1 Pathology and Laboratory Medicine, 2 Digestive Diseases, 3 Microbiology, Immunology and Molecular Genetics, 4 Molecular, Cellular and Developmental Biology, 5 Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA 6 VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA 7 Present address: Laboratory of Signal Transduction, and Division of Intramural Research, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA 8 Present address: Bristol-Myers Squibb Co., Princeton, NJ, USA
Correspondence to: J. Braun, Pathology and Medicine, UCLA, Box 951732, CHS 13-222, Los Angeles, CA 90095-1732, USA. E-mail: jbraun{at}mednet.ucla.edu
Transmitting editor: C. Terhorst
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Abstract |
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Keywords: autoimmunity, mucosa, signal transduction, T lymphocytes, TCR
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Introduction |
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Both in vitro and in vivo studies suggest that Gi proteins participate in immune function. Chemokines and immunomodulatory lipids are ligands for Gi-coupled receptors (810). Pharmacological interruption of Gi signaling by PT has been shown to promote TH1 immune responses (11), enhance IL-12 production (12), and augment immune mediated pathology in transgenic animals predisposed to autoimmunity (13,14). PT has been used as an adjuvant in experimental immunization protocols. In this setting, the immune enhancing effect of PT has been attributed G
i blockade resulting in enhanced production of IL-12 and the augmented expression of costimulatory molecules such as B7-1, B7-2 on antigen presenting cells (11,15). Finally, mice deficient in the G
i2 subunit due to gene targeting have an immune system biased towards the production of TH1 cytokines which predisposes them to the development of a spontaneous inflammatory bowel disease (1619).
While Gi signaling has been shown to be specifically involved in the regulation of IL-12 production from CD8
+ dendritic cells (12), little is known about the significance of G
i expression in other immune cell types. G
i subunits are present in T cells but the specific signaling role they have has not been fully characterized (20,21). In fact, divergent results are often obtained depending upon the mode of G
i protein inactivation, and the use of human or mouse cells (22,23).
In this study, we take advantage of the availability of gene knockout mice deficient in either the Gi2 (G
i2-/-) or the G
i3 (G
i3-/-) subunit to investigate the specific role of the two G
i subunits in lymphocyte activation and cytokine production. We find that the G
i2-/-, but not G
i3-/-, genotype renders T lymphocytes hyper-responsive for TCR signaling and cytokine production, with a relaxed costimulatory requirement. This disorder is a selective, intrinsic feature of the naïve CD4+ T cell subset, and is associated with a developmental cell physiologic abnormality in TCR-mediated calcium signaling. This TCR signaling abnormality may contribute to the CD4+ T cell-mediated autoimmunity characteristic of G
i2-/- mice.
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Methods |
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Antibodies and reagents
Monoclonal antibodies specific for CD3 (clone 145-2C11), CD28 (clone 37.51), CD44, CD62L, CD4 and goat anti-hamster IgG, were obtained from BD PharMingen (San Diego, CA). Anti-phosphotyrosine Ab (4G10) was from Upstate Biotechnology (Lake Placid, NY). Streptavidin, phorbol myristic acetate (PMA), and PT were from Sigma (St Louis, MO). Ionomycin was from Calbiochem (La Jolla, CA), and [3H]thymidine was from Amersham (Piscataway, NJ).
T cell purification
Purified total T cells were isolated from the spleen by negative selection using a T cell recovery column kit (Cedarlane Labs Limited, ON, CA) according to the manufacturers instructions. Purity ranged from 94 to 98% post isolation as determined by flow cytometry of cell surface CD3 expression.
In experiments where the naïve versus memory T cell population was required, CD4+ cells were purified from the mouse spleen using the mouse CD4+ T cell isolation kit according to the manufacturers instructions. (Miltenyi Biotech, Auburn, CA). The CD4+ T cells were separated into naïve and memory populations based on CD62L expression by using anti-CD62L microbeads (Miltenyi Biotech, Auburn, CA). Purity was determined by flow cytometry and ranged from 90 to 95% for CD4+ expression, and 80 to 85% and 0 to 2% for CD62L expression on the naïve and memory cell populations, respectively.
T cell stimulation and cytokine assays
Lymphocytes (12 x 106/ml) were isolated and cultured in complete RPMI 1640 medium supplemented with 1.0 mM sodium pyruvate, 1x non-essential amino acid, 100 U/ml penicillin, 0.1 mg/ml streptomycin (all from Gibco BRL, Grand Island, NY), 5% heat inactivated fetal calf serum (Hyclone, Logan, UT), and 0.05 µM 2-mercaptoethanol (Sigma), at 37°C in a 5% CO2-humidified atmosphere. Cells were stimulated with plate-bound anti-CD3, in the presence or absence of soluble anti-CD28 (1 µg/ml).
Supernatants for cytokine measurements were harvested after 24 or 48 h of cell culture, stored at 80°C, and assayed using the OptEIA ELISA kits according to manufacturers instruction (BD PharMingen, San Diego, CA). Plates were read on a microplate reader (Molecular Devices Corp., Sunnyvale CA). Cytokine concentrations were calculated by SoftMaxPro program based on the standard curves. Linear detection range was 3200 pg/ml for IL-2, 161000 pg/ml for IL-4, and 312000 pg/ml for IFN- and IL-10, respectively. For measurement of proliferation, splenocytes (106/ml) were cultured in 96-well plates for 48 h and [3H]thymidine (1 µCi/well) was added for the final 6 h. Cultures were harvested and counted on a micro cell harvester and radioactivity counted in a liquid scintillation counter (LKB 1205).
RNA preparation and RTPCR
Spleens and purified T cells were homogenized in TRIzol Reagent (Gibco BRL, Rockville, MD) with RNaseOUTTM Recombinant Ribonuclease Inhibitor (Gibco BRL). Chloro form extraction was used to separate phases and RNA was precipitated by isopropanol. RTPCR (Qiagen, Valencia, CA) was used to amplify GAPDH and Gi1-3 products. An aliquot of 0.5 µg of RNA was used for the RTPCR. All reactions were performed at 42°C, 30 min, 94°C, 2 min for 1 cycle; 94°C, 30 s, 55°C, 30 s, 72°C, 1 min for 50 cycles; and 72°C, 5 min for 1 cycle. The products were visualized on a 2% agarose gel. GADPH was used to ensure proper RNA preparation and equal loading. Primers for G
i1, 2 and 3 products were designed as described by Williams et al. (25). G
i1: 5' ATGAACCGAATGCATGAAAGCA 3' and 5'GTCCTTCCTT TTATTGAGGTCT 3'; G
i2: 5' GCCAACAAGTACGACGGCA 3' and 5' GTATCTCTCACGCTTCTTGTGCT 3'; G
i3: 5' ATG AACCGAATGCATGAAAGCA 3' and 5'TTTGGTGTCAGTGG CACAGGTA 3'; GADPH; 5' TGCTGAGTATGTCGTGGACTCT 3' and 5' ATGTGATCATACTTGGCAGGTTTC 3'. Product sizes were 228, 284, 273 and 500 nt, respectively.
Phosphoprotein immunoblotting
Splenic T cells from wild-type or Gi2/ mice were purified by negative selection using the T cell recovery column kit (Cedarlane Labs Limited). T cells (1 x 107) in 200 µl RPMI were incubated in the presence or absence of anti-CD3 antibody (5 µg/ml) for 30 min on ice, followed by crosslinking with rabbit anti-hamster IgG (1:200) at 37°C for 5 min. After stimulation, cell pellets were boiled in 2x reducing sample buffer. Cell lysates were resolved on 10% SDSPAGE, transferred to nitrocellulose membrane and immunoblotted with anti-phosphotyrosine antibody.
Flow cytometry and intracellular calcium flux assay
Splenic mononuclear cells (1 x 106) were stained with anti-CD4-PERCP and anti-CD8-PE, or with anti-CD44-FITC, -CD62L-PE and -CD4-PERCP. At least 10,000 events were collected and analyzed by a FACSCalibur using Cell Quest software (Becton Dickinson, Mountain View, CA).
For calcium flux analysis, purified CD4+ T cells were incubated with 1 µM Indo-1 acetoxy-methyl ester (Indo-1 AM; Molecular Probes, Eugene, OR) for 40 min at 37°C in complete media. Cells were then stained with anti-CD44-FITC, -CD62L-PE and -CD4-PERCP at room temperature to distinguish naïve and memory subsets. Cells (3 x 106 cells/ml) were equilibrated at 37°C for 10 min, and after a 25 s reading for baseline calcium levels, anti-CD3-biotin (15 µg/ml) followed by streptavidin crosslinking (100 µg/ml) were added, and intracellular calcium flux was monitored for 512 s. Cells were gated for the naïve CD4 population (CD4 + CD62LhiCD44lo), and intracellular calcium concentration was calculated based on the ratio of the fluorescence at 400 and 500 nm (analyzed by FLOWJO software; Treestar, San Carlos, CA). For assessment of PT effects, wild-type cells were preincubated with 1 µg/ml PT (Sigma) (or medium control) for 1 h on ice, then loaded with Indo-1 and stimulated as above with anti-CD3 and streptavidin.
Statistical evaluation
When applicable, means of several independent experiments would be calculated with SD, because all experiments were set up in triplicate wells, thus they would not need SEM to adjust. Two-tailed t-tests were performed when applicable, and the differences were significant if P was <0.05, and the important comparisons differed by >2 SD, assuming they were in Gaussian distribution.
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Results |
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Gi3-/- mice were healthy, and colony members were free of detectable disease by inspection or necroscopy even beyond 1 year of age (data not shown). A comparison of splenic T lymphocytes in G
i2-/- and G
i3-/- mice is shown in Fig. 2. The relative abundance of CD4+ and CD8+ T cells was comparable among these two null mutation mice, as well as wild-type littermate controls (Fig. 2A). There were also comparable absolute numbers of lymphocytes from spleen, and peripheral and mesenteric lymph nodes, among the three groups of animals (data not shown), indicating an overall similarity in T cell production and survival.
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The cytokine response of CD4+ T cells from these three groups of mice in response to TCR stimulation was compared in Fig. 1(BD). We found that Gi3-/- splenocytes produced similar amounts of IFN-
, IL-4 and IL-10 when compared with wild-type cells. In contrast, G
i2-/- splenocytes produced significantly increased levels of IFN-
and IL-4 but not IL-10, as previously reported (16,18). Although both subunits are expressed in T cells, these data specifically identify G
i2 but not G
i3 as having a role in regulating the magnitude of cytokine responses following stimulation of T cells through the TCR.
The costimulatory requirement for IL-2 production is relaxed in Gi2-/- T cells
The increased levels of cytokines produced by Gi2-/- splenocytes following activation with anti-CD3 mAb indicated that these cells were hyper-responsive to stimulation through the TCR, and prompted us to further characterize their activation requirements specifically for IL-2 production. Optimal T cell activation occurs through a combination of TCR occupancy and interaction with accessory receptors on the T cell to counter-ligands on the antigen presenting cell. This process, costimulation, is necessary for efficient recruitment of signaling molecules into the immune synapse, and a full amplitude and temporal duration of biochemical activity along the recruited signaling pathways (8,27). As shown in Fig. 3(A), activation of G
i2-/- splenocytes with anti-CD3 alone, notably 110 µg/ml, resulted in significantly more IL-2 production compared with wild-type cells. The addition of a costimulatory (anti-CD28) stimulus augmented the production of IL-2 from both G
i2-/- and wild-type cells. Figure 3(B) shows that when cell proliferation was measured, G
i2-/- cells proliferated to a much greater extent than wild-type cells correlating with the IL-2 data. Analysis of T cells from G
i2-/- and wild-type mice for CD3 or CD28 expression by flow cytometry did not reveal any major differences (data not shown). These results indicate that G
i2-/- T cells are less stringent in their requirement for costimulation for enhanced IL-2 production and proliferation.
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Agents that bypass the TCR do not result in Gi2-/- T cell hyper-responsiveness
To determine if the increased cytokine production by Gi2-/- T cells was restricted to signals initiated only through the TCR, we examined the levels of IL-2 and IFN-
following activation with PMA and ionomycin, agents that bypass the antigen receptor (29) (Table 1). Activation of purified T cells with PMA and ionomycin resulted in cytokine levels that were similar between wild-type and G
i2-/- cells. In contrast, stimulation with anti-CD3 resulted in markedly enhanced production of IL-2 and IFN-
. These results localize the alteration in G
i2-deficient T cells to TCR signaling events preceding the distal calcium and MAP-kinase induced biochemical pathways directly activated by these pharmacologic agents.
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TCR ligation initiates several protein kinase cascades contributing to calcium mobilization and its sequelae, as well as other direct pathways of biochemical activation and transcriptional regulation (8,35). We examined the global changes in TCR-induced protein tyrosine phosphorylation of Gi2-/- and wild-type T cells with anti-phosphotyrosine immunblots (Fig. 4D). Certain phosphoprotein bands were constitutively more prominent in G
i2-/- T cells (black arrowheads), but there was no obvious difference in TCR-induced phosphorylation (e.g. pp20). Moreover, immunoprecipitation and anti-phosphotyrosine immunoblot analysis of PLC-
, PKC-
, LAT and Lck also failed to reveal differences between these two groups of T cells (data not shown). It therefore appeared that the unusual calcium response in G
i2-/- cells was not associated with a proximal kinase pathway abnormality.
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Discussion |
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This study newly demonstrated that the Gi2 and G
i3 are the G
i subunits expressed in murine splenic T cells, extending previous reports concerning human blood T cells (21,36). Since G
i2 and G
i3 can have overlapping signaling roles (26), we were led to examine whether any abnormalities in TCR-mediated cytokine production were present in lymphocytes obtained from animals deficient in this G protein. However, unlike in G
i2-/- mice, G
i3-/- mice had normal production of IFN-
, IL-4 and IL-10. Thus, the G
i3 subunit did not appear to have a major role in regulating the magnitude of these cytokines in response to TCR engagement. The increased levels of cytokines produced by G
i2-/- cells also indicated that the G
i3 subunit could not functionally compensate for the absence of the other subunit. These data were obtained from splenocyte cultures, so the source of the cytokines may in part reflect non-T cells activated under these culture conditions. For example, the predominant source of IL-10 in such cultures after TCR stimulation is B lymphocytes (37). These results suggest that, at least in response to TCR stimuli, the G
i2 and G
i3 subunits have distinct, non-redundant signaling roles. Within this context, the G
i2 subunit is reported to be more abundantly expressed in T cells than the G
i3 subunit (20). Consequently, it is possible that the observed lack of signaling redundancy or functional compensation may relate in part to significantly lower expression levels of the G
i3 subunit in lymphocytes.
Our findings suggest an unappreciated intrinsic disorder in naïve CD4+ T cells. Enhanced cytokine production was observed with purified naïve CD4+ Gi2-/- T cells, indicating that this phenotype was not due to the preferential contribution of the expanded memory T cells or altered costimulatory cell activity in G
i2-/- spleen cell population. The naïve CD4+ T cell population in G
i2-/- mice was less stringent in its costimulatory requirement, with substantial cytokine production in the absence of costimulation at low to moderate levels of TCR ligation. It has been proposed that autoimmunity may occur when T cells no longer require a costimulatory or second signal to become optimally activated (38). In view of the exceptional colitis susceptibility of G
i2-/- mice, this hyper-responsiveness may represent an intrinsic T cell trait contributing to the autoimmune phenotype. Since IL-10 production was similar between wild-type and G
i2-/- splenocytes, it is likely that deficient IL-10 production by G
i2-/- mice in other in vitro and in vivo conditions reflects the skewed pro-inflammatory differentiation of antigen-presenting cell types associated with this null mutation.
The biochemical basis of TCR hyper-responsiveness in Gi2-/- T cells is perplexing. Activation with PMA and ionomycin revealed no difference between wild-type and G
i2-/- T cells. These agents bypass the antigen receptor to directly activate the Ca2+/calcineurin and protein kinase C pathways, suggesting that the TCR-mediated hyper-responsiveness in G
i2-/- T lymphocytes is due to an alteration involving proximal TCR signaling events. Accordingly, we observed that G
i2-/- CD4+ T cells responded to TCR stimulation with an augmented calcium response. However, other experiments did not reveal a concomitant elevation in activity of key upstream protein kinases. This suggests that the aberrant calcium response may involve distal steps in receptor-mediated calcium mobilization.
The products of activated PLC-, diacylglycerol and IP3, mobilize endoplasmic reticulum calcium ion stores through channels activated by the InsP3 receptor and perhaps ryanodine receptors (30,39,40). This event activates plasma membrane store operated calcium channels (4143), which mediate capacitative calcium entry. This calcium current (I-CRAC), and its augmentation by calcium-dependent potassium channels, is responsible for the sustained, oscillatory intracellular calcium concentration sufficient to induce the calcium-dependent pathways to cytokine gene expression and cell proliferation (35,44,45). It is thus plausible that the augmented calcium response in G
i2-/- T cells (accelerated kinetics, increased amplitude and delayed resequestration) might reflect increased activity of one of these channels. With advances in the understanding of these molecules and reagents for their study, this prediction is becoming experimentally testable.
How does Gi2 activity affect the calcium response? Our pertussis experiment suggests that G
i2 activity does not directly influence the process. These results with knockout mice contrast with some studies utilizing human peripheral T cells or the Jurkat T cell line (22,23,34). The divergent results may relate to the differences between human and mouse cells, and the divergent inhibitory and stimulatory biological effects of PT in different cell types. Instead, we surmise that a G
i2-dependent process attenuates the constitutive expression or activity of molecules in the pertinent phase of the receptor-mediated calcium response. This may be a remote differentiative effect on the naïve CD4+ T cell, and may not be easily amenable to study. However, genetic manipulations and adoptive transfer should make it possible to determine whether or not the process requires G
i2 expression at the level of the T cell.
Taken together, our results suggest that in the normal situation, Gi2 may function as a negative regulator of T cell activation and cytokine production, most likely by attenuating the expression or activity of molecules affecting the sustained calcium response to TCR signaling pathways. These findings further support the idea that G protein coupled receptors utilizing G
i2 signaling may be an important molecular context for physiologic immunoregulation, and a potential target for molecular pathophysiology and therapeutic immunomodulation.
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Acknowledgements |
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Abbreviations |
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IP31,4,5-inositol trisphosphate
PEphycoerythrin
PMAphorbol myristic acetate
PTpertussis toxin
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References |
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