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The Journal of Immunology, 2003, 170: 2871-2876.
Copyright © 2003 by The American Association of Immunologists

Forced Expression of the Fc Receptor {gamma}-Chain Renders Human T Cells Hyperresponsive to TCR/CD3 Stimulation 1

Madhusoodana P. Nambiar*,{dagger}, Carolyn U. Fisher*, Anil Kumar*, Christos G. Tsokos*, Vishal G. Warke* and George C. Tsokos2,*,{dagger}

* Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; and {dagger} Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High level expression of Fc{epsilon}RI{gamma} chain replaces the deficient TCR {zeta}-chain and contributes to altered TCR/CD3-mediated signaling abnormalities in T cells of patients with systemic lupus erythematosus. Increased responsiveness to Ag has been considered to lead to autoimmunity. To test this concept, we studied early signaling events and IL-2 production in fresh cells transfected with a eukaryotic expression vector encoding the Fc{epsilon}RI{gamma} gene. We found that the overexpressed Fc{epsilon}RI{gamma} chain colocalizes with the CD3{epsilon} chain on the surface membrane of T cells and that cross-linking of the new TCR/CD3 complex leads to a dramatic increase of intracytoplasmic calcium concentration, protein tyrosine phosphorylation, and IL-2 production. We observed that overexpression of Fc{epsilon}RI{gamma} is associated with increased phosphorylation of Syk kinase, while the endogenous TCR {zeta}-chain is down-regulated. We propose that altered composition of the CD3 complex leads to increased T cell responsiveness to TCR/CD3 stimulation and sets the biochemical grounds for the development of autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Fc{epsilon}RI{gamma} chain, a member of the {zeta}-chain family of proteins, is a component of Fc{epsilon}RI and has one cytoplasmic immunoreceptor tyrosine-based activation motif (1). It is expressed on mast cells, basophils, monocytes, and NK cells (2, 3). Unlike TCR {zeta}-chain, which mediates signaling through {zeta}-associated protein 70 (ZAP-70),3 Fc{epsilon}RI{gamma} mediates signaling by associating with the structurally homologous tyrosine phosphorylated protein kinase, Syk (4). It has been reported that the Syk kinase is 100-fold more potent compared with ZAP-70 (5), and it is preferentially recruited to the Fc{epsilon}RI{gamma} (6). In addition, Fc{epsilon}RI{gamma} is located very close to {zeta}-chain in chromosome 1q (7), an area that has been linked with the expression of systemic lupus erythematosus (SLE) in humans (8).

There is ample evidence that the Fc{epsilon}RI{gamma} chain can replace the TCR {zeta}-chain and facilitate TCR/CD3 complex-mediated signaling. Expression of Fc{epsilon}RI{gamma}, in lieu of TCR {zeta}-chain, has been reported in mouse large granular lymphocytes (9). Fc{epsilon}RI{gamma} chain supports T cell development and function in mice lacking endogenous TCR {zeta}-chain (10). T lymphocytes from tumor-bearing mice expressed TCR that completely lacked TCR {zeta}-chain-expressed Fc{epsilon}RI{gamma} chain (11, 12). Also, TCR {zeta}-deficient mice have been shown to express Fc{epsilon}RI{gamma} as part of the TCR-{gamma}{delta} complex (13, 14, 15).

We have recently shown that fresh T cells from patients with SLE express Fc{epsilon}RI{gamma} chain that is up-regulated and becomes part of the surface CD3 complex with complete ability to mediate signal transduction through the up-regulated Syk kinase (16). Notable is that this altered composition of CD3 in SLE T cells is associated with a higher magnitude signaling process, that is, with increased free intracytoplasmic calcium [Ca2+]i response and protein tyrosine phosphorylation responses (17, 18, 19). Increased Ag receptor-initiated signaling in a graft vs host model heralds the ignition of autoimmunity (20), and cognate Ag stimulation in the TCR transgenic MRL mouse leads to T cell hyperresponsiveness and autoimmune manifestations (21). Therefore, it seems that in both humans and mice increased lymphocyte excitability is associated with autoimmunity.

In this study we sought to test directly whether forced expression of Fc{epsilon}RI{gamma} chain in normal fresh T cells, which do not express this molecule, would lead to increased T cell responsiveness similar to that observed in SLE T cells. We capitalized on a recently developed efficient electroporation-based fresh T cell transfection protocol (22) and a transfection technique by nucleoporation, which enabled increased expression of Fc{epsilon}RI{gamma} chain encoded in an expression vector. We report that the Fc{epsilon}RI{gamma} chain containing CD3 complex leads to increased T cell responsiveness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

Anti-Fc{epsilon}RI{gamma} was a generous gift from Dr. J. P. Kinet (Beth Isreal Deaconess Medical Center, Boston, MA) and was raised to the peptide sequence CKHEKPPQ (aa 80–86) of the Fc{epsilon}RI{gamma} chain (1). Now, this Ab is available from Upstate Biotechnology (Lake Placid, NY), and it has been used in some experiments. OKT3 was from Orthoclone Biotech (Raritan, NJ). CD3-PE was from Sigma-Aldrich (St. Louis, MO). CD4-PE, CD8-PE, and CD14-PE were from Coulter (Miami, FL), CD16-PE was from BD PharMingen (San Diego, CA). Fluorescent isotype controls were purchased from either Sigma-Aldrich, Jackson ImmunoResearch Laboratories (West Grove, PA), or BD PharMingen. The TCR {zeta}-chain mAb, 6B10.2, recognizing the aa 31–45 of the polypeptide (N-terminal mAb) was purchased from BD PharMingen. The C-terminal TCR {zeta}-chain mAb, recognizing the amino acids from 145–161 and HRP-conjugated anti-phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology. Anti-ZAP-70 Ab and anti-Syk Ab (clone LR-10) and active kinase (clone 4D10) was from Upstate Biotechnology.

Isolation of T cells

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples by density gradient centrifugation using Ficoll, and T cells were isolated from PBMCs by negative selection by using magnetic separation (Miltenyi Biotec, Auburn, CA) as described previously (16). The purity of the T cells was usually >98% as measured by flow cytometry using anti-CD3-PE Ab.

RT-PCR of Fc{epsilon}RI{gamma} chain and expression cloning

Total RNA was isolated using an RNeasy mini kit (Qiagen, Santa Clarita, CA) from 5 million T cells according to the manufacturer’s directions. Single-stranded cDNA was synthesized from 1 µg total RNA by using the avian myeloblastoma virus reverse transcriptase-based reverse transcription system from Promega (Madison, WI) and oligo-dT primer. The primers for Fc{epsilon}RI{gamma} chain were synthesized by Sigma-Aldrich and are as follows: forward 5'-CCT GGG AGA GCC TCA GCT CTG CTA TAT C-3' (sense bp 79–106), reverse 5'-GAA TAT GAC CGC ATC TAT TGT AAA G-3' (antisense bp 311–287) (23). Reverse transcription products were PCR amplified with a high fidelity PCR system (Boehringer Mannheim, Indianapolis, IN). The PCR products were ligated to eukaryotic expression vector, pcDNA 3.1/CT-GFP-TOPO (Invitrogen, Carlsbad, CA). Recombinant plasmids were isolated and restriction mapped with EaeI and were then subjected to DNA sequencing. The Fc{epsilon}RI{gamma} clone was grown in 2-liter culture; plasmid was isolated by endonuclease free Qiagen Maxi kit (Qiagen) and was used for transfection. Empty plasmid pcDNA 3.1/CT-GFP-TOPO was used as control. Plasmid containing {beta}-galactosidase was used as a negative control, and the enhanced green fluorescence protein (GFP) in pIRES vector (Clontech Laboratories Palo Alto, CA) was used as a positive control for FACS analysis to determine transfection efficiency.

Transfection of T cells

After several modifications of the electroporation method, we established a method to transfect primary T cells routinely with 25–30% efficiency (22). Briefly, the PBMCs were incubated overnight at a density of 3 x 106/ml in RPMI 1640 containing penicillin/streptomycin and 10% fetal bovine serum or autologous serum and 1 µg/ml PHA for 18–20 h. T cells were isolated by negative selection of non-T cells by MACS separation as described above. Purified T cells were suspended in 500 µl Opti-MEM medium and transferred to Gene Pulser cuvette (Bio-Rad, Hercules, CA). Plasmid DNA (25 µg) was added, and the cells were electroporated at 300 V/1000 µF. Cells were washed and resuspended in AIM-V medium. Electroporated cells were incubated in the presence of 10 U of IL-2/ml at 37°C for 48–72 h for optimum gene expression.

T cells were also transfected by a very recently developed nucleoporation technique using a kit from Amaxa (Cologne, Germany). Purified T cells, 5 x 106–20 x 106, were suspended in 100 µl nucleofection reagent, mixed with 5 µg plasmid DNA, and electroporated using an optimized program U-14 in a nucleoporator. The T cells were then immediately transferred to flasks and cultured in complete medium. Because of the direct transfer of gene to nucleus, nucleoporated cells start expressing protein early, and the cells were analyzed after 18 h.

Measurement of free cytoplasmic Ca2+ concentration

Free cytoplasmic Ca2+ concentrations were estimated in indole-acetoxymethyl ester (INDO-1) loaded cells as previously described (17). Briefly, 1 x 106 cells were loaded with acetoxymethylester INDO-1 (Molecular Probes, Eugene, OR) (1 µg/ml) for 20 min at 37°C. Cells were analyzed using an Epics Altra (Coulter) flow cytometer equipped with a high power dual wavelength (365 nm and 488 nm) argon laser. In each run, first the cells were run unstimulated to record the baseline fluorescence ratio, which represents the resting intracellular Ca2+ concentration ([Ca2+]i) levels. After 40 s, either Ab OKT3 (10 µg/ml) or the isotype control mIgG2a, followed by 20 µg/ml goat anti-mouse Ab, was added to the tube, and the mean ratio of the fluorescence, which is directly proportional to the free cytosolic Ca2+, was recorded for a total of 10 min.

Western blotting and immunoprecipitation

Five million T cells transfected with control plasmid expressing {beta}-galactosidase or GFP and Fc{epsilon}RI{gamma}-GFP construct were lysed and separated by electrophoresis and blotted using the indicated Abs as previously described (24). The intensity of the bands in the autoradiogram was evaluated after scanning using GelPro software (Media Cybernetics, Silver Spring, MD). Normalization against ZAP-70 was done by dividing the densitometric values of each band with the corresponding values of ZAP-70, and the ratio was represented in arbitrary units. Detergent-insoluble membrane fraction was electrophoresed after lysis by mechanical agitation and heating in the presence of 4% SDS as described previously (24). For immunoprecipitation, total cell lysates from 20 x 106 transfected cells were precleared with protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) for 1 h. The precleared lysates were incubated with CD3{epsilon} mAb overnight at 4°C and immunoprecipitated with protein A-Sepharose. Immunoprecipitates were washed, resuspended in samples buffer, separated by electrophoresis, and immunoblotted with Fc{epsilon}RI Ab.

Flow cytometry

Cells were resuspended in staining buffer (PBS containing 2% FBS) and was mildly fixed with 50 µl of 1% paraformaldehyde in PBS for 10 min at 4°C. The cells were stained with labeled Abs essentially as described (16). Stained or GFP expressing cells were analyzed by a BD Biosciences FACScan using CellQuest software (BD Biosciences, San Jose, CA).

Confocal microscopy

Transfected cells (106) were adhered to poly-L-coated slides (Sigma-Aldrich) by incubating on ice for 1 h. Subsequently, capping of TCR was induced by incubating the cells at 37°C for 10 min with anti-CD3 IgM Ab. The reaction was stopped using 3.7% paraformaldehyde, and cells were washed three times with PBS (pH 7.4) followed by permeabilization using 0.1% saponin. Excessive amounts of blocking human IgG was added and incubated for 10 min followed by anti-Fc{epsilon}RI{gamma} Ab or TCR {zeta}-chain Ab or its isotype control for 45 min on ice. Cells were washed, blocked, and incubated with the secondary Abs labeled with FITC or tetramethylrhodamine isothiocyanate (TRITC), and anti-CD3{epsilon}-FITC was added for 30 min. Cells were washed four times with PBS, air dried for 1 min, and cover slips were mounted using Gel/Mount (Biomedia, Foster City, CA), and the edges were sealed. Samples were analyzed by laser scanning with a confocal fluorescence microscope (Zeiss, Oberkochen, Germany) using LSM 510 software.

T cell activation and IL-2 assay

Seventy-two hours after transfection, cells were activated via TCR/CD3 complex with 10 µg/ml anti-CD3 mAb plus 2.5 µg/ml anti-CD28 mAb for 24 h. Supernatants were collected, and IL-2 was measured by Quantikine ELISA kit (R&D Systems, Minneapolis, MN).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Fc{epsilon}RI{gamma} chain in transfected T cells

Human T cells were transfected using the electroporation method, and the transfection efficiency was determined by FACS analysis of the expressed GFP (22). Plasmid pIRES containing enhanced GFP was used as a positive control, and a vector containing {beta}-galactosidase gene was used as a negative control. The data showed that the electroporation method results in ~35–40% transfection in primary T cells 72 h after transfection (Fig. 1A). Primary T cells were also transfected by a recently developed nucleoporation technique. Nucleoporation resulted in 70–75% transfection efficiency in human T cells after 18 h (Fig. 1B). T cells were then transfected by electroporation or nucleoporation with pcDNA 3.1/CT-GFP carrying the Fc{epsilon}RI{gamma} gene. Empty vector pcDNA 3.1/CT-GFP was used as control. Immunoblotting of lysates of Fc{epsilon}RI{gamma}-transfected cells with an anti-Fc{epsilon}RI{gamma} Ab showed that significant levels of Fc{epsilon}RI{gamma} chain is expressed in transfected human primary T cells (Fig. 1C).



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  		FIGURE 1. Expression of Fc{epsilon}RI{gamma} chain in transfected primary human T cells. T cells were isolated from human donors and transfected by electroporation (A) or nucleoporation (B) using techniques described in Materials and Methods. Plasimd pIRES (Clontech Laboratories) containing enhanced GFP was used as a positive control. Mock transfected cells with a vector containing {beta}-galactosidase gene was used as negative control. Flow cytometry was used to assess the transfection efficiency. The results were highly reproducible throughout the completion of this study. C, Primary T cells were also transfected by electroporation or nucleoporation with the Fc{epsilon}RI{gamma} construct. Empty vector pcDNA 3.1/CT-GFP served as control. Immunoblotting of the electrophoretically separated lysates was performed using an Fc{epsilon}RI-specific mAb.

 
Overexpression of Fc{epsilon}RI{gamma} chain induces increased TCR/CD3-mediated [Ca2+]i responses in human T lymphocytes

Because Fc{epsilon}RI{gamma} has one immunoreceptor tyrosine-based activation motif, which confers signaling properties for this protein and can replace TCR {zeta} in certain cellular systems (9, 11, 13, 14, 15), we hypothesized that overexpression of Fc{epsilon}RI{gamma} in normal T cells may associate with TCR/CD3 complex and contribute to altered T cell signaling. To address this hypothesis, we measured the CD3-induced [Ca2+]i responses in cells transfected for 72 h with control vector or plasmid construct Fc{epsilon}RI{gamma}. As shown in Fig. 2A, the calcium response was significantly higher in Fc{epsilon}RI{gamma}-transfected cells compared with mock vector-transfected cells. The expression of TCR was similar in Fc{epsilon}RI{gamma}-transfected cells compared with the control vector-transfected cells. To compare the increase in the signaling between the TCR/CD3 complex with the Fc{epsilon}RI{gamma} and TCR/CD3 complex with TCR {zeta}-chain, we transfected equal amounts of TCR {zeta}-chain and Fc{epsilon}RI{gamma} plasmids under identical conditions and measured the [Ca2+]i response (Fig. 2B). TCR {zeta}-chain-transfected cells displayed a heightened [Ca2+]i response comparable to that of Fc{epsilon}RI{gamma}-transfected cells. The peak mean responses from four experiments were similar in {zeta}-chain- or Fc{epsilon}RI{gamma}-transfected cells (Fig. 2C). Although substantial amounts of the forcibly expressed Fc{epsilon}RI{gamma} associate with TCR, we predict some signaling in the Fc{epsilon}RI{gamma}-transfected cells could be mediated by TCR {zeta}-chain.



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  		FIGURE 2. Transfection with Fc{epsilon}RI{gamma} chain causes an increased TCR/CD3-mediated calcium response in human T cells. T cells were transfected with Fc{epsilon}RI (A), TCR {zeta}-chain (B), or empty vector pcDNA 3.1/CT-GFP (A and B) by electroporation. After 72 h, the cells were loaded with INDO-1, and [Ca2+]i levels were estimated as described in Materials and Methods. After the baseline fluorescence ratio which represents the resting [Ca2+]i levels was established, the cells were stimulated with either OKT3 (10 µg/ml) or the isotype control mIgG2a, followed by 20 µg/ml goat anti-mouse Ab. C, Histogram representing the peak [Ca2+]i response (280 s) in control vector, Fc{epsilon}RI and TCR {zeta}-chain-transfected cells; mean ± SEM (n = 4).

 
Overexpression of Fc{epsilon}RI{gamma} chain down-regulates the endogenous TCR {zeta}-chain expression in human T cells

The observed increase in TCR-mediated signaling in normal T cells forced to express Fc{epsilon}RI{gamma} chain could be attributed to a simple additive effect to the signaling process conducted through the endogenous TCR {zeta}-chain. As presented in Fig. 2, overexpression of TCR {zeta}-chain in T cells led to a further increase in [Ca2+]i responses. Therefore, we determined the levels of endogenous TCR {zeta}-chain in fresh cells forced to express Fc{epsilon}RI{gamma} chain. Transfected cells were lyzed and immunoblotted with {zeta}-chain-specific Abs. Because TCR {zeta}-chain is present in T cells either as a phosphorylated p21–23 or as a p16 band, we evaluated both forms in the lysates of transfected and control cells. The results show that the levels of both forms of {zeta}-chain are significantly (p < 0.05) decreased in Fc{epsilon}RI{gamma}-transfected cells (Fig. 3, A and B). Because {zeta}-chain undergoes ubiquitination following activation (25), and in SLE T cells we have reported increased levels of a ubiquitinated form of {zeta}-chain (24), we determined the levels of ubiquitinated {zeta}-chain in lysates from cells transfected with Fc{epsilon}RI{gamma} vector. As can be seen in Fig. 3, the decreased level of {zeta}-chain is not caused by increased ubiquitination in Fc{epsilon}RI{gamma}-transfected cells. A similar pattern of TCR {zeta}-chain expression was observed in the detergent-insoluble membrane fraction that includes the cytoskeletal and lipid raft-associated forms of the {zeta}-chain. This finding suggests that the expressed Fc{epsilon}RI{gamma} chain is responsible for the increased responsiveness of the T cells in a direct manner, and it does not simply confer an additive effect to the signaling process mediated through endogenous TCR {zeta}-chain.



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  		FIGURE 3. Suppression of TCR {zeta}-chain expression in Fc{epsilon}RI{gamma}-transfected human T lymphocytes. A, T cells 72 h after transfection by electroporation with Fc{epsilon}RI{gamma} or empty vector, were lysed with Nonidet P-40 lysis buffer containing various protease inhibitors, and 15 µg of protein from the detergent-soluble fractions was analyzed on 4–12% Nu-PAGE gel under reducing condition, transferred, and immunoblotted with {zeta}-chain mAb 6B10.2 and developed using a chemiluminescence kit. The membrane was stripped and sequentially reprobed with {zeta}-chain C-terminal mAb that recognizes the tyrosine phosphorylated and ubiquitinated forms of the {zeta}-chain, CD3 {epsilon}, and ZAP-70 Abs. The ubiquitinated form of TCR {zeta}-chain was confirmed by immunoblotting with anti-ubiquitin Ab. B, Immunoblots were analyzed by densitometry, and the levels of expression of {zeta}-chain were normalized to ZAP-70 and were plotted (mean ± SEM (n = 3)). C, Detergent-insoluble membrane fraction was electrophoresed after lysis by mechanical agitation and heating in the presence of 4% SDS and immunoblotted as described above.

 
To determine the mechanism of TCR {zeta}-chain down-regulation in Fc{epsilon}RI{gamma}-transfected cells, we analyzed the mRNA level by RT-PCR. Total RNA was isolated from nucleoporated cells after 18 h, reverse transcribed, and PCR amplified as described earlier (26). There was no significant difference between the level of TCR {zeta}-chain mRNA in control vector or Fc{epsilon}RI{gamma}-transfected cells (data not shown), suggesting that the decreased expression of {zeta}-chain is not the consequence of transcriptional down-regulation.

Fc{epsilon}RI{gamma} colocalizes with the CD3{epsilon} and {zeta}-chain on the surface membrane of T cells

To study whether the overexpressed Fc{epsilon}RI{gamma} associates with TCR and participates in T cell activation, we studied the colocalization of Fc{epsilon}RI{gamma} with {zeta}-chain and CD3{epsilon} chain after stimulating T cells with an IgM anti-CD3 Ab for 10 min. As shown in Fig. 4, in transfected cells Fc{epsilon}RI{gamma} staining colocalized with CD3{epsilon} after stimulation with TCR/CD3 for 10 min (Fig. 4Aa). Similarly, Fc{epsilon}RI{gamma} staining colocalized with {zeta}-chain after TCR/CD3 stimulation (Fig. 4Ac). The data overlay (last row of Fig. 4A, a, b, and c) show that the expressed Fc{epsilon}RI{gamma} colocalizes with the CD3{zeta} and CD3{epsilon} chain, and therefore it may be engaged in TCR-mediated signaling.



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  		FIGURE 4. Fc{epsilon}RI{gamma} colocalizes with CD3{epsilon} and {zeta}-chain in transfected cells. A, T cells were transfected with Fc{epsilon}RI{gamma} by electroporation and then activated with anti-CD3 IgM Ab for 10 min, fixed, permeabilized, and then double immunostained with either CD3{epsilon}-FITC plus Fc{epsilon}RI{gamma}-TRITC (a); or CD3{epsilon}-FITC plus TCR {zeta}-TRITC (b); or TCR {zeta}-FITC plus Fc{epsilon}RI{gamma}-TRITC-labeled CD3 {zeta}, CD3{epsilon}, and Fc{epsilon}RI{gamma} chain as described in Materials and Methods. The lower row "overlay" depicts merged images of the images shown in the first two rows, and the yellow-orange color indicates colocalization. B, Cells nucleoporated with Fc{epsilon}RI{gamma} or empty vector pcDNA 3.1/CT-GFP were lysed, and the proteins were immunoprecipitated with an anti-CD3{epsilon} mAb. The immunoprecipitates were resuspended in sample buffer, electrophoresed, and immunoblotted with Ab specific to Fc{epsilon}RI chain. The membranes were stripped and reprobed with an anti-CD3{epsilon} Ab.

 
In SLE T cells the spontaneously up-regulated Fc{epsilon}RI{gamma} chain coprecipitates with the CD3{epsilon} chain (16), suggesting that it becomes an integral part of the Ag recognition and signaling complex. To further prove that overexpressed Fc{epsilon}RI{gamma} chain associates with the TCR, we immunoprecipitated total cell lysates of transfected cells with a CD3{epsilon} chain Ab. The immunoprecipitates were electrophoresed and immunoblotted with Fc{epsilon}RI{gamma} chain Ab. As shown in Fig. 4B, the immunoprecipitated CD3{epsilon} was associated with Fc{epsilon}RI{gamma} in Fc{epsilon}RI{gamma}-transfected T cells.

TCR/CD3 mediated tyrosine phosphorylation of cellular substrates, and protein kinase Syk is up-regulated in Fc{epsilon}RI{gamma} chain-transfected cells

The first step in the signal transduction after TCR/CD3 engagement is the tyrosine phosphorylation of TCR {zeta}-chain followed by the phosphorylation of ZAP-70 or Syk and other cellular substrates. We predicted that the overexpression and association of Fc{epsilon}RI{gamma} chain would be associated with increased TCR/CD3-mediated tyrosine phosphorylation of cellular protein substrates. After 72 h of transfection, the cells were activated with 10 µg/ml OKT3 for 1 min, and the cell lysates were blotted with an anti-phosphotyrosine Ab. In Fc{epsilon}RI{gamma}-transfected cells, the tyrosine phosphorylation of cellular protein substrates was significantly increased compared with the control vector-transfected cells (Fig. 5A). Thus, consistent with the increase in the TCR/CD3-mediated [Ca2+]i response, the phosphorylation of cellular protein substrates were also up-regulated in Fc{epsilon}RI{gamma}-transfected cells.



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  		FIGURE 5. A, Fc{epsilon}RI{gamma}-transfected T cells display a higher intensity of TCR/CD3-mediated tyrosine phosphorylation of cellular protein substrates. Fresh unmanipulated cells were transfected with Fc{epsilon}RI{gamma} or empty plasmid pcDNA 3.1/CT-GFP by nucleoporation and then activated with 10 µg/ml OKT3 Ab for 1 min. Activation was stopped, and the cells were lysed, electrophoresed, and transferred to polyvinylidene difluoride membrane. The membranes were blocked and immunoblotted with anti-phosphotyrosine (4G10) Ab. B, Up-regulation of protein tyrosine kinase Syk phosphorylation in Fc{epsilon}RI{gamma}-transfected cells. Proteins (15 µg) from lysates of cells transfected with empty vector pcDNA 3.1/CT-GFP and Fc{epsilon}RI{gamma} by nucleoporation were electrophoresed, transferred, and immunoblotted with anti-Syk Ab. The membranes were stripped and reprobed with {beta}-actin, anti-CD3{epsilon}, and Syk active kinase Abs. C, Densitometric analysis of Syk expression in Fc{epsilon}RI{gamma}-transfected cells. The immunoblots were scanned and quantitated by densitometry, and the normalized levels of phosphorylated Syk to {beta}-actin was plotted in control and Fc{epsilon}RI{gamma}-transfected cells (mean ± SEM, n = 4). The transfection efficiency was ~30–34% in all experiments.

 
In mast cells, Fc{epsilon}RI{gamma} associates with the protein tyrosine kinase Syk, which apparently propagates the surface membrane-initiated activation signal (4, 27). To determine whether the overexpression of Fc{epsilon}RI{gamma} chain modulates the phosphorylation of Syk, we analyzed the level of Syk in Fc{epsilon}RI{gamma}-transfected cells. Control plasmid and Fc{epsilon}RI{gamma} chain-transfected cells were activated with OKT3 Ab for 1 min, lysed, and then equal amounts of proteins were separated electrophoretically and immunoblotted with the anti-phosphotyrosine mAb. The blots were stripped and probed with an anti-Syk Ab (clone LR). As shown in Fig. 5B, Syk phosphorylation was increased in Fc{epsilon}RI{gamma}-transfected cells stimulated with an anti-CD3 Ab. Densitometric analysis and normalization of the data against actin showed a significant increase in the levels of the phosphorylated Syk in Fc{epsilon}RI{gamma}-transfected cells (Fig. 5C). Increased phosphorylation of Syk was also revealed by another Ab that binds to activated Syk kinase (clone 4D10). Therefore, either spontaneous overexpression of Fc{epsilon}RI{gamma} chain, as it occurs in T cells from SLE patients, or forced expression of Fc{epsilon}RI{gamma} chain is associated with the increased phosphorylation of Syk kinase.

Overexpression of Fc{epsilon}RI{gamma}-induced TCR/CD3-mediated IL-2 production in human T cells

To determine whether transfection with Fc{epsilon}RI{gamma} induced the expression of IL-2, we stimulated cells transfected with Fc{epsilon}RI{gamma} for 72 h with 10 µg/ml anti-CD3 and for 24 h with 2.5 µg/ml anti-CD28 mAbs. The IL-2 levels in the culture supernatant were assayed by ELISA. As shown in Fig. 6, the amount of IL-2 production was significantly increased in Fc{epsilon}RI{gamma}-transfected cells compared with control vector, suggesting that TCR containing Fc{epsilon}RI{gamma} is functionally active in human T lymphocytes.



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  		FIGURE 6. Overexpression of Fc{epsilon}RI{gamma} up-regulates TCR/CD3-mediated IL-2 expression in T cells. T cells were transfected with Fc{epsilon}RI{gamma} or empty vector pcDNA 3.1 CT-GFP by electroporation. Seventy-two hours after transfection the cells were stimulated with 10 µg/ml OKT3 and 2.5 µg/ml anti-CD28 for 24 h. The supernatant was collected, and the IL-2 activity was measured by ELISA as described in Materials and Methods. Data are represented as the mean ± SEM from different subjects.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
That Fc{epsilon}RI{gamma} chain can be part of the TCR/CD3 complex in {alpha}{beta} or {gamma}{delta} T cells has been established. This study reveals several new points that may explain certain functional features of cells expressing Fc{epsilon}RI{gamma}. First, forced expression of Fc{epsilon}RI{gamma} is associated with a decrease in the expression of TCR {zeta}-chain. Second, expression of Fc{epsilon}RI{gamma} is followed by increased TCR/CD3-mediated phosphorylation of Syk, which mediates inward signaling. Third, expression of Fc{epsilon}RI{gamma} allows cells to display increased protein tyrosine phosphorylation, intracellular calcium, and IL-2 production responses.

Previously, it was considered that decreased expression or lack of TCR {zeta}-chain is followed by increased expression of the Fc{epsilon}RI{gamma} chain in a teleological manner to accomplish rewiring of the TCR, so it may retain its ability to transduce signal. In SLE T cells the decreased {zeta}-chain is replaced by Fc{epsilon}RI{gamma} (16). In mice that do not express {zeta}-chain, the Fc{epsilon}RI{gamma} contributes to the development of the T cell compartment (14, 15). In tumor infiltrating cells the degraded {zeta}-chain is replaced by Fc{epsilon}RI{gamma} (11). In this study we show that the opposite is also true. Forced expression of the Fc{epsilon}RI{gamma} chain results in down-regulation of the expression of the {zeta}-chain. The decreased expression is probably caused by the consequence of altered posttranscriptional regulation at the initial stages, and at later time points it may involve decreased transcription (Fig. 3). A similar posttranscriptional down-regulation of {zeta}-chain was also observed in human effector T cells generated in vitro that express increased levels of Fc{epsilon}RI{gamma} (S. Krishnan and D. Farber, unpublished results). Understanding the complementarity of the expression of the two chains will be an interesting task.

We had observed for the first time in SLE T cells the expression of Syk kinase (16) and found it to associate with the expressed Fc{epsilon}RI{gamma}. It was not clear at that point whether the overexpressed active Syk represented an independent aberration or whether it was a consequence of the expressed {gamma}-chain. The fact that only the {gamma}-chain-transfected normal T cells and not the control-transfected or {zeta}-chain-transfected (not shown) cells expressed the Syk kinase indicates that the Syk expression in SLE T cells is probably secondary to the overexpressed Fc{epsilon}RI{gamma} chain. The fact that ZAP-70 and Syk kinase are different in their ability to transfer the CD3-initiated signal, the latter being significantly stronger (5, 6), may explain the increased calcium and protein tyrosine phosphorylation responses that we observed in patients with SLE (19) and, in this study, in the Fc{epsilon}RI{gamma}-transfected cells.

Fc{epsilon}RI{gamma}-transfected cells display increased calcium and cytosolic protein tyrosine phosphorylation following cross-linking of CD3{epsilon}, features that have been ascribed to human SLE T cells (19). These observations indicate that the observed SLE T cell hyperresponsiveness is a result of overexpression of Fc{epsilon}RI{gamma} chain, which appears to play a central role in determining the magnitude of the response of the T cell. The fact that the signaling strength of Fc{epsilon}RI{gamma} chain and TCR {zeta}-chain-transfected cells is comparable can be explained by the fact that the latter express more {zeta}-chain and the former signal through Fc{epsilon}RI{gamma} and the residual {zeta}-chain. Our findings provide support to the position that cell excitability may predispose to autoimmunity. Vratsanos et al. (21) reported that T cells from MRL TCR transgenic mice responded better to cognate Ag than cells from control animals. Heightened responses have also been encountered among B cells from patients with SLE (28) and graft vs host mice that develop autoimmunity (20).

Although patients (29, 30, 31) and mice (32) with SLE produce decreased amounts of IL-2, Fc{epsilon}RI{gamma}-transfected cells produce IL-2 at levels comparable to cells transfected with {zeta}-chain (not shown). Because regulation of the production of IL-2 is complex, and in patients with SLE the decreased production is the result of increased expression of the transcriptional repressor cAMP response element modulator (33, 34), it is possible that increased production of IL-2 occurs at the ignition of the autoimmune response.

The molecular events that take place at the beginning of an autoimmune process are unclear. Evidence discussed in this study suggests that two factors determine the autoimmune response. First, the strength of the signal is higher than that observed in normal immune cells; and second, the molecules that are involved in the signaling process are different. It is not known, and this study did not address, whether the signaling process in autoimmune cells is sustained for a longer period of time either because suppressive molecules function inadequately or because the altered composition of the signaling apparatus leads to sustained responses. Molecular mimicry by altered biochemical modifications in proteins have been shown to contribute to a breakdown of immune tolerance and induce autoimmune responses (35, 36). Understanding the cellular biochemistry that leads to autoimmune responses will enable us to develop means to modulate the signaling process and treat autoimmunity.


   Acknowledgments
 
We thank Dr. G. M. Kammer for critical reading of the manuscript.


   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 study was supported by National Institutes of Health Grant RO1 AI42269. The opinions and assertions contained herein are private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Back

2 Address correspondence and reprint requests to: Dr. George C. Tsokos, Walter Reed Army Institute of Research, Building 503, Room 1A32, Robert Grant Avenue, Silver Spring, MD 20910-7500. E-mail: gtsokos{at}usuhs.mil Back

3 Abbreviations used in this paper: ZAP-70, {zeta}-associated protein 70; SLE, systemic lupus erythematosus; Syk, spleen tyrosine kinase; PBMCs, peripheral blood mononuclear cells; [Ca2+]i, intracellular Ca2+ concentration; GFP, green fluorescence protein; INDO-1, indole-acetoxymethyl ester; TRITC, tetramethylrhodamine isothiocyanate. Back

Received for publication September 17, 2002. Accepted for publication January 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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