Report

Role of the Guanosine Triphosphatase Rac2 in T Helper 1 Cell Differentiation

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Science  23 Jun 2000:
Vol. 288, Issue 5474, pp. 2219-2222
DOI: 10.1126/science.288.5474.2219

Abstract

T helper 1 (TH1) cells mediate cellular immunity, whereas TH2 cells potentiate antiparasite and humoral immunity. We used a complementary DNA subtraction method, representational display analysis, to show that the small guanosine triphosphatase Rac2 is expressed selectively in murine TH1 cells. Rac induces the interferon-γ (IFN-γ) promoter through cooperative activation of the nuclear factor kappa B and p38 mitogen-activated protein kinase pathways. Tetracycline-regulated transgenic mice expressing constitutively active Rac2 in T cells exhibited enhanced IFN-γ production. Dominant-negative Rac inhibited IFN-γ production in murine T cells. Moreover, T cells from Rac2−/− mice showed decreased IFN-γ production under TH1 conditions in vitro. Thus, Rac2 activates TH1-specific signaling and IFN-γ gene expression.

TH1 and TH2 cells can be differentiated in vitro from common naı̈ve precursor T cells during the course of a few days (1). During this time period, TH1 or TH2 regulatory proteins specific to each lineage are induced that are likely to play key roles in the differentiation process. To search for genes differentially expressed in TH1 or TH2 cells, we performed RDA (representational display analysis) (2) using in vitro–differentiated TH1 and TH2 cells. Using this procedure, we identified the transcription factor GATA3 as a TH2-specific gene and a key regulator of TH2 differentiation (3). Here we show that the small guanosine 5′-triphosphate (GTP)–binding protein Rac2 is a TH1-specific gene that plays a central role in TH1 development. A TH1 probe generated after three rounds of RDA subtraction with primary TH1 and TH2 cDNA was used to screen a TH1 cDNA library. Rac 2 was one of the genes identified. To confirm this result, we examined the expression level of Rac2 in day 4 TH1 and TH2 cells. A highly enriched Rac2 mRNA representation was found in primary TH1 cells (Fig. 1A) while, as expected, GATA3 showed enrichment in TH2 cells.

Figure 1

Identification of Rac2 as a TH1-specific gene. RDA analysis with cDNA from day 4 cultured TH1 and TH2 cells was performed. A TH1 probe was derived and used to screen a TH1 library, which was used to identify the Rac2 gene. (A) Slot-blot analysis of 50 ng of Rac2, GATA3, and γ-actin hybridized with radioactive labeled day 4 TH1 and TH2 initial representation probe. The bands were quantitated with a PhosphorImager (Becton-Dickinson). The Rac2 signal is sevenfold higher in the TH1 than in the TH2 sample, and the GATA3 signal is fivefold higher in the TH2 than in the TH1 sample. (B) The JNK and p38 MAP kinase in Rac2L61-transfected Jurkat cell extract were immunoprecipitated, and the MAP kinase activity was measured in the immune complex by protein kinase assay with recombinant c-Jun and ATF2 as substrate, respectively. (C) Jurkat cells were transfected with 3 μg of EBS-luciferase, collagenase AP1-luciferase, Chop-luciferase (Chop expression vector and pFR-luciferase construct), or κB-luciferase reporter plasmids with (shaded bars) or without (open and black bars) RacL61 and 100 ng of PRL-luciferase. PMA-ionomycin was added 4 hours after transfection (black bars). Dual luciferase assay was performed 24 hours after transfection.

Rac has been shown to activate both JNK, p38, and NF-κB pathways (4–6) in various cell types. Moreover, JNK and p38 are selectively activated in TH1 effector cells (7, 8). This suggested that the elevated level of Rac2 might be the cause of the selective activation of these pathways. Consistent with these observations, constitutively active Rac2 (L61, GTP-bound) activated JNK and p38 pathways and NFκB in Jurkat cells, as shown by both direct measurement of JNK and p38 kinase activity (Fig. 1B) and AP1-luciferase (9), Chop-luciferase (10), and κB-luciferase (11) reporter gene activation (Fig. 1C), while having no effect on a reporter construct bearing binding sites for the ets family of transcription factors. A similar result was observed with Rac1L61 (12).

We first examined whether Rac2 plays a role in TH1 cytokine gene expression. Constitutively active Rac2 cotransfected into Jurkat cells with an interferon-γ (IFN-γ) promoter reporter plasmid (13) induced a six- to sevenfold activation of the IFN-γ promoter, whereas CDC42L61 did not (Fig. 2A). To determine whether Rac2L61 activates IFN-γ expression in T cell clones, we cotransfected IFN-γ promoter reporter and the expression vector for Rac2L61 into the TH1 clone AE7 (14) and the TH2 clone D10 (15). Although Rac2L61 strongly activated the IFN-γ promoter in AE7 cells, it failed to activate the IFN-γ promoter in D10 cells (Fig. 2B).

Figure 2

IFN-γ promoter activation by Rac. Dual luciferase assay in Jurkat cells was performed as described in Fig. 1C (A, B, and D). IFNγluc (3 μg ) and expression vectors (6 μg) for active or negative mutants of Rho family small heterotrimeric GTP binding proteins (G proteins) were used. The values of the control samples were normalized as 1. (A) Cells were cotransfected with IFNγluc and Rac1L61, Rac2L61, or CDC42L61. (B) AE7 (a TH1 clone) (9) or D10 cells (a TH2 clone) (9) were transfected with IFNγluc reporter plasmid with or without the expression vector for Rac2L61. Sixteen hours later, cells were stimulated with APC and antigen for 24 hours, and dual luciferase activity was assayed. (C) Jurkat cells were transfected with IFNγluc with or without RacL61 expression vector and either cotransfected with 5 μg of plasmids expressing inhibitors of various signal transduction pathways or treated with 10 μM inhibitors 4 hours after transfection, as indicated. The cotransfected plasmids used express dominant-negative JNK1 and NF-κB super repressor; the inhibitors used are SB203580 for p38 and PD98059 for ERK. (D) Jurkat cells were cotransfected with IFNγluc together with plasmids that express constitutively active MKK6 (MKK6glu), constitutively active IKKβ (IKKCA), MKK7, or JNK1.

To examine which of these signaling pathways—JNK, p38, or NF-κB–is required for Rac-mediated IFN-γ activation, we blocked each pathway with specific inhibitors. Whereas dominant-negative JNK1 or the ERK inhibitor PD98059 has no effect on RacL61-induced IFN-γ promoter activation, both the NF-κB super repressor (16) and the p38 inhibitor SB203580 inhibited this activation completely (Fig. 2C). We next examined whether any of these pathways was sufficient for Rac-mediated IFN-γ activation. Whereas activation of the JNK pathway with MKK7 and JNK1 (17), the p38 pathway with MKK6glu (18), or NF-κB with a constitutively active form of IKKβ (19) alone was not sufficient to activate the IFN-γ promoter, activation of both the p38 pathway and NF-κB together induced IFN-γ promoter activity to a level similar to that obtained with RacL61 (Fig. 2D). The JNK pathway does not appear to contribute to IFN-γ promoter activity, because it did not synergize with either NF-κB or p38. Thus, both the p38 and NF-κB pathways are required for Rac-mediated IFN-γ activation, and these two pathways activate the IFN-γ promoter synergistically.

To investigate the role of Rac in TH1 differentiation in vivo, we used a reverse tetracycline-controlled transactivator (rtTA)–regulated transgenic mouse system (20,21) to express Rac2L61 in T cells (22). Although a low level of leaky expression was observed in transgene-positive mice before doxycycline induction, T and B lymphocyte development was normal (12). Strong induction of Rac2L61 transgene expression was observed after doxycycline treatment (Fig. 3A). No induction was observed in the transgene-negative littermates. Transgene expression was also confirmed by Western blot analysis with a monoclonal antibody against Myc tag (Fig. 3B).

Figure 3

Up-regulation of IFN-γ production in Rac2L61 transgenic mice. (A) The rtTA gene was fused with the second exon of the CD4 gene. The cDNA for Rac2L61 was placed under the control of the Tet promoter. A small intron between two small exons was inserted at the 3′-untranslated region. Primers derived from the second exon and the Rac2L61 coding region are used for screening the transgene mRNA in RT-PCR. T cells were isolated and cultured with APCs, Con A (2.5 μg/ml), IL-2 (30 U/ml), and doxycycline (1 μg/ml) for 2 days. RT-PCR was done with the primers described. Lanes 1 and 4, transgene positive; lanes 2 and lane 5, transgene positive without doxycycline; lanes 3 and 6, controls. The small intron in the 3′-untranslated region of the Rac2L61 gene results in a shorter PCR product from the cDNA than from the transgene. (B) T cells from transgene-positive (lanes 1 and 3) and transgene-negative mice (lanes 2 and 4) were treated as described in (A) and analyzed by Western blot with a monoclonal antibody against Myc tag. (C) Purified naı̈ve T cells were stimulated in vitro with APCs, ConA, and IL-2 in the presence of doxycycline for 6 days, followed by ribonuclease protection assay. (D) Supernatant from day 6 culture described above was used to measure IFN-γ and IL-4 expression by ELISA. (E and F) Mice were fed with doxycycline food (2 g per kilogram of food) for 7 days and immunized with 50 μg of KLH in CFA in each hind footpad. CD4 T cells in draining lymph nodes were isolated after 9 days and were cultured with KLH for 4 days. The average value of two ELISA readings is shown.

We examined the effect of transgene expression on expression of cytokine genes using a ribonuclease protection assay (Fig. 3C) (23). Production of the two TH1 cytokines IFN-γ and LTα was substantially enhanced in transgenic T cells compared with T cells from negative littermates, whereas lineage-nonspecific cytokines like tumor necrosis factor–α were unaffected. Quantitation by cytokine enzyme-linked immunosorbent assay (ELISA) showed a fourfold increase of IFN-γ in transgenic mice (Fig. 3D). In comparison, interleukin-4 (IL-4) levels were essentially unaffected. To determine whether Rac2L61 transgenic T cells were predisposed to produce IFN-γ in vivo, we immunized doxycycline-treated mice with keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA). Rac2L61 expression increased the level of IFN-γ production by T cells from draining lymph nodes significantly, but had little effect on IL-4 production or T cell proliferation. Thus, Rac activation is sufficient to induce IFN-γ production in T cells in vivo.

To determine whether Rac is necessary for IFN-γ production in T cells, we first expressed the dominant-negative (N17, guanosine diphosphate–bound) mutants of Rac1, which inhibits both Rac1 and Rac2, or CDC42N17 in Jurkat cells. Rac1N17 greatly inhibited phorbol 12-myristate 13-acetate (PMA)–ionomycin–induced IFN-γ promoter activation, whereas CDC42N17 had no significant effect (Fig. 4A). To examine the effect of Rac inactivation on IFN-γ expression in primary T cells, we generated recombinant retrovirus-expressing Rac1N17 under the control of the Moloney leukemia virus long-terminal repeat (MLVLTR). In vitro–stimulated primary CD4 T cells were activated, infected with virus and assayed after 5 days in culture (24). Equal proportions of cells positively stained for IFN-γ were observed in the infected and uninfected populations of T cells infected with control green fluorescent protein (GFP) vector (Fig. 4B). In cells infected with virus-expressing RAC1N17, however, IFN-γ was reduced to background levels (Rac1N17-GFP vector).

Figure 4

Dominant-negative Rac or Rac2 deficiency inhibits IFN-γ production in T cells. (A) Dual luciferase assay in Jurkat cells was done as described in Fig. 1C. Six micrograms of expression vectors for Rac1N17 or CDC42N17, and 3 μg of IFNγluc were used. (B) CD4 T cells from B6 mice stimulated with anti-CD3 and anti-CD28 for 24 hours were infected with recombinant virus supernatant and 5 days later restimulated with PMA-ionomycin, and IFNγ expression was analyzed with intracellular cytokine staining. The internal ribosome entry site (IRES-GFP) element in the vector allows for tracking of infected cells by fluorescence-activated cell sorting. (C and D) Naı̈ve CD4 T cells from Rac2Ko or wild-type littermate mice were stimulated with ConA plus APC (C) or plate-coated anti-CD3 plus anti-CD28 (D) in the presence of IL-12 and anti-IL-4 for 4 days. Differentiated cells were extensively washed and restimulated with ConA (C) or anti-CD3 (D) for 24 hours. Culture supernatant was collected and assayed for IFN-γ by ELISA.

Finally, we examined whether Rac2 plays a critical role in TH1 cytokine production under TH1-polarized conditions using Rac2 knockout mice (25). These animals have normal T lymphocyte development (25, 26). Wild-type CD4 T cells develop a strong TH1 response after primary culture with ConA, IL-12, antibodies to IL-4 (anti–IL-4), and antigen-presenting cells (APCs) and secondary stimulation with ConA. Stimulation of Rac2 knockout T cells under these conditions, however, led to a ∼twofold reduction of IFN-γ production (Fig. 4C). Further, when CD4 T cells were stimulated with plate-bound anti-CD3 plus anti-CD28 in the presence of IL-12 and anti-IL-4 for 4 days and restimulated with anti-CD3, IFN-γ production in Rac2 knockout mice was reduced by about two- to threefold compared with that in wild-type mice (Fig. 4D). Thus, the induction of Rac2 expression in TH1 cells provides these cells with the machinery to generate high-level IFN-γ, and in its absence the levels are reduced.

The molecular mechanisms controlling TH1 cell activation are poorly understood. Our recent reports have demonstrated the involvement of p38 and JNK2 in TH1 differentiation and IFN-γ production through specific activation of these pathways in TH1 cells (7, 8,27). How this specificity is achieved was previously not clear. Here we show that Rac2 is a TH1-specific gene that activates IFN-γ production both in vitro and in vivo through concomitant activation of both the NF-κB and p38 pathways. The finding that inactivation of Rac in primary T cells, either by a dominant-negative transgene or by gene targeting of Rac2, inhibits IFN-γ production demonstrates that Rac activation is required for IFN-γ production during normal T cell activation. Rac plays a role in regulating actin polymerization (5), and there is evidence supporting its role in T cell activation (28); here we demonstrate that Rac directly controls production of a key cytokine involved in TH cell differentiation. That both the inhibition of Rac signaling through dominant-negative transgenes, and the complete elimination of Rac2 by gene targeting, reduce but do not eliminate production of IFN-γ shows that Rac2 (this report) and the p38 mitogen-activated protein (MAP) [(7) and this report] kinase pathways play an amplifying role in the production of high-level IFN-γ at the effector phase of the T cell response. Such amplifying mechanisms are critical for the production of a vigorous immune response in mammals infected by pathogens. This finding and previous results showing the involvement of Rac in the reorganization of cytoskeletal structure at the site of T cell and APC contact (29) demonstrate a dual role for Rac in T cell activation.

  • * Present address: Pfizer, Groton, CT 06340, USA.

  • These authors contributed equally to this work.

  • Present address: University of Rochester, Center for Vaccine Biology and Immunology, Post Office Box 609, Rochester, NY 14623, USA.

  • § Present address: Medizinische Klinik III, Immunologie Labor, Gluckstrabe 41, 91054 Erlangen, Germany.

  • || Present address: Lilly Research Laboratory, Eli Lilly and Company, Indianapolis, IN 46285, USA.

  • Present address: The Walter Eliza Hall Institute, Melbourne, Australia.

  • # To whom correspondence should be addressed: E-mail: fran.manzo{at}yale.edu

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