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Ubiquitin-like polypeptide inhibits cAMP-induced p38 MAPK activation in Th2 cells
Immunobiol. 208, 439 ± 444 (2004) Elsevier-Urban & Fischer www.elsevier-deutschland.de/imbio
Ubiquitin-like polypeptide inhibits cAMP-induced p38 MAPK activation in Th2 cells Morihiko Nakamuraa , Yoshinori Tanigawab a b
Cooperative Medical Research Center Department of Biochemistry, Shimane Medical University, Japan
Received: October 10, 2003 ¥ Accepted: January 8, 2004
Abstract Ubi-L, an isoform of the monoclonal nonspecific suppressor factor (MNSF), is an 8.5-kDa ubiquitin-like polypeptide. Ubi-L exhibits an antigen-nonspecific immunosuppressive function on various target cells including murine T helper type 2 (Th2) clone, D10 cells. Ubi-L specifically binds to cell surface receptors on D10 cells. In this study, we observed that Ubi-L inhibited cAMP-induced IL-5 mRNA expression in D10 cells but not in thymoma cell line EL4. In addition, Ubi-L effectively inhibited cAMP-induced p38 MAPK activation in D10 cells. Ubi-L also showed inhibitory activity on IL-5 and IL-13 production by D10 cells stimulated with phorbol ester plus dibutyryl cAMP. Furthermore, Ubi-L inhibited IL-4 production in Th2 cells derived from primary CD4 T cells. Abbreviations: bt2cAMP dibutyryl cyclic AMP; MAPK mitogen-activated protein kinase; MNSF monoclonal nonspecific suppressor factor; p38 p38 MAPK; Ubi-L ubiquitinlike polypeptide
Introduction Interleukin-5 (IL-5) is a T cell cytokine involved in type 2 diseases and is commonly described as being coordinately regulated with other type 2 cytokines, such as IL-4 and IL-13. It has been reported that direct stimulation of cAMP pathways with dibutyryl cAMP (bt2cAMP) induces IL-5 mRNA in murine T helper type 2 (Th2) clone, D10 cells (Chen et al., 2000). Mitogen-activated protein kinases (MAPKs) transduce a variety of extracellular signals to the transcriptional machinery via a cascade of protein phosphorylation. There are three genetically distinct MAPKs in
mammals, consisting of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK (p38). p38 is activated by dual specificity MAP kinase kinases, including MKK3, MKK6, and JNKK1, that phosphorylate p38 on threonine 180 and tyrosine 182 (Lin et al., 1995). cAMP activates p38 in Th2 but not in Th1 cells (Chen et al. 2000). Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by a murine T cell hybridoma, possesses pleiotropic nonspecific suppressive functions (Kondoh et al., 1999; Nakamura et al., 1996; Suzuki et al., 1996). MNSFb (a subunit of MNSF) is a 14.5 kDa fusion protein
Corresponding author: Morihiko Nakamura, PhD, Cooperative Medical Research Center, Shimane University, School of Medicine, 89-1 Enya-cho, Izumo 693, Japan, Tel: 81-853-20-2916, Fax: 81-853-20-2913, e-mail: [email protected]
0171-2985/04/208/5-439 $ 15.00/0
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consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30 (Nakamura et al., 1995a). The ubiquitin-like segment of MNSFb (Ubi-L) is an 8 kDa polypeptide with MNSF-like activity. Ubi-L shows an antigen-nonspecific immunosuppressive action on various target cells including D10 cells. We have mentioned that Ubi-L inhibits both the IL-4 secretion and the proliferation of concanavalin A-activated D10 cells (Nakamura et al., 1995b). We have demonstrated that Ubi-L binds specific receptor proteins on Con A-activated D10 cells (Nakamura & Tanigawa, 1999). Ubi-L receptor expression is limited to lymphoid cells. We have demonstrated that tyrosine phosphorylation may be a key step in the initiation of the Ubi-L receptor-mediated transmembrane signaling (Nakamura & Tanigawa, 2000). Interestingly, ubiquitin interferes with Ubi-L activity and high-dose ubiquitin mimics the action of Ubi-L (Nakamura et al., 1996). In addition, ubiquitin shows a slight but significant inhibition of the Ubi-L binding to activated D10 cells (Nakamura & Tanigawa, 1999). Furthermore, Ubi-L inhibits the proliferative response of T cells in vivo (Kondoh et al., 1999). In this study, we investigated whether Ubi-L is involved in the cAMP-mediated signal transduction in D10 cells.
Northern blot analysis Total cellular RNA was isolated, fractionated and transferred to ECL membrane as described (Nakamura et al., 1994). Hybridization, labeling of cDNA probe and detection were done according to the ECL-GENE Detection System (Amersham Biosciences, Piscataway, NJ). The mouse IL-5 cDNA probe was a 450-bp SacI-AccI fragment. Analysis of MAP kinase activation Twenty mg of soluble proteins were run in 10% SDSPAGE and transferred onto polyvinylidene fluoride (PVDF). Phosphorylated MAPK were detected by Western blot analysis using specific antibodies for the phosphorylated forms of p38, ERK, and JNK (Santacruz Biotechnology, Santa Cruz, CA). The membranes were blocked with 5% dry milk in PBS for 1 hour and then washed with PBS containing 0.1% Tween 20. First antibodies were revealed using anti-rabbit secondary antibodies conjugated to horseradish peroxidase and an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences). The same membranes were reprobed with anti-kinase antibodies. Quantification of cytokines
Materials and Methods BALB/c mice were obtained from Clea Japan, Inc. and were reared in the Laboratory of the Institute for Experimental Animals, Shimane Medical University.
Murine cytokines were measured using sandwich ELISA. The lower limits of detection for the cytokines were IL-5, 10 pg/ml (Endogen, Woburn, MA); IL-13, 2 pg/ml (R&D Systems, Minneapolis, MN); IFNg and IL-4, 2 and 3 pg/ml, respectively (BioSource International, Camarillo, CA).
Tumor cell lines
Preparation of CD4 Tcells
D10 cells (T helper type 2 clone) were maintained with biweekly stimulation with 100 mg/ml conalbumin in the presence of 0.5 U/ml recombinant human IL-2. Cells were used 10 ± 12 days after stimulation with antigen. EL4 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).
Splenocytes from 6- to 8-wk-old BALB/c mice were purified by RBC lysis. CD4 T cells were isolated by positive selection using CD4 Dynabeads followed by detachment with DETACHaBEAD¾ (Dynal Biotech, Oslo, Norway). The complete medium consisted of RPMI 1640 supplemented with 10% FBS plus penicillin-streptomycin, sodium pyruvate, and glutamine to final concentration of 100 mg/ml. In addition, 2-ME was added to a final concentration of 50 mM and gentamicin to 50 ng/ml.
Mice
Ubi-L preparation Recombinant Ubi-L was obtained as described previously (Nakamura et al., 1995a). Briefly, Ubi-L was expressed as a fusion protein with glutathioneS-transferase using the pGEX-2T vector (Pharmacia, Piscataway, NJ). Ubi-L was cleaved from the fusion partner by thrombin and purified by using antiUbi-L Ab coupled to Sepharose 4B (Pharmacia).
In vitro differentiation of CD4 T cells CD4 T cells (2 106 cells/ml) were stimulated in vitro with plate-bound anti-CD3 monoclonal antibody (2C11) at 3 mg/ml and anti-CD28 monoclonal antibody (37.51) at 1 mg/ml plus IL-2 at 20 U/ml
Ubiquitin-like protein inhibits p38 activation
(nonskewing condition). In addition, cells were incubated with IL-12 at 5 ng/ml and anti-IL-4 monoclonal antibody (11B11) at 3 mg/ml (Th1skewing condition) or IL-4 at 500 U/ml and antiIL-12 (C17.8) at 5 mg/ml (Th2-skewing condition). Differentiation proceeded in the presence or absence of Ubi-L. Four days post-stimulation, the cultures were expanded 2-fold with fresh medium, cytokines, and antibodies. Three days later, the cells were harvested, washed five times with RPMI 1640 and counted, and equal numbers of cells were restimulated with plate-bound anti-CD3 at 1 mg/ml. Twenty-four hours post-restimulation, supernatants were collected and cytokine levels were measured by ELISA.
Results Ubi-L inhibits cAMP-induced IL-5 mRNA in D10 cells To determine whether Ubi-L inhibits cAMP induced-IL-5 mRNA in Th2 effector cells, Northern blot analysis was carried out. D10 cells were employed in this study because this helper T cell line has been extensively studied in our laboratory. Fig. 1 shows that 10 ng/ml of Ubi-L strongly inhibited the cAMP-induced IL-5 mRNA expression in D10 cells. As can be seen in Fig. 2A, Ubi-L dosedependently inhibited the cAMP-induced IL-5 mRNA expression. No cytotoxic effect of Ubi-L on D10 cells was observed (data not shown). Ubi-L at doses greater than 1 ng/ml significantly inhibited the cAMP-induced IL-5 mRNA. This effect was abro-
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Fig. 1. Northern blot analysis of IL-5 mRNA in D10 cells. D10 cells were stimulated with 1 mM bt2cAMP for 6 hours. Ubi-L (10 ng/ml) was added at the initiation of the cultures. For controls, PBS was added in place of Ubi-L. Total RNA preparations from control and treated cells were subjected to Northern blot analysis using a cDNA probe specific for murine IL-5.
gated by anti-Ubi-L receptor serum (IgG). To determine whether the COOH-terminal Gly-Gly doublet of Ubi-L is necessary to Ubi-L action, we employed a mutant Ubi-L containing a substitution of Gly-74 with Ala at the COOH terminus (Ubi-LG74A) (Nakamura et al., 2002). It is evident that the COOH-terminal Gly-Gly doublet of Ubi-L is necessary to the conjugation of Ubi-L to target proteins. As shown in Fig. 2A, this mutant inhibited IL-5 mRNA expression to a similar extent as wild-type Ubi-L, implying that the C-terminal Gly-Gly doublet is not required to the Ubi-L binding to D10 cells. We also determined whether ubiquitin affects the cAMP-induced IL-5 mRNA expression. It should be noted that Ubi-L shows 38% identity with ubiquitin (Nakamura et al., 1995a). As can be seen in Fig. 2A, ubiquitin did not show any suppressive effects on the IL-5 gene induction (Fig. 2A), al-
Fig. 2. Kinetics of Ubi-L suppression of IL-5 mRNA expression. (A) bt2cAMP (1 mM)-stimulated D10 cells were incubated with various concentrations of Ubi-L, mutant Ubi-L, or ubiquitin. In some experiments, Ubi-L and anti-Ubi-L receptor serum (IgG) (anti-Ubi-L R) were added at the initiation of the cultures. The levels of IL-5 mRNA were determined as described in the legend to Fig. 1. (B) Ubi-L (10 ng/ml) was added at indicated times after the addition of 1 mM bt2cAMP to D10 cell cultures. The data represent one of three independent experiments with similar results.
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though high does of ubiquitin (1 mg/ml) showed a slight but significant suppression (data not shown). To effectively inhibit cAMP-mediated IL-5 mRNA induction, Ubi-L was needed to be added to the D10 cell cultures at the time of stimulation with bt2cAMP. No significant effect could be detected when it was added during the later culture period (Fig. 2B), thereby suggesting that Ubi-L acts on D10 cells at an early stage. Ubi-L does not inhibit cAMP-induced IL-5 mRNA in EL4 cells The involvement of the PKA pathway in cAMPinduced IL-5 gene expression has been shown in thymoma line EL4 (Lee et al., 1993). Thus, we investigated whether Ubi-L affects cAMP-induced IL-5 mRNA expression in EL4 cells. As can be seen in Fig. 3, Ubi-L did not show any effect on the IL-5 mRNA induction. In addition, Ubi-L did not affect the cAMP-induced IL-5 secretion by EL4 cells (data not shown). Hence, it is feasible that the inhibitory mechanism of Ubi-L may differ between D10 and EL4 cells. Ubi-L inhibits cAMP-mediated p38 MAPK but not JNK activation It is well established that P38 MAPK (p38), JNK, and ERK are involved in T cell activation. In addition, cAMP has been reported to induce p38 activation in Th2 cells. Thus, we tested whether UbiL inhibits cAMP-mediated p38 activation in D10 cells. As shown in Fig. 4A, Ubi-L strongly inhibited the cAMP-induced p38 phosphorylation. In addition, the pharmacological inhibitor of p38 (SB203580) suppressed IL-5 mRNA induction by cAMP-stimulated D10 cells (Fig. 4B). We did not observe any effect of cAMP on the activation of JNK or ERK (data not shown), although the basal level of phospho-JNK was detected (Fig. 4C). The addition
Fig. 4. Analysis of MAP kinase activation. (A) D10 cells were stimulated with bt2cAMP (1 mM) in the presence or absence of UbiL (10 ng/ml) for 30 minutes at 37 8C, and then cell lysates were prepared. Phosphorylated p38 was detected by Western blot as described in Materials and Methods. (B) SB203580 (1 and 10 mM) was added to bt2cAMP-stimulated D10 cell cultures. The levels of IL5 mRNA were determined as described in the legend to Fig. 1. (C) Ubi-L (100 ng/ml) was added to unstimulated D10 cell cultures. After 30 minutes of treatment, phospho-ERK and -JNK were detected by the use of specific antibodies.
of Ubi-L (100 ng/ml) to the cultures did not affect the JNK phosphorylation at any time points examined (from 5 to 60 minutes) (Fig. 4C and data not shown). Ubi-L inhibits IL-5 and IL-13 production
Fig. 3. Northern blot analysis of IL-5 mRNA in EL4 cells. EL4 cells were stimulated with PMA (100 ng/ml) plus bt2cAMP (0.5 mM) for 6 hours before mRNA was collected. Northern blot analysis of IL-5 mRNA was performed as described in the legend to Fig. 1.
We next examined whether Ubi-L affects cAMPinduced IL-5 and IL-13 production in D10 cells. PMA plus bt2cAMP was used in this experiment because the combination is a more potent stimulus for IL-5 production (Zhang et al., 1997). As depicted in Fig. 5, Ubi-L effectively inhibited both IL-5 and
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Fig. 5. ELISA for IL-5 and IL-13. D10 cells were stimulated with PMA (100 ng/ml) plus bt2cAMP (0.5 mM) in the presence or absence of Ubi-L (3 and 10 ng/ml) for 48 hours. IL-5 (A) and IL-13 (B) in the culture supernatants were measured using a sandwich ELISA as described in Materials and Methods. The data represent one of three independent experiments with similar results.
IL-13 production in PMW- and bt2cAMP-stimulated D10 cells. Ubi-L inhibits cytokine production in Th2 cells derived from primary CD4 T cells In addition to testing the effects of Ubi-L on established cell lines, we also investigated whether Ubi-L affects the in vitro Th differentiation of activated CD4 T cells. As shown in Fig. 6, Ubi-L inhibited IL-4 but not IFNg production during Th differentiation in activated CD4 Tcells. In addition, Ubi-L did not alter IL-4 production in the unskewed population (Fig. 6B) which produced as much IL-4 as the Th2-polarized cells. The IL-4 production might be due to the genetic background of the mice used, BALB/c, which develop a Th2 response preferentially.
Fig. 6. Effect of Ubi-L on Th differentiation of activated CD4 T cells. CD4 T cells were stimulated in vitro under non-polarizing, Th1-polarizing, or Th2-polarizing conditions in the presence or absence of 10 ng/ml of Ubi-L. Cytokine production was measured by ELISA 7 days after the start of differentiation. The data represent one of three independent experiments with similar results.
Discussion cAMP induces IL-5 gene expression in a PKAindependent manner via the p38 MAPK pathway in D10 cells. In contrast, PKA activates the IL-5 promoter in EL4 cells (Lee et al. 1993), which express both Th1- and Th2-type cytokines including IL-2, IL-3, IL-4, IL-10, and GM-CSF. Ubi-L inhibited cAMP-induced IL-5 mRNA expression in D10 but not in EL4 cells (Fig. 1 and Fig. 3). In addition, p38 phosphorylation was not significantly induced
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in cAMP-stimulated EL4 cells in our experimental system (data not shown). In this context, the difference observed between the two cell lines may be due to the differential effects of cAMP in these cells. Alternatively, the low level of Ubi-L receptor expression on EL4 cells might be implicated in the difference (Nakamura & Tanigawa, 1999). JNK and p38 have a common upstream prerequisite, MEKK, as a MAPK kinase kinase. In contrast to p38, JNK was not activated by cAMP in our system (not shown), although a low level of basal phospho-JNK was detected. A large amount of Ubi-L (100 ng/ml) did not affect the basal level of phosphorylation of JNK (Fig. 4B). The intracellular mechanism of action of ubiquitin-like proteins remains unknown. ISG15 is a ubiquitin-like protein that conjugates to a number of proteins in cells treated with interferon or lipopolysaccharide (Narasimhan et al., 1996). It has been reported that Jak1 and ERK1 are modified by ISG15 (Malakhov et al., 2003), suggestive of a role for ISG15 in the regulation of multiple signal transduction pathways. The p38 pathway has been implicated to play a critical role in apoptosis (Xia et al., 1995). Most recently, we have found that Ubi-L conjugates to intracellular acceptor protein, a novel pro-apoptotic member of the Bcl-2 family (Nakamura & Tanigawa, 2003). Further investigations are underway to determine more precisely the intracellular mechanism of action of Ubi-L.
References Chen CH, Zhang DH, LaPorte JM and Ray A. 2000. Cyclic AMP activates p38 mitogen-activated protein kinase in Th2 cells: phosphorylation of GATA-3 and stimulation of Th2 cytokine gene expression. J. Immunol. 165: 5597 ± 5605. Kondoh T, Nakamura M, Nabika T, Yoshimura Y and Tanigawa Y. 1999. Ubiquitin-like polypeptide inhibits the proliferative response of T cells in vivo. Immunobiol. 200: 140 ± 149. Lee HJ, Koyano-Nakagawa N, Naito Y, Nishida J, Arai N, Arai K and Yokota T. 1993. cAMP activates the IL-5 promoter synergistically with phorbol ester through the signaling pathway involving protein kinase A in mouse thymoma line EL-4. J. Immunol. 151: 6135 ± 6142. Lin A, Minden A, Martinetto H, Claret FX, LangeCarter C, Mercurio F, Johnson GL and Karin M. 1995. Identification of a dual specificity kinase that
activates the Jun kinases and p38-Mpk2. Science 268: 286 ± 290. Malakhov MP, Kim KI, Malakhova OA, Jacobs BS, Borden EC and Zhang DE. 2003 High-throughput immunoblotting. Ubiquitin-like protein ISG15 modifies key regulators of signal transduction. J. Biol. Chem. 278: 16608 ± 16613. Nakamura M and Tanigawa Y. 1999. Biochemical analysis of the receptor for ubiquitin-like polypeptide. J. Biol. Chem. 274: 18026 ± 18032. Nakamura M and Tanigawa Y. 2000. Protein tyrosine phosphorylation induced by ubiquitin-like polypeptide in murine T helper clone type 2. Biochem. Biophys. Res. Commun. 274: 565 ± 570. Nakamura M and Tanigawa Y. 2003. Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl-2 family. Eur. J. Biochem. 270: 4052 ± 4058. Nakamura M, Xavier RM and Tanigawa Y. 1994. Monoclonal non-specific suppressor factor (MNSF) inhibits the IL4 secretion by bone marrow-derived mast cell (BMMC). FEBS Lett. 339: 239 ± 242. Nakamura M, Xavier RM, Tsunematsu T and Tanigawa Y. 1995a. Molecular cloning and characterization of a cDNA encoding monoclonal nonspecific suppressor factor. Proc. Natl. Acad. Sci. USA 92: 3463 ± 3467. Nakamura M, Xavier RM and Tanigawa Y. 1995b. Monoclonal nonspecific suppressor factor beta inhibits interleukin-4 secretion by a type-2 helper T cell clone. Eur. J. Immunol. 25: 2417 ± 2419. Nakamura M, Xavier RM and Tanigawa Y. 1996. Ubiquitin-like moiety of the monoclonal nonspecific suppressor factor beta is responsible for its activity. J. Immunol. 156: 532 ± 538. Nakamura M, Tsunematsu T and Tanigawa Y. 2002. Biochemical analysis of a T cell receptor alpha-like molecule involved in antigen-nonspecific suppression. Biochem. Biophys. Acta 1589: 196 ± 202. Narasimhan J, Potter JL and Haas AL. 1996. Conjugation of the 15-kDa interferon-induced ubiquitin homologue is distinct from that of ubiquitin. J. Biol. Chem. 271: 324 ± 330. Suzuki K, Nakamura M, Nariai Y, Dekio S and Tanigawa Y. 1996. Monoclonal nonspecific suppressor factorb (MNSFb) inhibits the production of TNF-a by lipopolysaccharide-activated macrophages. Immunobiol. 195: 187 ± 198. Xia Z, Dickens M, Raingeaud J, Davis RJ and Greenberg ME. 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326 ± 1331. Zhang DH, Cohn L, Ray P, Bottomly K and Ray A. 1997. Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272: 21597 ± 21603.
Ubiquitin-like polypeptide inhibits cAMP-induced p38 MAPK activation in Th2 cells Morihiko Nakamuraa , Yoshinori Tanigawab a b
Cooperative Medical Research Center Department of Biochemistry, Shimane Medical University, Japan
Received: October 10, 2003 ¥ Accepted: January 8, 2004
Abstract Ubi-L, an isoform of the monoclonal nonspecific suppressor factor (MNSF), is an 8.5-kDa ubiquitin-like polypeptide. Ubi-L exhibits an antigen-nonspecific immunosuppressive function on various target cells including murine T helper type 2 (Th2) clone, D10 cells. Ubi-L specifically binds to cell surface receptors on D10 cells. In this study, we observed that Ubi-L inhibited cAMP-induced IL-5 mRNA expression in D10 cells but not in thymoma cell line EL4. In addition, Ubi-L effectively inhibited cAMP-induced p38 MAPK activation in D10 cells. Ubi-L also showed inhibitory activity on IL-5 and IL-13 production by D10 cells stimulated with phorbol ester plus dibutyryl cAMP. Furthermore, Ubi-L inhibited IL-4 production in Th2 cells derived from primary CD4 T cells. Abbreviations: bt2cAMP dibutyryl cyclic AMP; MAPK mitogen-activated protein kinase; MNSF monoclonal nonspecific suppressor factor; p38 p38 MAPK; Ubi-L ubiquitinlike polypeptide
Introduction Interleukin-5 (IL-5) is a T cell cytokine involved in type 2 diseases and is commonly described as being coordinately regulated with other type 2 cytokines, such as IL-4 and IL-13. It has been reported that direct stimulation of cAMP pathways with dibutyryl cAMP (bt2cAMP) induces IL-5 mRNA in murine T helper type 2 (Th2) clone, D10 cells (Chen et al., 2000). Mitogen-activated protein kinases (MAPKs) transduce a variety of extracellular signals to the transcriptional machinery via a cascade of protein phosphorylation. There are three genetically distinct MAPKs in
mammals, consisting of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK (p38). p38 is activated by dual specificity MAP kinase kinases, including MKK3, MKK6, and JNKK1, that phosphorylate p38 on threonine 180 and tyrosine 182 (Lin et al., 1995). cAMP activates p38 in Th2 but not in Th1 cells (Chen et al. 2000). Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by a murine T cell hybridoma, possesses pleiotropic nonspecific suppressive functions (Kondoh et al., 1999; Nakamura et al., 1996; Suzuki et al., 1996). MNSFb (a subunit of MNSF) is a 14.5 kDa fusion protein
Corresponding author: Morihiko Nakamura, PhD, Cooperative Medical Research Center, Shimane University, School of Medicine, 89-1 Enya-cho, Izumo 693, Japan, Tel: 81-853-20-2916, Fax: 81-853-20-2913, e-mail: [email protected]
0171-2985/04/208/5-439 $ 15.00/0
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consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30 (Nakamura et al., 1995a). The ubiquitin-like segment of MNSFb (Ubi-L) is an 8 kDa polypeptide with MNSF-like activity. Ubi-L shows an antigen-nonspecific immunosuppressive action on various target cells including D10 cells. We have mentioned that Ubi-L inhibits both the IL-4 secretion and the proliferation of concanavalin A-activated D10 cells (Nakamura et al., 1995b). We have demonstrated that Ubi-L binds specific receptor proteins on Con A-activated D10 cells (Nakamura & Tanigawa, 1999). Ubi-L receptor expression is limited to lymphoid cells. We have demonstrated that tyrosine phosphorylation may be a key step in the initiation of the Ubi-L receptor-mediated transmembrane signaling (Nakamura & Tanigawa, 2000). Interestingly, ubiquitin interferes with Ubi-L activity and high-dose ubiquitin mimics the action of Ubi-L (Nakamura et al., 1996). In addition, ubiquitin shows a slight but significant inhibition of the Ubi-L binding to activated D10 cells (Nakamura & Tanigawa, 1999). Furthermore, Ubi-L inhibits the proliferative response of T cells in vivo (Kondoh et al., 1999). In this study, we investigated whether Ubi-L is involved in the cAMP-mediated signal transduction in D10 cells.
Northern blot analysis Total cellular RNA was isolated, fractionated and transferred to ECL membrane as described (Nakamura et al., 1994). Hybridization, labeling of cDNA probe and detection were done according to the ECL-GENE Detection System (Amersham Biosciences, Piscataway, NJ). The mouse IL-5 cDNA probe was a 450-bp SacI-AccI fragment. Analysis of MAP kinase activation Twenty mg of soluble proteins were run in 10% SDSPAGE and transferred onto polyvinylidene fluoride (PVDF). Phosphorylated MAPK were detected by Western blot analysis using specific antibodies for the phosphorylated forms of p38, ERK, and JNK (Santacruz Biotechnology, Santa Cruz, CA). The membranes were blocked with 5% dry milk in PBS for 1 hour and then washed with PBS containing 0.1% Tween 20. First antibodies were revealed using anti-rabbit secondary antibodies conjugated to horseradish peroxidase and an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences). The same membranes were reprobed with anti-kinase antibodies. Quantification of cytokines
Materials and Methods BALB/c mice were obtained from Clea Japan, Inc. and were reared in the Laboratory of the Institute for Experimental Animals, Shimane Medical University.
Murine cytokines were measured using sandwich ELISA. The lower limits of detection for the cytokines were IL-5, 10 pg/ml (Endogen, Woburn, MA); IL-13, 2 pg/ml (R&D Systems, Minneapolis, MN); IFNg and IL-4, 2 and 3 pg/ml, respectively (BioSource International, Camarillo, CA).
Tumor cell lines
Preparation of CD4 Tcells
D10 cells (T helper type 2 clone) were maintained with biweekly stimulation with 100 mg/ml conalbumin in the presence of 0.5 U/ml recombinant human IL-2. Cells were used 10 ± 12 days after stimulation with antigen. EL4 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).
Splenocytes from 6- to 8-wk-old BALB/c mice were purified by RBC lysis. CD4 T cells were isolated by positive selection using CD4 Dynabeads followed by detachment with DETACHaBEAD¾ (Dynal Biotech, Oslo, Norway). The complete medium consisted of RPMI 1640 supplemented with 10% FBS plus penicillin-streptomycin, sodium pyruvate, and glutamine to final concentration of 100 mg/ml. In addition, 2-ME was added to a final concentration of 50 mM and gentamicin to 50 ng/ml.
Mice
Ubi-L preparation Recombinant Ubi-L was obtained as described previously (Nakamura et al., 1995a). Briefly, Ubi-L was expressed as a fusion protein with glutathioneS-transferase using the pGEX-2T vector (Pharmacia, Piscataway, NJ). Ubi-L was cleaved from the fusion partner by thrombin and purified by using antiUbi-L Ab coupled to Sepharose 4B (Pharmacia).
In vitro differentiation of CD4 T cells CD4 T cells (2 106 cells/ml) were stimulated in vitro with plate-bound anti-CD3 monoclonal antibody (2C11) at 3 mg/ml and anti-CD28 monoclonal antibody (37.51) at 1 mg/ml plus IL-2 at 20 U/ml
Ubiquitin-like protein inhibits p38 activation
(nonskewing condition). In addition, cells were incubated with IL-12 at 5 ng/ml and anti-IL-4 monoclonal antibody (11B11) at 3 mg/ml (Th1skewing condition) or IL-4 at 500 U/ml and antiIL-12 (C17.8) at 5 mg/ml (Th2-skewing condition). Differentiation proceeded in the presence or absence of Ubi-L. Four days post-stimulation, the cultures were expanded 2-fold with fresh medium, cytokines, and antibodies. Three days later, the cells were harvested, washed five times with RPMI 1640 and counted, and equal numbers of cells were restimulated with plate-bound anti-CD3 at 1 mg/ml. Twenty-four hours post-restimulation, supernatants were collected and cytokine levels were measured by ELISA.
Results Ubi-L inhibits cAMP-induced IL-5 mRNA in D10 cells To determine whether Ubi-L inhibits cAMP induced-IL-5 mRNA in Th2 effector cells, Northern blot analysis was carried out. D10 cells were employed in this study because this helper T cell line has been extensively studied in our laboratory. Fig. 1 shows that 10 ng/ml of Ubi-L strongly inhibited the cAMP-induced IL-5 mRNA expression in D10 cells. As can be seen in Fig. 2A, Ubi-L dosedependently inhibited the cAMP-induced IL-5 mRNA expression. No cytotoxic effect of Ubi-L on D10 cells was observed (data not shown). Ubi-L at doses greater than 1 ng/ml significantly inhibited the cAMP-induced IL-5 mRNA. This effect was abro-
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Fig. 1. Northern blot analysis of IL-5 mRNA in D10 cells. D10 cells were stimulated with 1 mM bt2cAMP for 6 hours. Ubi-L (10 ng/ml) was added at the initiation of the cultures. For controls, PBS was added in place of Ubi-L. Total RNA preparations from control and treated cells were subjected to Northern blot analysis using a cDNA probe specific for murine IL-5.
gated by anti-Ubi-L receptor serum (IgG). To determine whether the COOH-terminal Gly-Gly doublet of Ubi-L is necessary to Ubi-L action, we employed a mutant Ubi-L containing a substitution of Gly-74 with Ala at the COOH terminus (Ubi-LG74A) (Nakamura et al., 2002). It is evident that the COOH-terminal Gly-Gly doublet of Ubi-L is necessary to the conjugation of Ubi-L to target proteins. As shown in Fig. 2A, this mutant inhibited IL-5 mRNA expression to a similar extent as wild-type Ubi-L, implying that the C-terminal Gly-Gly doublet is not required to the Ubi-L binding to D10 cells. We also determined whether ubiquitin affects the cAMP-induced IL-5 mRNA expression. It should be noted that Ubi-L shows 38% identity with ubiquitin (Nakamura et al., 1995a). As can be seen in Fig. 2A, ubiquitin did not show any suppressive effects on the IL-5 gene induction (Fig. 2A), al-
Fig. 2. Kinetics of Ubi-L suppression of IL-5 mRNA expression. (A) bt2cAMP (1 mM)-stimulated D10 cells were incubated with various concentrations of Ubi-L, mutant Ubi-L, or ubiquitin. In some experiments, Ubi-L and anti-Ubi-L receptor serum (IgG) (anti-Ubi-L R) were added at the initiation of the cultures. The levels of IL-5 mRNA were determined as described in the legend to Fig. 1. (B) Ubi-L (10 ng/ml) was added at indicated times after the addition of 1 mM bt2cAMP to D10 cell cultures. The data represent one of three independent experiments with similar results.
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though high does of ubiquitin (1 mg/ml) showed a slight but significant suppression (data not shown). To effectively inhibit cAMP-mediated IL-5 mRNA induction, Ubi-L was needed to be added to the D10 cell cultures at the time of stimulation with bt2cAMP. No significant effect could be detected when it was added during the later culture period (Fig. 2B), thereby suggesting that Ubi-L acts on D10 cells at an early stage. Ubi-L does not inhibit cAMP-induced IL-5 mRNA in EL4 cells The involvement of the PKA pathway in cAMPinduced IL-5 gene expression has been shown in thymoma line EL4 (Lee et al., 1993). Thus, we investigated whether Ubi-L affects cAMP-induced IL-5 mRNA expression in EL4 cells. As can be seen in Fig. 3, Ubi-L did not show any effect on the IL-5 mRNA induction. In addition, Ubi-L did not affect the cAMP-induced IL-5 secretion by EL4 cells (data not shown). Hence, it is feasible that the inhibitory mechanism of Ubi-L may differ between D10 and EL4 cells. Ubi-L inhibits cAMP-mediated p38 MAPK but not JNK activation It is well established that P38 MAPK (p38), JNK, and ERK are involved in T cell activation. In addition, cAMP has been reported to induce p38 activation in Th2 cells. Thus, we tested whether UbiL inhibits cAMP-mediated p38 activation in D10 cells. As shown in Fig. 4A, Ubi-L strongly inhibited the cAMP-induced p38 phosphorylation. In addition, the pharmacological inhibitor of p38 (SB203580) suppressed IL-5 mRNA induction by cAMP-stimulated D10 cells (Fig. 4B). We did not observe any effect of cAMP on the activation of JNK or ERK (data not shown), although the basal level of phospho-JNK was detected (Fig. 4C). The addition
Fig. 4. Analysis of MAP kinase activation. (A) D10 cells were stimulated with bt2cAMP (1 mM) in the presence or absence of UbiL (10 ng/ml) for 30 minutes at 37 8C, and then cell lysates were prepared. Phosphorylated p38 was detected by Western blot as described in Materials and Methods. (B) SB203580 (1 and 10 mM) was added to bt2cAMP-stimulated D10 cell cultures. The levels of IL5 mRNA were determined as described in the legend to Fig. 1. (C) Ubi-L (100 ng/ml) was added to unstimulated D10 cell cultures. After 30 minutes of treatment, phospho-ERK and -JNK were detected by the use of specific antibodies.
of Ubi-L (100 ng/ml) to the cultures did not affect the JNK phosphorylation at any time points examined (from 5 to 60 minutes) (Fig. 4C and data not shown). Ubi-L inhibits IL-5 and IL-13 production
Fig. 3. Northern blot analysis of IL-5 mRNA in EL4 cells. EL4 cells were stimulated with PMA (100 ng/ml) plus bt2cAMP (0.5 mM) for 6 hours before mRNA was collected. Northern blot analysis of IL-5 mRNA was performed as described in the legend to Fig. 1.
We next examined whether Ubi-L affects cAMPinduced IL-5 and IL-13 production in D10 cells. PMA plus bt2cAMP was used in this experiment because the combination is a more potent stimulus for IL-5 production (Zhang et al., 1997). As depicted in Fig. 5, Ubi-L effectively inhibited both IL-5 and
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Fig. 5. ELISA for IL-5 and IL-13. D10 cells were stimulated with PMA (100 ng/ml) plus bt2cAMP (0.5 mM) in the presence or absence of Ubi-L (3 and 10 ng/ml) for 48 hours. IL-5 (A) and IL-13 (B) in the culture supernatants were measured using a sandwich ELISA as described in Materials and Methods. The data represent one of three independent experiments with similar results.
IL-13 production in PMW- and bt2cAMP-stimulated D10 cells. Ubi-L inhibits cytokine production in Th2 cells derived from primary CD4 T cells In addition to testing the effects of Ubi-L on established cell lines, we also investigated whether Ubi-L affects the in vitro Th differentiation of activated CD4 T cells. As shown in Fig. 6, Ubi-L inhibited IL-4 but not IFNg production during Th differentiation in activated CD4 Tcells. In addition, Ubi-L did not alter IL-4 production in the unskewed population (Fig. 6B) which produced as much IL-4 as the Th2-polarized cells. The IL-4 production might be due to the genetic background of the mice used, BALB/c, which develop a Th2 response preferentially.
Fig. 6. Effect of Ubi-L on Th differentiation of activated CD4 T cells. CD4 T cells were stimulated in vitro under non-polarizing, Th1-polarizing, or Th2-polarizing conditions in the presence or absence of 10 ng/ml of Ubi-L. Cytokine production was measured by ELISA 7 days after the start of differentiation. The data represent one of three independent experiments with similar results.
Discussion cAMP induces IL-5 gene expression in a PKAindependent manner via the p38 MAPK pathway in D10 cells. In contrast, PKA activates the IL-5 promoter in EL4 cells (Lee et al. 1993), which express both Th1- and Th2-type cytokines including IL-2, IL-3, IL-4, IL-10, and GM-CSF. Ubi-L inhibited cAMP-induced IL-5 mRNA expression in D10 but not in EL4 cells (Fig. 1 and Fig. 3). In addition, p38 phosphorylation was not significantly induced
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in cAMP-stimulated EL4 cells in our experimental system (data not shown). In this context, the difference observed between the two cell lines may be due to the differential effects of cAMP in these cells. Alternatively, the low level of Ubi-L receptor expression on EL4 cells might be implicated in the difference (Nakamura & Tanigawa, 1999). JNK and p38 have a common upstream prerequisite, MEKK, as a MAPK kinase kinase. In contrast to p38, JNK was not activated by cAMP in our system (not shown), although a low level of basal phospho-JNK was detected. A large amount of Ubi-L (100 ng/ml) did not affect the basal level of phosphorylation of JNK (Fig. 4B). The intracellular mechanism of action of ubiquitin-like proteins remains unknown. ISG15 is a ubiquitin-like protein that conjugates to a number of proteins in cells treated with interferon or lipopolysaccharide (Narasimhan et al., 1996). It has been reported that Jak1 and ERK1 are modified by ISG15 (Malakhov et al., 2003), suggestive of a role for ISG15 in the regulation of multiple signal transduction pathways. The p38 pathway has been implicated to play a critical role in apoptosis (Xia et al., 1995). Most recently, we have found that Ubi-L conjugates to intracellular acceptor protein, a novel pro-apoptotic member of the Bcl-2 family (Nakamura & Tanigawa, 2003). Further investigations are underway to determine more precisely the intracellular mechanism of action of Ubi-L.
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