also unusually resistant to apoptosis (5). The intracellular defect(s) responsible for this paradoxical behavior have not been fully elucidated, although chronic TNF-α exposure may be an etiological factor in the anergic phenotype (6), and cytokines may protect the cells from apoptosis (7). It is important to understand these features of the disease, however. The morbidity and mortality of RA are largely secondary to infections and malignancies, reflecting the immunosuppression that is intrinsic to the disease (8). It is also possible that a defect in the regulation of apoptosis contributes to the pathogenesis of RA (9).

During development of the immune system, a number of processes occur that ensure that mature lymphocytes do not become activated by self-antigens and cause autoimmune disease. Clonal T-cell anergy represents one of these mechanisms (10). Full T-cell activation requires appropriate stimulation through both the specific T-cell receptor for antigen (TCR) and the costimulatory molecule CD28. In contrast, defective costimulation or qualitatively altered TCR signals may result in anergy, whereupon the cell becomes refractory to subsequent activating stimuli. There are a number of in vitro models of anergy induction. These include the presentation of cognate peptide on chemically fixed antigen-presenting cells (APC) (11); the pharmacological blockade of costimulatory interactions between CD28 and B7 (12); and T-cell stimulation by altered peptide ligands, which differ structurally from the cognate ligand by the introduction of a limited number of amino acid substitutions (13). Anergy can also be induced by polyclonal stimulation of T cells using bacterially derived superantigens (14), or anti-CD3 mAb’s (15), or by clonal stimulation with high concentrations of cognate peptide antigen in the presence or absence of professional APCs. Thus, the human CD4⁺ T-cell clone, HA1.7, becomes anergic when incubated with high concentrations of its ligand, residues 306-318 of influenza virus hemagglutinin (HA 306-318) (16). Bidirectional T cell-to-T cell antigen presentation may be the critical event when anergy is induced by high concentrations of peptide, and T-cell apoptosis is an alternative outcome under these circumstances (17).

The critical feature of anergic T cells is their inability to produce IL-2 and, hence, their defective autocrine stimulation upon encounter with antigen. RA T cells share this characteristic (18), but the associated signaling defects are not yet fully characterized in either situation. Most work has been performed in anergy induced by defective costimulation in which there is evidence for defective coupling of the TCR to downstream signaling pathways with a block in ERK-1, ERK-2, and p21ras activation (19, 20). On the other hand, anergy induced by exposure to high concentrations of cognate peptide may differ from other models in that predominant defects in the calcium/calcineurin-signaling pathway have been demonstrated (21, 22), although the Ras/MAP kinase pathway has not been extensively characterized in this model. This again is reminiscent of RA T cells, in which defective calcium signaling has also been clearly demonstrated (23, 24). Although more marked in the synovium, calcium defects are also present in RA peripheral blood (25).

A clearer understanding of the molecular events associated with anergy induction on the one hand and the behavior of RA T cells on the other may suggest novel therapeutic strategies for autoimmune diseases in general and RA in particular. A number of techniques now permit a comprehensive comparison of gene transcription between tissues or cells in different states of differentiation. These include differential cDNA hybridization using cDNA microarrays (26), differential display RT-PCR (DDRT-PCR) (27), and serial analysis of gene expression (SAGE) (28). We have used DDRT-PCR to analyze changes in gene transcription in the human T-cell clone HA1.7 during anergy induction by its cognate peptide HA 306-318. In this model, APC are not present during anergy induction, considerably simplifying data interpretation. We subsequently used a differential hybridization technique to examine levels of anergy-associated transcripts in RA and, as a control, in reactive arthritis (ReA) synovium. The latter is a self-limiting condition, not characterized by defects in T-cell activation or apoptosis (29). We thereby identified transcriptional changes common to both anergic T cells and to RA synovial T cells, which may provide clues to the underlying cellular defects in RA.

Induction of anergy in the human CD4⁺ T-cell clone HA1.7. The human CD4⁺ T-cell clone, HA1.7, was incubated with peptide HA 306-318 (50 μg/ml) for 24 hours. The anergic state of the cells was tested subsequently by an immunogenic challenge (Figure 1). Peptide-preincubated cells did not proliferate when stimulated with HA 306-318 in the presence of APC, but their viability was confirmed by responsiveness to exogenous IL-2. In contrast, control cells proliferated to both antigenic challenge and to IL-2. The inability of peptide-preincubated cells to respond to an immunogenic challenge confirmed their anergic state.

Figure 1

Induction of anergy in the human CD4⁺ T-cell clone HA1.7. HA1.7 (10⁶/ml) was incubated with peptide HA 306-318 (50 μg/ml) for 24 hours or with medium alone. The anergic state of the cells was subsequently tested by an immunogenic challenge. Peptide-preincubated cells (dark bars, 10⁴/well) did not proliferate when stimulated with HA 306-318 in the presence of APC, but their viability was confirmed by responsiveness to exogenous IL-2. In contrast, control cells (light bars, 10⁴/well) proliferated after either antigenic challenge or IL-2. Neither control nor anergic cells proliferated when incubated with medium, APC, or peptide alone. The figure shows proliferation after 48 hours of stimulation, measured as [³H]thymidine incorporation.

Multiple transcriptional changes occur during anergy induction. To identify transcriptional events occurring during the early stages of anergy induction, we used DDRT-PCR to compare RNA extracted from cells after 2 hours of incubation with peptide and after 2 hours’ incubation in medium alone (control incubation). Figure 2 provides a summary of our overall strategy. We identified 79 bands that differed absolutely or quantitatively between the two conditions: 56 of these were upregulated by peptide incubation and 23 were downregulated. We decided to focus on the 40 upregulated and 12 downregulated bands representing absolute differences in transcription, and the DNA contained in these was extracted, reamplified, and cloned. Because a DDRT-PCR band often comprises more than one cDNA moiety (34), three subclones were analyzed from each clonable band. Twelve bands could not be cloned, and we ultimately identified 55 different cDNA sequences. Twenty-two corresponded to known genes and 33 were expressed sequence tags (ESTs), of which 20 gave no database matches at the nucleotide level.

Figure 2

A schematic of the DDRT-PCR and confirmatory analysis. After differential display (DDRT-PCR) analysis of T cells undergoing anergy induction, 55 distinct cDNA sequences were isolated. These formed the basis of the array that was used to confirm differential gene expression and compare transcription in RA and ReA synovium.

Confirmation of differential gene transcription and analysis of transcription at 24 hours. Differential transcription was confirmed by a reverse Northern dot blot technique. PCR was used to amplify a cDNA fragment of approximately 200 bp from each of the 55 sequences and of the housekeeping transcript GAPDH. PCR products were sequence verified and then immobilized onto duplicate nylon-membrane filters. Filters were subsequently hybridized to radiolabeled first-strand cDNAs prepared from peptide-incubated or control cells after 2 or 24 hours of culture. Figure 3 shows an example of a filter representing the 2-hour time point. Of the 55 sequences, 35 gave hybridization signals during reverse Northern analysis. Transcription of target genes was initially related to that of GAPDH, before a ratio was obtained for relative gene transcription between anergic and control resting T cells. In all, 25 (45%) of our DDRT-PCR–derived cDNAs were confirmed to represent differentially expressed transcripts as determined by the reverse Northern technique. It was noteworthy that the hybridization-based technique gave relatively modest differences in gene transcription compared with the large transcriptional differences originally identified by DDRT-PCR analysis. There are a number of factors that may contribute to this apparent discrepancy. These include the amplification step that is intrinsic to the DDRT-PCR procedure as well as the complexity of the bands in DDRT-PCR, which often contain several distinct cDNAs, including some false positives.

Figure 3

Confirmation of DDRT-PCR results by reverse Northern blotting analysis. Replicate nylon-filter membranes were prepared, comprising 200-bp cDNA fragments (2 μg of each) representing each differentially expressed transcript. Radiolabeled cDNA probes were prepared from 5 μg total RNA extracted from (a) resting or (b) anergic T cells. The probes were hybridized to the filters overnight, and, after extensive washing, the filters were exposed for 3 days to Phosphor Screens. Transcripts downregulated during anergy induction are highlighted by white arrows. Transcripts upregulated are highlighted by black arrows. The blot represents all 55 transcripts that were considered to be differentially expressed after the initial DDRT-PCR screen, as well as the housekeeping control transcript, GAPDH.

Transcription of anergy-related transcripts in inflamed synovium. We next compared transcription of the genes identified as anergy related in synovial tissue from patients in the early stages of RA and ReA. In their initial stages, RA and ReA resemble each other histologically, although RA synovium may contain more T cells than ReA (29). The latter resolves rapidly, however, and serves as a disease control when investigating RA. Radiolabeled first-strand cDNAs from RA and ReA synovial biopsies were used to probe duplicate filters representing differentially transcribed genes. Of the 25 confirmed anergy-related transcripts, 11 were expressed in synovial tissues (Table 1). These were generally more abundant in RA or equally transcribed in the two conditions, perhaps corresponding to the generally higher number of T cells in RA synovium. Three genes were clearly downregulated in RA, however, in comparison with ReA. These were CAS, Calmodulin, and BF626661. CAS encodes for the cellular apoptosis susceptibility protein that plays a dual role in promoting growth or apoptosis at the mitotic G2/M checkpoint (35). Apoptosis is known to be defective in RA synovial T cells (5), whereas anergic T cells are unduly susceptible to apoptosis (17) and upregulate CAS (Table 1). Calmodulin is a calcium-binding protein that binds intracellular free calcium and regulates other calcium-dependent proteins (33). BF626661 was also downregulated in both RA and in anergy. Of these three genes, the most marked difference was seen with Calmodulin, whose transcription in RA was less than 1% of that in ReA. In contrast, transcription of BF626661 was tenfold lower in RA, and there was only a fourfold difference in CAS transcription (Table 1). We also studied Calmodulin and CAS transcript levels in individual biopsies from RA and ReA patients using real-time PCR quantification and confirmed the large difference in Calmodulin transcription and modest difference in CAS transcription as well as the paucity of CAS transcripts in both types of synovitis (Figure 4). Calmodulin was also downregulated during anergy induction, potentially linking the anergic state in HA1.7 with the hyporesponsiveness of RA T cells. Genes that were equally transcribed or upregulated in RA compared with ReA included those coding for PMA-ionophore–linked activation factor of T cells (PILOT), T-plastin, calumenin, eucaryotic elongation factor 1-δ, and ADP ribosylation-like factor-1 (ARL-1). ESTs corresponding to BF626660 and BF626663 were both relatively abundant in RA compared to ReA (Table 1).

Figure 4

Calmodulin and CAS transcription in individual biopsies. First-strand cDNA was prepared from synovial tissue from individuals with RA and with ReA. Real-time PCR was used to quantitate transcription of (a) Calmodulin and (b) CAS relative to the housekeeping transcript GAPDH. P = 0.002 for Calmodulin and 0.035 for CAS, for RA vs. ReA (unpaired t test).

Table 1

Transcripts common to anergic T cells and inflamed synovium

Transcription of Calmodulin and BF626661 in synovial fluid mononuclear cells. Whole synovium contains a variety of cell types including fibroblasts, B cells, and plasma cells. We therefore compared transcription of Calmodulin and BF626661 in matched synovial fluid mononuclear cells (SFMCs) and PBMCs from four patients with RA. We employed real-time quantitative PCR, relating transcription of our target genes to that of the housekeeping gene GAPDH. Figure 5 illustrates pooled data for four patients and confirms that both Calmodulin and BF626661 are downregulated in SFMCs compared with PBMCs. This variation between transcript levels in RA SFMCs and PBMCs is consistent with the proliferative and signaling defects previously documented in these cell types (4, 23–25).

Figure 5

Quantitation of gene transcription in mononuclear cells. First-strand cDNA was prepared from PBMCs and SFMCs from RA patients. Real-time PCR was used to quantitate transcription of Calmodulin and BF626661 relative to GAPDH as a housekeeping transcript. The figure shows the ratio of target gene to GAPDH transcription in SFMCs and PBMCs (mean and SD of four patients). P = 0.050 for Calmodulin and 0.026 for BF626661 for PBMC vs. SFMC (paired t test).

Upregulation of Calmodulin transcription following anti–TNF-α therapy of RA. The hyporesponsiveness of RA PBMCs is reduced after TNF-α blockade (36). We therefore hypothesized that, if calmodulin is related to the hyporesponsiveness of RA T cells, its transcription should increase in PBMCs after therapeutic TNF-α blockade. This is illustrated in Figure 6. Six patients were studied using quantitative real-time PCR. In every case Calmodulin transcription increased 2 weeks after a single infusion of 3 mg/kg Infliximab three- to tenfold. In contrast, transcription of CAS demonstrated no consistent transcriptional variation after therapy.

Figure 6

Transcription of Calmodulin and CAS in PBMCs isolated from RA patients before and after treatment with anti–TNF-α mAb. First-strand cDNA was prepared from PBMCs from six patients before and 2 weeks after receiving 3 mg/kg Infliximab. Real-time PCR was used to quantitate transcription of (a) Calmodulin and (b) CAS relative to GAPDH. The figure shows the ratio of transcripts in the posttreatment samples relative to the pretreatment samples for each patient.

Calmodulin inhibition impairs antigen-specific proliferation of PBMCs. The data above suggest that the hyporesponsiveness of RA T cells may be related to low levels of calmodulin. We therefore studied the effect of calmodulin blockade on antigen-specific proliferation of PBMCs from four control individuals. In each case, the pharmacological calmodulin antagonist TFP inhibited proliferation of T cells to PPD in a dose-dependent manner (Figure 7).

Figure 7

Inhibition of antigen-specific PBMC proliferation by calmodulin blockade. PBMCs were prepared from normal controls. Proliferation assays were established using PPD as a test antigen in the presence or absence of TFP, a pharmacological antagonist of calmodulin. The figure illustrates pooled data for four individuals of the percentage inhibition (mean ± SD) of the proliferation index at each dose of TFP. The proliferation index was calculated as the ratio of counts between stimulated and unstimulated cells.

Synovial fluid and peripheral blood T cells in RA exhibit a number of unusual characteristics. First, despite the fact that RA is an autoimmune disease, they are hyporesponsive to both mitogens and to recall antigens (4). Second, despite high levels of proapoptotic cytokines, such as TNF-α, synovial fluid T cells are unduly resistant to apoptosis (5). Synoviocytes themselves also exhibit an apoptotic defect, which may contribute to the pathogenesis of the disease (9). Over the past two decades, the perception of RA as a disabling but nonfatal condition has changed dramatically, and it is now recognized to be a condition associated with considerable mortality. Death is frequently secondary to infection, and there is also an increased risk of malignancy, particularly of the immune system (8). Parallels can be drawn between the T-cell hyporesponsiveness and apoptotic defects on the one hand, and infection and malignancy risk on the other, which make it important for us to understand the basic cellular defects in RA.

A number of powerful methods are now available to compare transcription between different tissues or between cells in different states of differentiation. SAGE (28) provides a quantitative analysis of total gene transcription but is less suitable for rapidly comparing multiple similar samples and gives minimal sequence information for each cDNA (typically 9–13 bp). cDNA microarrays (26) provide rapid analytical methods, but may be biased toward the identification of high-abundance transcripts. In contrast, DDRT-PCR frequently identifies rare species and provides around 200 bp of 3′ sequence information. We have applied DDRT-PCR to a clonal T-cell population in its resting and anergic state and subsequently used a hybridization approach to compare the transcription of anergy-related genes in RA and ReA synovial tissue. Our rationale was that because RA T cells share some characteristics with anergic T cells, an analysis of the latter may provide insights into molecular defects in the former. There are a number of in vitro models of anergy, but peptide-induced anergy provides two significant advantages. First, APC are not required, which greatly facilitates interpretation of the DDRT-PCR data. Second, in contrast to other anergy models, calcium-signaling pathways may be defective in peptide-induced anergy (21, 22) as they are in RA synovial T cells (23–25). We chose two time points to examine differential gene transcription: at 2 hours to represent anergy induction and at 24 hours to represent established anergy. The trigger of the anergic phenotype in RA is unknown, and it is unclear which of these time points is most relevant to the differentiation state of synovial T cells. On the other hand, the synovium contains a heterogeneous population of T cells at different stages of differentiation, and both time points may therefore be pertinent.

We identified a number of differentially regulated transcripts in anergic T cells, which were also present in RA and ReA synovium (Table 1). ReA serves as a useful disease control when investigating RA because the two conditions are histologically similar in their early phase. The former resolves without causing permanent damage, however, whereas the latter persists. From the perspective of the current analysis, some reports suggest that T cells are more numerous in RA than in ReA synovium (29). Additionally, synovium is a heterogeneous tissue, complicating the analysis of transcripts which are T-cell specific. We therefore focused our attention on three anergy-related transcripts that demonstrated reduced transcription in RA compared with ReA. One of these has not been described previously and is currently undergoing further analysis. The other two transcripts were those for Calmodulin and CAS, and downregulation of these was confirmed in individual biopsies. Calmodulin transcription was greatly reduced in RA, by up to 99%. Furthermore, its transcription in PBMCs increased after in vivo TNF-α blockade, a treatment known to reverse the proliferative abnormality of RA T cells. Additionally, transcript levels were lower in RA SFMCs compared with RA PBMCs, mirroring the known proliferative and signaling defects (25). Last, pharmacological calmodulin inhibition impaired antigen-specific proliferation of PBMCs in a dose-dependent manner. While not proving causality, these observations provide compelling evidence linking the hyporesponsiveness of RA T cells to reduced Calmodulin transcription. Thus, calmodulin binds free calcium in cells and is critical in T-cell activation (33). It regulates the influx of extracellular calcium (37), and, although recent evidence has implicated intracellular calcium stores in the RA signaling abnormality, that does not exclude an additional and possibly synergistic defect in extracellular calcium entry (25). Furthermore, calmodulin regulates other calcium-dependent proteins including calcineurin, a phosphatase critical to the NFAT T-cell signaling pathway (38), and calcium/calmodulin-dependent protein kinase, which has also been linked to the anergic state (39). It is also implicated in cytoskeletal changes associated with T-cell activation (40) and in phosphatidyl-serine synthesis (41). Ultimately, calmodulin regulates genes central to T-cell proliferation (42), and it is clear that downregulation of Calmodulin transcription is consistent with the T-cell phenotype in RA.

CAS is the human homologue of the yeast chromosome segregation gene CSE1, which determines cell fate at the G2/M mitotic checkpoint by influencing the decision between cell growth and apoptosis (35).

Its expression correlates with malignant potential in human tumors (43) but, notably, reduced levels are associated with resistance to TNF-α–mediated apoptosis (44). Reduced transcription in RA compared with ReA may, therefore, contribute to the defect in apoptosis observed in RA synovium in the face of high TNF-α levels. Both RA synoviocytes and synovial T cells demonstrate a resistance to apoptosis, however (5, 9). Additional analyses are therefore needed before attributing either defect to reduced CAS transcription, particularly in view of the relatively modest difference in transcript levels between RA and ReA and the overall paucity of CAS transcripts (Table 1 and Figure 4).

A number of transcripts were more abundant in RA compared with ReA synovium, or were equally transcribed (Table 1). These included PILOT, an Egr gene family member that increases FasL promoter activity and whose expression is sensitive to cyclosporine A (45). Transcription of this gene increased transiently during anergy induction and was similar in RA and ReA. The gene encoding T-plastin also was equally transcribed in the two types of inflamed synovium and showed a transient and slight rise in transcription during anergy induction. This is a calcium-binding protein containing both a calmodulin-like and an actin-binding domain. Its structure would allow it to transduce signals from the cell membrane to the cytoskeleton, a process shown recently to be critically important during T-cell activation (46). Notably, T-plastin has previously been described only in mesenchymal cells, which may account for its expression in synovium. Its isoform, L-plastin, has been linked to T-cell activation (47), but we used a T-plastin–specific sequence for hybridization. Another calcium-binding protein, calumenin, was transiently downregulated during anergy induction but displayed only minor differences between the two types of inflammatory arthritis. It is a member of the EF-hand protein family and, in contrast to calmodulin, is present predominantly in the lumen of the endoplasmic reticulum and Golgi apparatus (48). The ESTs BF626663 and BF626660 showed similar transcription patterns to calumenin, although the former remained downregulated in established anergy. The gene encoding aortic carboxypeptidase-like protein (ACLP) was transiently upregulated during anergy induction but was highly transcribed in both forms of synovitis (Table 1). ACLP contains a discoidin domain and a COOH-terminal region homologous to carboxypeptidase E. It has been linked previously to vascular smooth muscle differentiation (49), which may reflect its high level of transcription in inflammatory synovitis (Table 1) where neovascularization is abundant (50). Lastly, ARL-1 is a guanine nucleotide-binding protein that generates high-affinity membrane-binding sites for the heterotetrameric AP-1 adaptor complex on the Golgi apparatus (51). Transcription was transiently increased during anergy induction and was higher in RA than in ReA.

The ESTs BF626660 and BF626663 are currently undergoing further analysis in our laboratory. These could represent unrecorded sequences of known transcripts because cDNA fragments isolated by DDRT-PCR preferentially locate to the 3′-untranslated region of transcripts, which are sometimes omitted from databases. To address this possibility we have prepared cDNA libraries from T-cell lines to enable us to further characterize these apparently novel cDNA fragments. It is also important to emphasize that our DDRT-PCR analysis could not have identified posttranscriptional events. These have been shown to influence cytokine secretion by anergic HA1.7 cells (52) and are also likely to be active in synovial fluid T cells as a result of the oxidative nature of the local microenvironment (53). Notwithstanding the above, we have identified a number of genes whose transcription changes during anergy induction and differs between RA and ReA synovium. In particular, regulation of Calmodulin and CAS could explain some of the characteristic features of RA T cells and synovium, and further experiments are currently underway to more precisely define their roles. These may improve our understanding of the immunosuppression that is intrinsic to RA (8) and also the apoptotic defects that appear to play a critical role in RA pathogenesis (5, 9).

J.D. Isaacs was a UK Medical Research Council (UK MRC) Clinician Scientist during this work and M. Ali was supported by the West Riding Medical Research Trust. J.R. Lamb is supported by the UK MRC, the Wellcome Trust, and the British Lung Foundation. P. Emery is supported by the UK Arthritis Research Campaign. A.F. Markham is supported by the West Riding Medical Research Trust, the Wellcome Trust, the UK MRC, and the Candlelighters. The authors are grateful to Sue Douglas for technical assistance and to David Carruthers for helpful discussions.

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