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The Journal of Immunology, 2006, 176: 949-956.
Copyright © 2006 by The American Association of Immunologists

DNA Microarray Gene Expression Profile of T Cells with the Splice Variants of TCR{zeta} mRNA Observed in Systemic Lupus Erythematosus1

Kensei Tsuzaka2,*,{dagger}, Kyoko Nozaki{dagger}, Chika Kumazawa{dagger}, Kiyono Shiraishi{dagger}, Yumiko Setoyama*, Keiko Yoshimoto*, Katsuya Suzuki*, Tohru Abe* and Tsutomu Takeuchi*

* Division of Rheumatology, Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, Saitama, Japan; and {dagger} Project Research Division, Research Center for Genomic Medicine, Saitama Medical School, Saitama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have reported that the TCR{zeta} mRNA with alternatively spliced 3' UTR ({zeta} mRNA/as-3'-untranslated region (UTR)) and {zeta} mRNA lacking exon 7 ({zeta} mRNA/exon 7–) observed in systemic lupus erythematosus patient T cells can lead to down-regulation of both {zeta} and TCR/CD3 complexes. To determine whether these T cells expressing decreased {zeta} exhibit differential transcription patterns, we transfected retrovirus vectors containing wild-type {zeta} cDNA, {zeta} cDNA/as-3' UTR, and {zeta} cDNA/exon 7– into murine T cell hybridoma MA5.8 cells which lack {zeta} expression to construct the MA5.8 mutants WT, AS3' UTR, and EX7–, respectively. FACS analyses demonstrated reduced cell surface expression of {zeta} and TCR/CD3 complexes on the AS3' UTR mutant and the EX7– mutant in comparison to that on the WT mutant. Total RNA was collected after stimulating the MA5.8 mutants with anti-CD3 Ab. Reverse-transcribed cDNA was applied to the mouse cDNA microarray containing 8691 genes, and the results were confirmed by real-time PCR. The results showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-{beta}2, were down-regulated in both the AS3' UTR mutant and the EX7– mutant. Another 16 genes were up-regulated in both, and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus receptor-related 2, syndecan-1, and granzyme A. Increased protein expression of these genes was confirmed by Western blot and FACS analyses. Identification of these responsive genes in T cells in which the {zeta} and TCR/CD3 complexes were down-regulated may help to better understand the pathogenesis of systemic lupus erythematosus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE)3 is a systemic autoimmune disease of unknown etiology characterized by multiorgan involvement and abundant production of a variety of autoantibodies (1, 2, 3). T cells are considered central to the pathogenesis of SLE, because a dysfunction in their regulatory action may be responsible for the altered immune responses and overproduction of autoantibodies (4). We and other groups have reported that a decrease in tyrosine phosphorylation and the diminished expression of the TCR {zeta}-chain ({zeta}) in the peripheral blood T cells (PBTs) of SLE patients may be responsible for the pathogenesis of SLE (5, 6, 7). SLE patients have several alterations in the {zeta} mRNA open reading frame (8). We previously reported the aberrant form of {zeta} mRNA lacking exon 7 ({zeta} mRNA/exon 7–) observed in SLE patients (5). Moreover, we and other groups have demonstrated that an aberrant form of the {zeta} mRNA 3'-untranslated region (3' UTR), which is alternatively spliced and 562 bp shorter than the WT 3' UTR, is predominantly expressed in SLE T cells ({zeta} mRNA/as-3' UTR) (9, 10). Predominant expression of these two {zeta} mRNA splice variant forms ({zeta} mRNA/as-3' UTR and {zeta} mRNA/exon 7–) leads to down-regulation not only of {zeta} but of the other TCR/CD3 components because of the instability of these {zeta} mRNA splice variant forms (11, 12, 13). Several molecules corresponding to the TCR costimulatory pathway have been reported to be up-regulated in SLE T cells and play crucial roles in the pathogenesis of SLE. To identify the genes encoding membranous proteins or TCR costimulatory molecules that are induced secondarily by the expression of the {zeta} mRNA splice variant forms, we used a cDNA microarray analysis to compare the gene expression profile of an AS3' UTR mutant (MA5.8 cells (murine T cell hybridoma lacking of {zeta}) transfected with {zeta} mRNA/as-3' UTR) (11) and an EX7– mutant (MA5.8 cells transfected with {zeta} mRNA/exon 7–) (12) with a WT mutant (MA5.8 cells transfected with wild-form {zeta} mRNA) (11, 12). The results showed up-regulation of membrane proteins, including syndecan-1 (Sdc1) and poliovirus receptor-related 2 (Pvrl2), and down-regulation of ILs and chemokines, in both the EX7– and AS3' UTR mutants.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

The MA5.8 (lacking endogenous {zeta} expression) and 2B4.11 cells were provided by Dr. T. Saito (RIKEN, Research Center for Allergy and Immunology, Yokohama, Japan), and the RetroPack PT67 (BD Biosciences) was used as the dual tropic packaging cell line.

Construction of WT, AS3' UTR, EX7–, and NEG mutants

WT, AS3' UTR, EX7–, and NEG mutants were constructed by the method described previously (11, 12). Briefly, full-length wild-type human {zeta} cDNA, {zeta} cDNA/as-3' UTR (10), and {zeta} cDNA/exon 7– (5) were ligated into SalI-cut pDON-AI (Takara Bio), and each pDON-AI with insert DNA and pDON-AI without any insert DNA were then transfected into RetroPack PT67 cells with a cationic liposome kit (TransFast Transfection Reagent; Promega). Supernatant containing the same amount of the vector retrovirus was subsequently used to infect 1.0 x 107 MA5.8 cells to construct the WT, AS3' UTR, EX7–, and NEG mutants, respectively.

Microarray analyses

Total RNA was prepared with a GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). A 5-µg sample of total RNA was reverse-transcribed into a cDNA probe with oligo(dT) primer and labeled nucleotides (14). The reaction was conducted at 42°C for 1 h in a solution containing 50 µM dATP/dGTP/dTTP, 25 µM dCTP, and 25 µM cyanine 3 (Cy3)-dCTP (for the WT sample), or cyanine 5 (Cy5)-dCTP (for the AS3' UTR or EX7– sample) (Amersham Biosciences; Ref.15), and Moloney murine leukemia virus reverse transcriptase. The labeled cDNA probes were applied to the mouse cDNA microarray (Agilent Technologies) and hybridized at 65°C for 17 h. After washing, the microarray was scanned on a G2565BA scanner (Agilent Technologies), and the image was analyzed with Feature Extraction software (Agilent Technologies). The average signal intensity was subtracted from the median background intensity and output with GenBank descriptors to a Microsoft Excel data spreadsheet. Relative expression levels were evaluated as the ratio between two samples. Up-regulated genes were defined as those with a ratio >3, and down-regulated genes were defined as those with a ratio <1:3, in accordance with the previous reports (16, 17, 18). Three independent experiments were performed to assess the reproducibility of the observed changes.

Preparation of the template DNA for real-time PCR

Whole mRNA was isolated from 2B4.11 cells and converted to whole cDNA with reverse transcriptase according to a previously described method (8). The whole cDNA was quantified and used as the template DNA standard for real-time PCR.

Real-time PCR

The primers and TaqMan probes were designed with Primer Express software (Applied Biosystems). The sequences for the primers and probes are listed in Table I. In addition, the forward primer of 5'-GGCCAACCGTGAAAAGATGA-3' (+419 to +438) and the reverse primer of 5'-CACGCTCGGTCAGGATCTTC-3' (+669 to +650) for the murine {beta}-actin cDNA were designed in exons 3 and 4, respectively. The TaqMan probe for the murine {beta}-actin cDNA was 5'-TTTGAGACCTTCAACACCCCAGCCA-3' (+450 to +474). Amplification and detection of specific products were performed by using an ABI PRISM 7700 sequence detection system (Applied Biosystems) according to a previously described amplification protocol (11). The amount of standard DNA construct per well was adjusted to 10 pg and then serially diluted to yield samples containing 10, 1, 10–1, 10–2, and 10–3 pg, which were then used to construct standard plots.


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  		Table I. Primers and TaqMan probes used for real-time PCR

 
Western blot analysis

Cells were lysed with the lysis buffer and disrupted by sonication according to a previously described method (19). After centrifuging at 10,000 x g for 5 min, the supernatant was loaded on a 12.5% SDS-PAGE gel by a reducing method. The proteins were electrophoretically blotted onto polyvinylidene difluoride membranes (Millipore), and the membranes were soaked at 37°C for 1 h in blocking agents (Blockace; Dainippon Pharmaceuticals). The blots were then probed with a rat anti-mouse Sdc1 mAb (281-2) (BD Biosciences), a goat anti-mouse nectin-2 Ab (N-20; Santa Cruz Biotechnology), or a goat anti-mouse granzyme A (Gzma) Ab (D-15) (Santa Cruz Biotechnology) at 16°C for 1 h. The Abs were visualized with peroxidase-conjugated anti-rat (for 281-2) or anti-goat IgG (for N-20 or D-15; Amersham Biosciences). After washing three times, the signals were detected with chemiluminescence-enhancing reagents (Amersham Biosciences). The treated membranes were visualized on ECL x-ray film (Amersham Biosciences).

Flow cytometric analysis

The flow cytometric analysis procedure has been described previously (19). Briefly, the MA5.8 mutants were stained with an FITC-conjugated Armenian hamster anti-mouse CD3{epsilon} mAb (145-2C11; Coulter Immunology), an FITC-conjugated mouse anti-human {zeta} mAb (TIA-2; Coulter Immunology), or a PE-conjugated rat anti-mouse Sdc1 mAb (281-2; BD Biosciences). The analysis was performed using a FACScan flow cytometer and consort-30 software. An FITC-conjugated Armenian hamster anti-mouse IgG (Coulter Immunology), an FITC-conjugated mouse anti-human IgG (Coulter Immunology), and a PE-conjugated rat anti-mouse IgG (Coulter Immunology) were used as negative controls.

Ab stimulation and IL-2 quantification

Anti-mouse CD3 mAb (KT3) (10 µg/ml; Coulter Immunology) was bound for 16 h to a 24-well, flat-bottom plate in PBS. The wells were rinsed with fresh PBS three times before the addition of the cells. Fifty microliters of transfected cells (1.0 x 106 cells/ml) were added to each well and incubated at 37°C in 7.0% CO2. Cells and culture supernatants were collected at 24 h after stimulation. The supernatants were assayed using a standard IL-2 assay. Recombinant murine IL-2 (BD Pharmingen) was used as a standard.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lower expression of {zeta} and TCR/CD3 complex expression on the cell surface of the AS3' UTR and EX7– mutants

To investigate the expression of {zeta} protein and the TCR/CD3 complex on the cell surface, MA5.8 and its mutants were stained with an FITC-conjugated anti-mouse CD3{epsilon} mAb (145-2C11) (black profiles in Fig. 1A) or an FITC-conjugated anti-human {zeta} mAb (TIA-2) (black profiles in Fig. 1B) and analyzed by flow cytometry (Fig. 1). Although the expression of {zeta} protein on the cell surface of the AS3' UTR and EX7– mutants (mean channel fluorescence value, 29.79 and 34.43, respectively) seemed to be up-regulated compared with the MA5.8 (12.56) and NEG (14.35) cells, it was much lower than that of the WT mutants (108.56). CD3{epsilon} was weakly positive on the cell surface of MA5.8 cells (8.00) and the NEG mutant (7.07). Interestingly, whereas the expression of CD3{epsilon} on the cell surface of the AS3' UTR and EX7– mutants (10.23 and 18.32) was also likely to be higher than those on the MA5.8 cells (8.00) and the NEG mutant (7.07), it was much lower than that on the WT mutants (42.21).



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  		FIGURE 1. Flow cytometric analysis of the {zeta} and TCR/CD3 complexes on the MA5.8 mutants. The surface expression of (A) the TCR/CD3 complex and (B) the {zeta} protein on MA5.8 cells and its mutants (NEG, WT, EX7–, AS3' UTR) was quantified using FITC-conjugated anti-mouse CD3{epsilon} mAb (145-2C11) (filled profiles in A) and FITC-conjugated anti-human {zeta} mAb (TIA-2) (filled profiles in B), respectively. An FITC-conjugated Armenian hamster anti-mouse IgG (open profiles in A) or an FITC-conjugated mouse anti-human IgG (open profiles in B) was used as the negative control. The mean channel fluorescence value is indicated within the figures at the top right.

 
Microarray analysis of gene expression profiles in AS3' UTR and EX7– mutants

The WT, AS3' UTR, EX7–, and NEG mutants were stimulated with anti-CD3 Ab for 24 h and collected, respectively. To confirm that this activation by anti-CD3 Ab was induced exactly, IL-2 production in the WT mutant, which has demonstrated the TCR/CD3 complex on its cell surface by FACS analysis in this study, was compared with that in the NEG mutant, which has demonstrated low level cell surface expression of TCR/CD3 complex by FACS analysis. As a result, IL-2 production in the WT mutant (56.25 ± 0.25 ng/ml) was significantly (p < 0.01) higher than that in the NEG mutant (4.50 ± 0.71 ng/ml), demonstrating the accuracy of this activation by anti-CD3 Ab.

Total RNA was prepared from the WT, AS3' UTR, and EX7– mutants after stimulating with anti-CD3 Ab, respectively and reverse-transcribed into a cDNA probe (WT, AS3' UTR, and EX7– probe, respectively). After microarray analysis with Agilent cDNA microarrays that contained 8691 probe sets (genes), all hybridization spots on the image were quantified. The expression level of each gene in the AS3' UTR and EX7– mutants was evaluated as the ratio to its expression in the WT mutant. In genes expressed in the AS3' UTR and EX7– mutants, 8553 (98.4%) and 8568 (98.6%), respectively, of 8691 genes were judged to be detectable. In the AS3' UTR mutant, 48 (0.6%) and 187 (2.2%) of 8553 genes were up-regulated and down-regulated, respectively. In the EX7– mutant, in contrast, 88 (1.0%) and 189 (2.2%) of 8568 genes were up-regulated and down-regulated, respectively, secondarily to the down-regulation of {zeta}.

Because the AS3' UTR and EX7– mutants are representative transfectants associated with down-regulated {zeta} expression (11, 12), they were evaluated for gene expression profiles that were increased or decreased in both of them and the results showed that 36 genes were down-regulated in both the AS3' UTR and EX7– mutants (Table II). Interestingly, expression of the genes associated with inflammatory activity, including genes encoding cytokines and chemokines, was significantly decreased. These genes whose expression was decreased included the genes for IL-2, IL-18, IL-13, IL-15, CCL9, CCL3, CCL1, CCL5, and C chemokine ligand 1 (XCL1). Expression of genes encoding molecules related to signal transduction and cell growth, such as TGF{beta}-2, guanine nucleotide binding protein {beta}4, and MEK kinase 8, was also down-regulated due to decreased expression of {zeta}. Expression of genes that encode membranous proteins, including member 11 of solute carrier family 21, member 1 of solute carrier family 14, CD24a, CD52, and member 11 of the TNF superfamily, known as RANKL, was also diminished.


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  		Table II. Down-regulated genes in both the AS3' UTR mutant and EX7– mutant

 
In contrast, 16 genes were up-regulated in both the AS3' UTR and EX7– mutants (Table III), and they included the genes associated with membranous proteins, including cell adhesion molecules (Pvrl2, Sdc1, Wiskott-Aldrich syndrome-like (Wasl), solute carrier family 4 (Slc4a8)) and genes related to the cell damage molecules (granzyme A (Gzma) and matrix metalloproteinase 11 (Mmp11)). Expression of the genes encoding molecules related to the lipid transport and metabolism, including phosphomannomutase 1 (Pmm1), lipocalin 2 (Lcn2), and fatty acid desaturase 2 (Fads2), was also up-regulated in both mutants.


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  		Table III. Up-regulated genes in both the AS3' UTR mutant and EX7– mutant

 
Real-time PCR confirms the microarray results

To verify the findings obtained with the cDNA microarray, selected genes that were up-regulated or down-regulated in both the AS3' UTR and EX7– mutants were further studied by real-time PCR. As genes down-regulated secondary to the decreased expression of {zeta}, we focused on the expression level of the genes encoding cytokines IL-13, IL-15, IL-18, IL-2, and TGF{beta}-2. As up-regulated genes, we verified the expression level of the genes encoding Fads2, glutathione S-transferase {alpha}4 (Gsta4), Gzma, Lcn2, max dimerization protein 3 (Mad3), Mmp11, Pmm1, protein tyrosine phosphatase 4a3 (Ptp4a3), Pvrl2, Sdc1, selenium binding protein 1 (Selenbp1), Slc4a8, T cell-specific transcription factor 7 (Tcf7), and Wasl.

One microgram of whole mRNA was isolated from 2B4.11 cells and each of the MA5.8 mutants (WT, AS3' UTR, and EX7– mutants) and converted to whole cDNA with reverse transcriptase. A 5-µl sample of the whole cDNA obtained from the WT, AS3' UTR, or EX7– mutant was used as the template, and {beta}-actin cDNA and each of the cDNA above was quantified by real-time PCR. The standard curve for each cDNA was constructed from the whole cDNA of 2B4.11 cells to validate the real-time PCR. The critical threshold cycle for each cDNA was inversely proportional to the logarithm of the initial amount of the standard template DNA (correlation coefficients of the genes, 0.911–0.999). Then the threshold cycle of each cDNA was measured by real-time PCR, and the relative expression level of each gene was evaluated as its ratio to the level of {beta}-actin cDNA. Then the expression level of each transcript in the AS3' UTR and EX7– mutants was estimated relative to that in the WT mutant, which was assigned a value of 1.0. The cDNAs were measured by three separate experiments, and statistical significance was calculated by using the Student t test.

The results of the real-time PCR confirmed that the expression of IL-13, IL-15, IL-18, IL-2, and TGF{beta}-2 in both the AS3' UTR and EX7– mutants was significantly reduced (p < 0.01) compared with expression in the WT mutant (Table IV). Interestingly, the relative expression level of the IL-2 transcript was markedly (<1/10) decreased in the MA5.8 mutants with reduced {zeta} expression (AS3' UTR and EX7– mutants).


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  		Table IV. Genes whose expression level was confirmed by real-time PCR

 
In contrast, real-time PCR confirmed that the expression level of Gsta4, Gzma, Lcn2, Mad3, Pmm1, Ptp4a3, Pvrl2, Sdc1, Selenbp1, Slc4a8, Tcf7, and Wasl in both the AS3' UTR and EX7– mutants was significantly (p < 0.01) increased compared with the WT mutant (Table IV). However, the transcriptional level of Fads2 in the EX7– mutant, and of Mmp11 in the AS3' UTR mutant, did not differ from the level in the WT mutant.

Granzyme A, syndecan-1, and Pvrl2 protein expression in AS3' UTR and EX7– mutants

We focused on the cytotoxic granules and cell membrane proteins, including adhesion molecules, that were up-regulated due to decreased expression of {zeta}, because these up-regulated cell membrane proteins may be related to the pathogenesis of SLE, and confirmed the protein expression level of Gzma, Sdc1, and Pvrl2.

MA5.8 cells and its mutants (WT, AS3' UTR, EX7–, NEG) were stimulated with anti-CD3 Ab for 24 h and collected. As shown in Fig. 2, Western blot analysis of the cell lysate with an anti-mouse Sdc1 mAb (281-2) showed markedly increased production of Sdc1 protein (43 kDa) in the AS3' UTR and EX7– mutants in comparison with the WT mutant. In contrast, production of Pvrl2 protein (53 kDa) was diminished in the WT mutant, but it was clearly visualized in the AS3' UTR and EX7– mutant by Western blot with anti-mouse nectin-2 Ab (N-20). In addition, production of Gzma, which was detected by anti-mouse Gzma Ab (D-15), was increased in the AS3' UTR and EX7– mutants in comparison with the WT mutant. Production of Sdc1, Pvrl2, and Gzma after the anti-CD3 Ab stimulation in the AS3' UTR and the EX7– mutants was up-regulated as similarly in MA5.8 cells as the untransduced control and the NEG mutant that was transduced with empty vector, demonstrating that transduction itself would not alter the transcription of these proteins.



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  		FIGURE 2. Western blot analysis of syndecan-1, Pvrl2, and granzyme A expressed in the MA5.8 mutants. MA5.8 cells and its mutants (WT, NEG, AS3' UTR, and EX7–) were stimulated with anti-CD3 Ab for 24 h and collected. Cell lysates were electrophoresed on 12.5% SDS-polyacrylamide gels and blotted onto a polyvinylidene difluoride membrane. The membranes were then incubated at 16°C for 1 h with a rat anti-syndecan-1 mAb, a goat anti-mouse nectin-2 Ab, or a goat anti-mouse granzyme A Ab. The Abs were visualized with peroxidase-conjugated anti-rat or anti-goat IgG. After washing three times, the signals were detected by chemiluminescence enhancing. The treated membranes were visualized on ECL x-ray film.

 
To investigate expression of Sdc1 protein on the cell surface, the MA5.8 mutants were stimulated with anti-CD3 Ab for 24 h, stained with a PE-conjugated anti-mouse Sdc1 mAb (281-2), and were analyzed by flow cytometry. The results as shown in Fig. 3 indicated that Sdc1 expression on the cell surface of the AS3' UTR (mean channel fluorescence value, 47.12) and EX7– mutants (36.70) was up-regulated compared with the WT mutant (26.90).



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  		FIGURE 3. Flow cytometric analysis of Sdc1 on the MA5.8 mutants. MA5.8 mutants (WT, AS3' UTR, and EX7–) were stimulated with anti-CD3 Ab for 24 h and their cell surface expression of Sdc1 was stained with a PE-conjugated rat anti-mouse Sdc1 mAb (281-2). The analysis was performed using a FACScan flow cytometer and consort-30 software. A PE-conjugated rat anti-mouse IgG (Coulter Immunology) was used as negative controls. The mean channel fluorescence value is indicated within the figures at the top right.

 
Based on the above observations, we can conclude that the protein expression level of Sdc1, Pvrl2, and Gzma was also confirmed to be up-regulated in the T cells expressing the splice variant forms of {zeta} mRNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have reported for the first time the gene expression profiles of T cells associated with reduced expression of {zeta}. The AS3' UTR mutant (11) is one of the MA5.8 mutants transfected with the alternative splice form of {zeta} mRNA, whereas the EX7– mutant (12) is one of the MA5.8 mutants transfected with the exon7-deleted {zeta} mRNA. Cell surface expression not only of {zeta}, but also of the TCR/CD3 complex, was confirmed to be absent or decreased in both of these MA5.8 mutants in this study. Thus, the up-regulated or down-regulated gene expression profiles observed in both of these MA5.8 mutants with the splice variant forms of {zeta} mRNA in this study may be strongly associated with down-regulation of {zeta}. Also the decreased expression of certain genes could simply reflect reduced Ag-receptor mediated signaling intensity. We have already reported significantly reduced IL-2 production after anti-CD3 Ab treatment in both of these AS3' UTR and EX7– mutants (11, 12). Our results in the study presented here showed down-regulation not only of IL-2 but also of other ILs (IL-13, IL-15, and IL-18), chemokines (CCL3, CCL5, CCL9, XCL1), and a chemokine receptor (CCR7) in both the AS3' UTR and EX7– mutants, indicating that down-regulation of {zeta} may lead to aberrant signal transduction followed by reduced expression of these cytokines and chemokines. Expression of {zeta} in the PBT of cancer patients or in tumor-infiltrating T cells has also been reported to be decreased (20, 21, 22, 23), and, interestingly, cytokine expression of IL-2 and IL-4 in these T cells has been observed to be down-regulated. DNA microarray analyses of the gene expression profiles of SLE patient T cells has revealed down-regulation of several TCRs, such as TCR{alpha}{beta}, TCR{delta}, and TCR{gamma} (24, 25, 26), indicating reduced expression of the TCR/CD3 complex in SLE patient T cells, the same as observed in the AS3' UTR and EX7– mutants (11, 12).

Impressive data on genes that were up-regulated in relation to the decreased expression of {zeta} were observed in this study. Not only was the expression of Sdc1 and Pvrl2 genes at the transcriptional level in the AS3' UTR and EX7– mutants in comparison with the WT mutant significantly increased, but it was also significantly increased at the protein level. Sdc1 is a family of highly conserved type I transmembrane heparan sulfate proteoglycans that is expressed in a developmental and cell type-specific inflammation (27, 28, 29, 30). Sdc1 is known to bind several ligands, including chemokines and cytokines, growth factor like FGF-2, cell adhesion molecules like L-selectin, and extracellular matrix molecules like fibronectin, via its heparan sulfate chains, to act as coreceptors in a variety of physiological processes (28). Seagal et al. (31) reported that Fas- and IgM transmembrane tail exon-deficient mice (µMT/lpr) produce autoantibodies and develop severe lymphoproliferation accompanied by up-regulation of Sdc1, suggesting a relationship between autoimmunity and increased Sdc1 expression. Dhodapkar et al. (32) also demonstrated that Sdc1 leads to a striking induction of apoptosis and inhibition of cell growth in vitro. Pvrl2, also known as nectin-2, is a member of the Ig superfamily proteins that plays a pivotal role in homophilic adhesion. It consists of an NH2-terminal signal sequence, three extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic tail and is known to be involved in cell-cell and cell-extracellular matrix interactions (33, 34). Thus, the T cells with reduced expression of {zeta} described in this paper may exhibit aberrant cell adhesion. Also the cytotoxic molecules, like Gzma or MMP-11, which were observed to be up-regulated in these T cells, may be responsible for the tissue damage. MMP-3 and MMP-9 transcripts have also been found to be increased in SLE patient PBTs by methods using cDNA microarrays (24, 35). Several molecules responsible for lipid transport and metabolism (Pmm1, Lpn2, Fads2) were also found to be increased in the study presented here, indicating the potential importance of lipids in the pathogenesis of SLE. Several reports have mentioned up-regulated genes in tumor-related T cells with reduced {zeta} expression. Takahashi et al. (20) reported increased expression of caspase-3 in gastric cancer patients. {zeta}-KO mice, in contrast, show increased expression of IFN-{gamma}. IL-10 secretion has also been reported to be up-regulated in T and NK cells isolated from a patient with ovarian carcinoma (36). These reports were based on analyses of just a small number of molecules, whereas we evaluated the expression of a wide variety of genes by using cDNA microarrays. Several IFN-inducible genes have been reported to increase in SLE patient T cells. However, no significant up-regulation of IFN-inducible genes was observed in the AS3' UTR or EX7– mutant in comparison with the WT mutant in our study (data not shown). Expression of the up-regulated genes observed in these MA5.8 mutants with diminished {zeta} should be confirmed in SLE patient T cells. These projects are now under way in our laboratory. In conclusion, identification of the responsive genes in T cells with down-regulation of TCR/CD3 complex, including {zeta}, may help to better understand the mechanism underlying T cell dysfunction and the pathogenesis of SLE.


   Acknowledgments
 
We thank Dr. Takashi Saito (RIKEN, Research Center for Allergy and Immunology, Yokohama, Japan) for providing the MA5.8 and 2B4.11 cells.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants-in-Aid for Scientific Research (C), the Ministry of Education, Science and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Kensei Tsuzaka, Division of Rheumatology, Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, Kamoda 1981, Kawagoe, Saitama 350-8550, Japan. E-mail address: kentsu{at}saitama-med.ac.jp Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; PBT, peripheral blood T cell; UTR, untranslated region. Back

Received for publication August 11, 2005. Accepted for publication October 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Mills, J. A.. 1994. Systemic lupus erythematosus. N. Engl. J. Med. 30: 1871-1879.
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