Published online before print March 22, 2006
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* Department of Biomedical Sciences, C. W. Post Campus, Long Island University, Brookville, New York; and
University of Miami, School of Medicine, Microbiology and Immunology, Florida
1Correspondence: Department of Biomedical Sciences, School of Health Professions and Nursing, C. W. Post Campus, Long Island University, 720 Northern Blvd., Brookville, NY 11548. E-mail: stamma{at}liu.edu
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Key Words: STAT-6 • Bcl-2 • Bcl-xL
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-CD3 in the absence of CD28 costimulation produce only interleukin (IL)-2 and interferon-
, whereas the addition of -CD28 monoclonal antibody (mAb) induced IL-4 [2
, 3
]. Consequently, CD28 knockout mice have preserved T helper cell type 1 (Th1)-mediated cytotoxic T lymphocyte and cellular immunity but impaired Th2-dependent immunoglobulin (Ig) production [4
]. Thus, CD28 signaling may regulate the balance of inflammatory/humoral (Th1/Th2) responses during an immune reaction. CD28 costimulation lowers the threshold required for T cell activation, increases the expression of lymphokine mRNAs, in particular, those for IL-2 and IL-4 [5
6
7
], and regulates the expression of Bcl-xL [8
] and CD154 {CD40 ligand (CD40L) [9
]}, all of which contribute to successful progression of T cell responses.
Moriggl et al. [10
] have previously shown that human CD4+ 45RO– T cells could be primed for a Th2 phenotype, independent of IL-4, if they were activated by anti-CD28 mAb plus IL-2 and that anti-CD28/IL-2-primed Th2 cells expressed high levels of activated signal transducer and activator of transcription (STAT)-6. IL-4 had a synergistic effect with anti-CD28 on the generation of Th2 cells in memory T cells. IL-4 was also shown to be the major determining factor in the differentiation of naive T cells into the Th2 phenotype and facilitating the humoral immune response [11
, 12
]. Within the intracellular domains of human IL-4 receptor (hIL-4R), STAT-6 interacts with the second, third, and fourth conserved tyrosines (Y575, Y603, Y631) [13
14
15
16
]. STAT-6 is recruited to the IL-4R complex by binding to any of three phosphotyrosines (Pty) in IL-4R. It becomes tyrosyl-phosphorylated at its C terminus through the action of Janus kinases 1 and/or 3. Phosphorylated STAT-6 dimerizes, migrates to the nucleus, and binds to specific DNA elements, and together with other transcription factors, activates transcription of some IL-4-induced genes [17
18
19
20
]. There is growing evidence now that Bcl-2 plays a unique, functional role in extending the cellular survival by inhibiting the cell death induced by various apoptotic stimuli [14
, 18
, 21
22
23
24
25
26
27
].
We and others have shown evidence that jacalin, a plant lectin, competes with human immunodeficiency virus (HIV)-1 gp120 in binding to the CD4 molecule and have shown that jacalin binds to CD4 and inhibits in vitro HIV infection [28
, 29
]. Our findings suggest that jacalin might be a useful, surrogate marker for quantitative as well as qualitative deficiency of CD4+ T cells in HIV-1 infection [28
]. Based on the effect of jacalin on CD4+ T cells and the effect of interaction of CD4, gp120, and jacalin on CD4+ T cells, we investigated the ability of jacalin to transmit intracellular signals associated with T cell activation. We analyzed early signaling events such as p56lck, p59fyn, 
-associated protein 70, p95 vav, phospholipase C-1, and ras activation. We further analyzed the downstream events of ras such as phosphorylation of extracellular signal-regulated kinase 2 and Jun N-terminal kinase. We have shown that jacalin may be used as a possible tool for the study of CD4-mediated signal transduction and HIV-impaired T cell activation. We have also shown that jacalin induces moderate levels of IL-2 [30
]. Based on our previous findings that jacalin, a lectin, is a CD4+ T cell mitogen and induces intracellular signaling and moderate levels of IL-2 secretion [30
], we examined whether stimulation with jacalin would induce IL-4 secretion. We observed that jacalin stimulation alone resulted in insignificant levels of IL-4 when compared with that induced by phorbol 12-myristate 13-acetate (PMA) plus ionomycin stimulation. Similarly, CD28 stimulation alone failed to induce IL-4 secretion. We asked whether costimulation would induce Th2 cytokine IL-4 in jacalin-stimulated cells. Our study demonstrates that costimulation would result in IL-4 secretion (S. M. L. Tamma S. Balan, K. N. Chung, S. Pahwa, manuscript accepted for publication). We also observed a positive relationship between activation of p38 mitogen-activated protein kinase (MAPK) and IL-4 synthesis.
It has been shown by others that a Th2 phenotype would protect the cell from undergoing apoptosis [31 32 33 34 35 36 37 38 39 40 41 42 43 ]. We have shown in the present study that normal T cells in the presence of jacalin plus CD28 costimulation exhibit STAT-6 phosphorylation, as well as expression of Bcl-2 and Bcl-xL. Then, we asked if jacalin blasts would respond to CD28 cross-linking (CD28XL) in the absence of further stimulation with jacalin to express IL-4 message, STAT-6 phosphorylation, and Bcl-2 and Bcl-xL expression. We observed that jacalin blasts in the absence of further stimulation with jacalin and in the presence of CD28 express IL-4 message as well STAT-6 phosphorylation and Bcl-2 and Bcl-xL expression. We examined whether p38 MAPK plays an important role in the expression of IL-4, phosphorylation of STAT-6, and expression of Bcl-2 and Bcl-xL by using p38 MAPK inhibitor SB203580. Our data suggest that p38 may play an important role, and we propose that CD28-mediated signaling in jacalin blasts results in IL-4 synthesis, and p38 may be one of the molecules involved in this pathway.
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Isolation and purification of cells
Peripheral blood mononuclear cells were isolated from healthy volunteers by Ficoll-Hypaque (Lymphoprep, Nycomed, UK) density gradient centrifugation. T cells were enriched by double rosetting with neuraminidase-treated sheep red blood cells. Adherent cells were removed by incubation in the petri dishes for 2 h at 37°C. CD4+ T cells were purified by negative selection with anti-CD8 mAb-coated magnetic beads (Dynal, Great Neck, NY).
All cell cultures were carried out in RPMI-1640 medium supplemented with penicillin G (100 IU/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), and 10% fetal calf serum (FCS).
Generation of jacalin-stimulated T cell blasts
All cell cultures were carried out in RPMI-1640 medium supplemented with penicillin G (100 IU/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), 10% FCS, and recombinant (r)hIL-2 (100 U/ml). CD4+ T cells were cultured 3 x 105 cells/ml with 10% monocytes and jacalin (200 µg/ml) in six-well tissue-culture plates in a total volume of 6 ml culture medium. After 5 days of culture, jacalin-stimulated T cells were further fed every 3 or 4 days with the culture medium with 100 U/ml rIL-2. On Day 12, stimulated T cells were washed extensively, and viable cells were obtained by Ficoll Hypaque gradient and used for further studies.
Phosphorylation studies
To delineate its role in CD28-mediated IL-4 secretion, phosphoactivation of the MAPK p38 in response to CD28 engagement and its contribution to Th2 cell differentiation were studied. For signal transduction studies, CD4+ T cells blasts (3–5x106 cells) were washed and stimulated with CD28 antibody (5 µg/1x106 cells) for 30 min at 4°C on a rotator or with murine IgG1 as a control antibody, cells were brought to room temperature, and then jacalin was added and incubated at 37°C for 10 min. Cells were also stimulated with jacalin alone or with CD28 antibody alone. Treatment with PMA (50 ng/106 cells) + ionomycin (400 ng/106cells) is used as a positive control for T cell activation and phosphorylation of p38 MAPK. Where indicated, cells were pretreated with p38 MAPK (SB203580, 0.4 µM, Calbiochem) inhibitor or for control, with the solvent of the inhibitors (dimethyl sulfoxide) for 1 h (45 min at 37°C and 15 min on ice). There were no significant differences in cell viabilities at the time of sample collection for analysis. Reactions were terminated by washing cells in ice-cold phosphate-buffered saline (PBS) + EDTA + sodium orthovanadate and lysing in lysis buffer (0.04 mol/L Tris-HCl, 0.276 mol/L NaCl, 20% glycerol, 2% Nonidet P-40, 0.002 mol/L sodium orthovanadate, 0.02 mol/L NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.004 mol/L EDTA, and 1 mmol/L phenylmethylsulfonyl fluoride). Lysed samples were microcentrifuged for 20 min at 14,000 revolutions per minute at 4°C. Supernatants were aliquoted and stored at –80°C until ready to use. The protein content in the supernatant was determined by the Bio-Rad protein assay. The supernatants, with equal amount of protein for each sample (50 µg/lane), were mixed with sample buffers and boiled, and aliquots were size-fractionated in SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto nitrocellulose membranes. Blots were washed once in PBS and blocked in 5% nonfat dry milk in PBS for 1 h. Blot was incubated with phosphor-p38 antibody, washed, treated with enzyme-conjugated secondary antibody, incubated, washed, developed with the substrate as described before or peroxidase-conjugated second antibody, and developed by an enhanced chemiluminescence system.
Analysis of STAT-6 expression in CD28-stimulated normal T cells or jacalin blasts
CD4+ T cells were stimulated with CD28 antibody (5 µg/1x106 cells) for 30 min at 4°C on a rotator, cells were brought to room temperature, and then jacalin was added and cultured for 72 h. Cells were washed and lysed, and lysates were immunoprecipitated with anti-STAT-6 antibody and immunoblotted with STAT-6 antibody or anti-Pty antibody. PMA plus ionomycin treatment was used as positive control. Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody for 24 h in the presence or absence of p38 MAPK inhibitor (SB203580, 400 nM, Calbiochem). Cells were washed and lysed, and lysates were immunoprecipitated with anti-STAT-6 antibody and immunoblotted with STAT-6 antibody or anti-Pty antibody. Stimulated cell lysate samples were also analyzed directly with phosphor-STAT-6 antibody in a Western blot.
Analysis of expression of Bcl-2 and Bcl-xL
CD4+ T cells were cultured for different periods with anti-CD28XL plus jacalin (0, 10, 20, 30, 40, or 50 h), washed, and lysed, and lysates were immunoprecipitated with anti-Bcl-2 or -Bcl-xL or anti-Bcl-2-associated X protein (BAX) antibody and immunoblotted with appropriate antibody.
Normal CD4+ T cells were stimulated overnight with anti-CD28 antibody plus jacalin in the presence of various doses of SB203580 to assess expression of Bcl-2, Bcl-xL, and BAX. Based on the response, we chose SB203580 at 0.4 µM concentration as the optimum concentration, where p38 phosphorylation was inhibited. There were no significant differences in cell viabilities at the time of sample collection for analysis. The protein content in the supernatant was determined by the Bio-Rad protein assay. The supernatants, with equal amount of protein for each sample (50–100 µg/sample), were used for immunoprecipitation.
Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody. Cell lysates were prepared, immunoprecipitated with anti-Bcl-2 or -Bcl-xL or anti-BAX antibody, and immunoblotted with appropriate antibody.
Similarly, jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody overnight in the presence or absence of SB203580 (0.4 µM). Cell lysates were prepared, immunoprecipitated with anti-Bcl-2 or anti-Bcl-xL, separated by PAGE, and immunoblotted with appropriate antibody.
RNA isolation and RT-PCR analysis of IL-4 mRNA
Cells were stimulated for 24 h, and total cellular RNA was isolated using the Ambion kit according to the manufacturer’s instructions. RNA (1 µg) was reverse-transcribed with oligo(dT). PCR amplification was then performed for 35 cycles with specific primers for the hIL-4 [456 base pair (bp) PCR product] and the human actin (661 bp PCR product) protein, respectively. The PCR products were then subjected to agarose gel electrophoresis and stained with Sybergreen.
IL-4 secretion and cell proliferation
Normal CD4+ T cells were stimulated for 48 h with various doses of SB203580, and culture supernatants were harvested and analyzed for IL-4 using a commercial kit from Biosource International, according to the manufacturer’s instructions. Normal CD4+ T cells were stimulated for 72 h with various doses of SB203580, and proliferative responses were analyzed as described before [30
].
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Figure 1. CD28XL-induced phosphorylation of p38 MAPK in jacalin blasts. Jacalin-stimulated blasts were generated, washed, and stimulated with CD28 antibody in the presence or absence of p38 MAPK inhibitor (SB203580) as described in Materials and Methods. Stimulated cells were lysed, lysates were probed with phosphor-p38 antibody in Western blot, and data were analyzed by densitometry: Lane 1 = 260; lane 2 = 300; lane 3 = 1500. Data are from one representative experiment of at least three independent experiments.
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Figure 2. (A) CD28XL plus jacalin stimulation induced STAT-6 phosphorylation in normal CD4+ T cells, which were cultured for 72 h, washed, and lysed, and lysates were immunoprecipitated with anti-STAT-6 antibody and immunoblotted with STAT-6 antibody or anti-Pty antibody (AB). Densitometric readings of scanned immunoblots with anti-Pty antibody (upper panel) were as follows: Lane 1 = 90; lane 2 = 1380; lane 3 = 220; lane 4 = 1780; lane 5 = 510. Data are from one representative experiment of at least three independent experiments. (B) CD28XL induced STAT-6 phosphorylation in jacalin blasts. Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody for 24 h in the presence or absence of p38 MAPK inhibitor (SB203580). Densitometric readings of scanned immunoblots with anti-Pty antibody were as follows: Lane 1 = 210; lane 2 = 1660; lane 3 = 1450. Data are from one representative experiment of at least three independent experiments.
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Induction of IL-4 mRNA by CD28XL in jacalin blasts
Earlier studies have shown that jacalin plus CD28 costimulation resulted in IL-4 secretion in normal CD4+ T cells (S. M. L. Tamma et al., manuscript accepted for publication). Here, we examined whether stimulation of jacalin blasts by CD28XL in the absence of stimulation with jacalin would result in induction of IL-4 message. Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody overnight in the presence or absence of the p38 MAPK (SB203580) inhibitor. Total cellular RNA was isolated and reverse-transcribed with oligo(dT), and PCR amplification was then performed for 35 cycles with specific primers for the hIL-4 and the human-actin protein, respectively. The PCR products were then subjected to agarose gel electrophoresis and stained with Sybergreen. As shown in Figure 3
, CD28XL of jacalin blasts resulted in IL-4 gene transcription, which was inhibited significantly by the p38 MAPK inhibitor. The data suggest that p38 MAPK plays an important role in the pathway leading to IL-4 synthesis. Actinomycin D completely inhibited CD28 plus jacalin-induced increase of IL-4 mRNA (data not shown).
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Figure 3. Induction of IL-4 mRNA by CD28XL in jacalin blasts. Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody overnight in the presence or absence of p38 MAPK inhibitor (SB203580). Total cellular RNA was isolated and reverse-transcribed with oligo(dT), and PCR amplification was then performed for 35 cycles with specific primers for the hIL-4 and the human-actin protein, respectively. The PCR products were then subjected to agarose gel electrophoresis and stained with Sybergreen. Data are from one representative experiment of at least three independent experiments.
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Figure 4. (A) Expression of Bcl-xL in CD28 plus jacalin-stimulated CD4+ T cells, which were cultured for different time periods as indicated, washed, and lysed, and lysates were immunoprecipitated and immunoblotted with anti-Bcl-xL antibody. Densitometric readings of scanned immunoblots with anti-Bcl-xL: Lane 1 = 80; lane 2 = 550; lane 3 = 1680; lane 4 = 1700; lane 5 = 1500; lane 6 = 380. Data are from one representative experiment of at least three independent experiments. (B) Expression of Bcl-2 and BAX in CD28 plus jacalin-stimulated CD4+ T cells, which were cultured for different periods as indicated, washed, and lysed, and lysates were immunoprecipitated and immunoblotted with anti-Bcl-2 antibody (upper panel). Densitometric readings of scanned immunoblots with anti-Bcl-2: Lane 1 = 70; lane 2 = 1890; lane 3 = 1780; lane 4 = 1520; lane 5 = 450. Data are from one representative experiment of at least three independent experiments. Lower panel shows expression of BAX (lane 1=260; lane 2=280; lane 3=380; lane 4=410; lane 5=960). (C) CD28XL induced Bcl-2 and Bcl-xL expression in jacalin blasts. Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody as indicated. Cell lysates were prepared, immunoprecipitated with anti-Bcl-2 or anti-Bcl-xL or BAX antibody, and immunoblotted with appropriate antibody. Densitometric readings of scanned immunoblots with anti-Bcl-2 (top panel): Lane 1 (10 h) = 1760; lane 2 (20 h) = 1850; lane 3 (30 h) = 1690. Data are from one representative experiment of at least three independent experiments. Densitometric readings of scanned immunoblots with anti-Bcl-xL (middle panel): Lane 1 (10 h) = 1500; lane 2 (20 h) = 1030; lane 3 (30 h) = 1790. Densitometric readings of scanned immunoblots with anti-BAX (bottom panel): Lane 1 (10 h) = 1100; lane 2 (20 h) = 1260; lane 3 (30 h) = 1710. Data are from one representative experiment of at least three independent experiments.
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p38 MAPK inhibitor (SB203580) down-regulates Bcl-2 expression in normal CD4+ T cells and jacalin blasts stimulated by CD28 antibody
Normal CD4+ T cells were stimulated overnight with anti-CD28 antibody plus jacalin in the presence or absence of various doses of SB203580 (0.1, 0.4, 1, 5, and 10 µM concentration) as shown in Figure 5A
. There were no significant differences in cell viabilities. Cells were lysed, and lysates were immunoprecipitated with anti-Bcl-2 or anti-Bcl-xL or anti-BAX antibodies and immunoblotted with the appropriate antibodies. As shown in Figure 5A
, bottom panel, there is an increase in BAX expression in a dose-dependent manner, whereas expression of Bcl-2 and Bcl-xL was down-modulated (Fig. 5A
, top and middle panels, respectively).
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Figure 5. (A) Dose response to SB203580. Normal CD4+ T cells were stimulated overnight with anti-CD28 antibody plus jacalin in the presence of different doses of SB203580 as indicated, and expression of Bcl-2, Bcl-xL, and BAX was assessed. Densitometric readings were as follows: Top panel, lane 1 = 150; lane 2 = 210; lane 3 = 190; lane 4 = 380; lane 5 = 420. Middle panel, lane 1 = 140; lane 2 = 160; lane 3 = 210; lane 4 = 390; lane 5 = 200. Bottom panel, lane 1 = 470; lane 2 = 590; lane 3 = 570; lane 4 = 1580; lane 5 = 1530. (B) p38 MAPK inhibitor (SB203580) down-regulates Bcl-2 expression with normal CD4+ T cells and jacalin blasts. Normal CD4+ T cells were stimulated overnight with anti-CD28 antibody plus jacalin in the presence or absence of SB203580 (0.4 µM). Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody overnight. Cell lysates were prepared, immunoprecipitaed with anti-Bcl-2, separated by PAGE, and immunoblotted with anti-Bcl-2 antibody. Densitometric readings of scanned immunoblots with anti-Bcl-2: Lane 1 = 90; lane 2 = 1510; lane 3 = 390 (normal CD4+ T cells); lane 4 = 420; lane 5 = 1800; lane 6 = 680 (jacalin blasts). Data are from one representative experiment of at least three independent experiments. (C) p38 MAPK inhibitor (SB203580) down-regulates Bcl-xL expression in jacalin blasts. Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody overnight in the presence or absence of SB203580 (0.4 µM). Cell lysates were prepared, immunoprecipitaed with anti-Bcl-xL, separated by PAGE, immunoblotted with anti-Bcl-xL antibody, and analyzed by densitometry: Lane 1 = 1780; lane 2 = 750; lane 3 = 400; lane 4 = 1610. Data are from one representative experiment of at least three independent experiments.
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Figure 5B , lanes 4–6, represents jacalin blasts. Similar to the response by normal CD4+ T cells, there is significant inhibition of Bcl-2 by SB203580 in jacalin blasts (1810±120 without SB20358 treatment and 870±100 with SB20358 treatment). The data appear to suggest that p38 MAPK plays a role in expression of Bcl-2.
p38 MAPK inhibitor (SB203580) down-regulates Bcl-xL expression in jacalin blasts stimulated with CD28 antibody
Jacalin-stimulated blasts were generated, washed, and stimulated with anti-CD28 antibody overnight in the presence or absence of SB203580. Cell lysates were prepared, immunoprecipitated with anti-Bcl-xL, separated by PAGE, immunoblotted with anti-Bcl-xL antibody, and analyzed by densitometry. As shown in Figure 5C
, SB203580 inhibited Bcl-xL expression induced by jacalin (1790±120 vs. 740±60) or CD28XL (1650±90 vs. 390±40) in jacalin blasts.
IL-4 secretion and cell proliferation
Normal CD4+ T cells were stimulated in the presence of various concentrations of SB203580 for 48 h or 72 h to analyze IL-4 secretion and proliferation, respectively. We observed a dose-dependent inhibition of IL-4 secretion (Fig. 6A
) and proliferative response (Fig. 6B)
.
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Figure 6. Inhibition of IL-4 synthesis and proliferation by SB203580. Normal CD4+ T cells were stimulated for 48 h with various doses of SB203580, and culture supernatants were harvested and analyzed for IL-4 using a commercial kit from Biosource International, according to the manufacturer’s instructions. Normal CD4+ T cells were stimulated for 72 h with various doses of SB203580, and proliferative responses were analyzed as described before [30
]. CPM, Counts per minute.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As it has been shown by previous studies that a Th2 phenotype would protect cells from undergoing apoptosis [31 32 33 34 35 36 37 38 39 40 41 42 43 , 45 ], we analyzed the expression of Bcl-2 and Bcl-xL in this culture system. We have shown in the present study that normal T cells in the presence of jacalin plus CD28 costimulation exhibit STAT-6 phosphorylation (Fig. 2A) , as well as expression of Bcl-2 and Bcl-xL (Fig. 4 , A and B, upper panel). We have also observed expression of BAX by 40 h (Fig. 4B , lower panel). Then, we examined whether jacalin blasts would respond to CD28XL in the absence of further stimulation with jacalin to express IL-4 message, STAT-6 phosphorylation, and Bcl-2 and Bcl-xL expression. We observed that jacalin blasts, in the absence of further stimulation with jacalin and in the presence of CD28, express IL-4 message (Fig. 3) as well as STAT-6 phosphorylation (Fig. 2B) and Bcl-2 and Bcl-xL expression (Fig. 4C) . We examined whether p38 MAPK plays an important role in the expression of IL-4, phosphorylation of STAT-6, and expression of Bcl-2 and Bcl-xL by using p38 MAPK inhibitor SB203580 (Fig. 5) . Our data suggest that p38 may play a role, and we propose that CD28-mediated signaling in jacalin blasts results in IL-4 synthesis, and p38 may be one of the molecules involved in this pathway.
Brown and co-workers [46 ] reported that CD28-deficient mice produce less antigen-specific IL-4 after in vivo immunization with keyhole limpet hemocyanin (KLH). CD28-deficient mice bred onto the BALB/c or C57BL/6 background and CD28-positive controls were immunized with KLH. The authors reported that in both genetic backgrounds, the absence of CD28 reproducibly resulted in a ten- to 20-fold decrease in IL-4 production upon antigen stimulation of draining lymph node cells compared with their CD28-positive controls (Fig. 1 ; ref. [46 ], page 805), in agreement with our findings that CD28 costimulation may be critical for IL-4 secretion. Their results suggest that Th2 responses were impaired more severely than Th1 responses. These authors also found differences in IL-4 production between C56BL/6 and BALB/c strains under similar conditions. Brown et al. [46 ] also concluded that signals that allow for efficient CD28-independent Th2 development in vivo cannot always be fully recapitulated in vitro. These authors concluded that genetic factors can influence the need for costimulation.
Zhu et al. [20 ] found that STAT-6 is necessary for IL-4-induced functions in CD4+ T cells and that STAT-6 is not only necessary but also sufficient for the IL-4 effects in Th2 differentiation and cell expansion. We have shown that in naïve T cells, stimulation with CD28 plus jacalin resulted in IL-4 secretion (S. M. L. Tamma et al., manuscript accepted for publication), whereas in jacalin-stimulated blasts (memory cells), CD28 stimulation alone resulted in p38 phosphorylation (Fig. 1) , IL-4 gene transcription (Fig. 3) , and STAT-6 activation (Fig. 4C) . To delineate whether p38 activation exerts its effect on Th2 cell differentiation predominantly by inducing IL-4 production [47 ], jacalin blasts were generated from resting T cells and were stimulated further with CD28 in the presence or absence of the p38 MAPK inhibitor SB203580. Our data suggest that p38 MAPK plays a role in IL-4 induction. However, IL-4 could not compensate for the inhibitory effects of SB203580 if added along with the inhibitor (data not shown).
We examined the levels of Bcl-2 and Bcl-xL proteins by immunoblotting in resting and activated T cells and found that Bcl-2 and Bcl-xL levels were increased in resting cells when they were stimulated with jacalin plus CD28XL with anti-CD28 antibody (Fig. 4A
and 4B)
, whereas in jacalin T cell blasts (memory cells), stimulation with CD28 or IL-4 (data not shown) was sufficient to induce up-regulation of Bcl-2 and Bcl-xL. Control cells failed to express either of these molecules. This continued high level of Bcl-2 and Bcl-xL in activated T cells is dependent on continued exposure to CD28 or IL-4, which provides apoptosis-suppressing signals in agreement with studies by Broome et al. [48
]. These authors [48
] suggested that Bcl-2 levels are probably important for maintaining the survival of resting T cells, whereas Bcl-xL may preferentially regulate the survival of activated T cells. Two of the IL-4 promoter regulatory elements, PRE-I and P1, are shown to confer CD28 stimulation-induced transactivation [7
]. Three independent, experimental approaches revealed that Vav/protein kinase C-
-derived signals selectively target the P1 and positive PRE-I elements contained within the hIL-4 promoter [7
]. It has been suggested from other studies [49
] that anti-CD28-induced T cell help is delivered via a CD40L-dependent process.
In summary, we have provided evidence that Th2 cytokines can be induced by signaling through CD28 in the absence of TCR ligation and hence, in an antigen-independent manner in human peripheral blood memory but not in naive T cells. Engagement of CD28 alone initiated IL-4 gene transcription in memory but not in naive T cells. Our studies also indicate that the activation of p38 MAPK may protect cells from apoptosis by inducing IL-4 production and up-regulation of antiapoptotic markers Bcl-2 and Bcl-xL, as assessed by inclusion of the p38 MAPK inhibitor in the culture. Previous studies also support that blocking the CD28-B7 interaction blocks the induction of IL-4 synthesis, indicating that CD28 costimulation is important in regulation of IL-4 synthesis [4 , 11 , 36 , 50 ]. TCR-independent generation of Th2 effectors might provide a mechanism to control Th1-dominated cellular inflammation [44 ]. We propose that jacalin plus CD28XL induces Th2 cytokine IL-4 via activation of p38 MAPK and may play a role in cell survival.
Received September 12, 2005; revised January 5, 2006; accepted February 11, 2006.
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RI expression through a STAT6-dependent mechanism J. Immunol. 161,6915-6923
B: direct association and synergistic activation of interleukin-4-induced transcription Mol. Cell. Biol. 18,3395-3404This article has been cited by other articles:
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