Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0905512 on January 24, 2006

Published online before print January 24, 2006
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(Journal of Leukocyte Biology. 2006;79:876-880.)
© 2006 by Society for Leukocyte Biology

The lectin jacalin plus costimulation with anti-CD28 antibody induces phosphorylation of p38 MAPK and IL-4 synthesis-I

Seetha M. Lakshmi Tamma*,1, Satya Priya Balan*, Ken Wook Chung* and Savita Pahwa{dagger}

* Department of Biomedical Sciences, C. W. Post Campus, Long Island University, Brookville, New York; and
{dagger} University of Miami, School of Medicine, Microbiology and Immunology, Florida

1Correspondence: Department of Biomedical Sciences, C. W. Post Campus, Long Island University, Brookville, NY 11548. E-mail: stamma{at}liu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Costimulatory signals play an important role in the development of T helper cell type 1 (Th1) or Th2 type. Little is known about jacalin plus CD28-mediated signaling and cytokine secretion. In the present study, we analyzed the intracellular signaling events following stimulation of CD4+ T cells with jacalin plus CD28 cross-linking (CD28XL) with anti-CD28 antibody. Our results indicate enhanced phosphorylation of Tec and linker for activation of T cells when compared with stimulation with jacalin alone or CD28XL alone. Stimulation with jacalin or CD28XL appears to be insufficient to induce interleukin (IL)-4 secretion; however, CD28XL followed by stimulation with jacalin resulted in enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and increased secretion of IL-4. However, compared with stimulation with phorbol 12-myristate 13-acetate plus ionomycin, jacalin plus CD28XL resulted in decreased levels of tumor necrosis factor {alpha} secretion. Addition of p38 inhibitor, SB203580, inhibited p38 phosphorylation and IL-4 secretion. These data suggest that jacalin stimulation alone appears to be insufficient for Th2 development, and addition of CD28 costimulation induced Th2 generation. We propose that jacalin plus CD28XL induces Th2 differentiation via activation of p38 MAPK.

Key Words: Th1 • CD28XL • PMA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on their distinctive cytokine secretion pattern and effector functions, human CD4+ T cells can be divided into at least two different subsets. T helper cell type 1 (Th1) cells mediate cellular immunity by secreting the proinflammatory cytokines such as interferon-{gamma} (IFN-{gamma}) and interleukin (IL)-2 and promoting macrophage activation. Th2 cells, in contrast, down-modulate macrophage effector functions by secreting the anti-inflammatory cytokines IL-4, IL-5, and IL-13 and also facilitate immunoglobulin E (IgE) secretion [1 , 2 ]. Two distinct signals are required for optimal T cell activation, one of which is transduced through the polymorphic T cell receptor (TCR), and extensive studies have demonstrated that the glycoprotein CD28 is one of the major costimulatory molecules involved in T cell activation [3 ]. 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 [4 5 6 ], and regulates the expression of Bcl-xL [7 ] and CD154 (CD40 ligand) [8 ], all of which contribute to successful progression of T cell responses.

CD28 engagement has been shown to induce phosphorylation of intracellular substrates such as phosphatidylinositol-3 kinase (PI-3K) [9 ] and induce activation of the mitogen-activated protein kinase (MAPK) cascades [10 ]. It has been shown that CD28 costimulation may promote the production of Th2 cytokines directly [11 ] in mice [12 13 14 15 ] and humans [16 17 18 ]. IL-4 is a pleiotropic cytokine with a wide rage of biological effects on many hematopoietic and nonhematopoietically derived cells and tissues; IL-4 is also a well-known cytokine, which plays a central role in the pathogenesis of allergic inflammation by promoting Ig class switching to IgE [19 ].

Purified, naive human T cells stimulated with {alpha}-CD3 in the absence of CD28 costimulation produce only IL-2 and IFN-{gamma}, whereas the addition of {alpha}-CD28 monoclonal antibodies (mAb) induced IL-4 [16 ]. The Tec protein tyrosine kinase (PTK) family includes Btk, Itk/Tsk/Emt, Tec, Rlk/Txk, and Bmx, which are involved in signals mediated by various surface receptors. It has been shown previously [20 ] that Tec is involved in T cell signaling in a way distinct from Itk. The data from these studies suggest that CD28 activates Tec via Src family PTK, and transcription of the IL-4 promoter is enhanced by overexpression of wild-type Tec but inhibited by overexpression of a kinase-dead version of Tec following CD28 activation. These results imply that Tec can modulate transcription of Th1 and Th2 cytokines in a kinase-dependent manner. Furthermore, it has been shown that overexpression of kinase-dead Lck can block Tec-induced cytokine expression following CD28 ligation [21 ]. Up-regulation of the IL-4 production by CD28 signaling may be one of the mechanisms that promotes Th2 differentiation. Given the significance of CD28 in T cell activation and differentiation, analysis of the signaling pathways involved in transducing CD28-generated signals in the presence and absence of the CD4+ T cell mitogen jacalin might provide insights into the understanding of molecular mechanisms involved in T cell activation and, importantly, in regulating Th2 cell differentiation in an antigen-independent manner. We analyzed the intracellular signaling events following stimulation of T cells with jacalin plus CD28 cross-linking (CD28XL) with anti-CD28 antibody and have shown that CD28-mediated signaling induces p38 and IL-4 secretion, thus in an antigen-independent mechanism. Our study suggests that in the presence of CD28-induced costimulation, jacalin induces secretion of IL-4.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
The following reagents were used: anti-CD3 antibody and anti-CD28 antibody from BD PharMingen (San Jose, CA); antiphosphotyrosine (4G10), anti-linker for activation of T cells (LAT), anti-Tec, anti-Vav, phosphor-extracellular signal-regulated kinase, and phosphor-Jun N-terminal kinase (JNK) antibodies (Upstate Biotechnology, Lake Placid, NY); sodium dodecyl sulfate (SDS) gradient gels and polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA); jacalin (E-Y Labs, San Mateo, CA); phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO); ionomycin (Calbiochem, La Jolla, CA); phospho-p38 antibody (Cell Signaling Technologies, Beverly, MA); IL-4 kit (Biosource International, Camarillo, CA); reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Ambion, Austin, TX); Sybergreen (Molecular Probes, Eugene, OR; and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolim bromide (MTT) assay kit (Chemicon International, Temecula, CA).

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), 10% fetal calf serum, and recombinant human IL-2 (10 U/ml).

Cell culture
PMA (50 ng/1x106 cells) + ionomycin (400 ng/1x106cells), anti-CD28 mAb (5 µg/1x106 cells), and jacalin (50 µg/1x106 cells) were used to stimulate the cells.

Cell activation and lysis
For signal transduction studies, CD4+ T cells were washed and stimulated with PMA (50 ng/106 cells) + ionomycin (400 ng/106 cells) as a positive control for T cell activation and tyrosine phosphorylation of various kinases. For stimulation with CD28 plus jacalin, cells were washed and incubated with anti-CD28 antibody for 30 min at 4°C on a rotator, 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. Where indicated, cells were pretreated with p38 MAPK (SB203580) 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). Reactions were terminated by washing cells in ice-cold phosphate-buffered saline + EDTA + sodium orthovanadate and lysed 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 rotations per minute (RPM) at 4°C. Supernatants were aliquoted and stored at –80°C until ready to use.

Immunoprecipitation and immunoblotting
Cell lysates were precleared with protein A-agarose and were incubated overnight at 4°C with appropriate antibodies, as indicated in figure legends, on a rotator, mixed with second antibody-coated protein A-agarose beads and allowed to precipitate at 4°C on a rotator overnight. The immunoprecipitates were washed extensively with lysis buffer, and pellets were boiled with sample buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) using 4–20% gradient gels (Bio-Rad), followed by immunoblotting with specific antibody or with antiphosphotyrosine antibody (4G10), followed by alkaline phosphatase-conjugated secondary antibody, followed by bromo-4-chloro-3-indolylphosphate p-toluidine salt-nitroblue tetrazolium chloride or a peroxidase-conjugated secondary antibody, and developed by an enhanced chemiluminescence system.

RNA isolation and RT-PCR analysis of IL-4 mRNA
Cells were stimulated for 24 h; total cellular RNA extraction and RT-PCR were performed according to the manufacturer’s instructions (Ambion). RNA was isolated and subsequently subjected to RT-PCR analysis using primers specific for the human IL-4 and the human ß-actin cDNA as an internal control. cDNA samples were isolated on agarose gels visualized by SyberGreen and photographed.

Enzyme-linked immunosorbent assay
Cells treated as described above were cultured for 48 h, and culture supernatants were harvested and analyzed for the cytokines IL-4 and tumor necrosis factor {alpha} (TNF-{alpha}) using commercial kits from Biosource International, according to the manufacturer’s instructions.

Lymphoproliferation assay
Cells stimulated with anti-CD3 antibody, PMA + ionomycin, CD28 plus jacalin, and jacalin or CD28 alone were cultured for 3 days, and proliferation was assessed by a MTT assay kit, according to the manufacturer’s instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD28XL induces phosphorylation of LAT and Tec
Cells in medium alone were used as negative control, and cells stimulated with PMA plus ionomycin were used as positive controls. CD4+ T cells (10x106) were incubated with anti-CD28 antibody (5 µg/106 cells) for 30 min at 4°C on a rotator, cells were brought to room temperature, jacalin (50 µg/ml) was added to the cells, and cells were incubated at 37°C for 10 min. Cells were transferred quickly to ice and washed with ice-cold washing buffer. Cell pellets were lysed for 30 min on ice with lysis buffer, and lysed samples were microcentrifuged for 20 min at 14,000 RPM at 4°C. Supernatants were immunoprecipitated with appropriate antibodies and immunoblotted as indicated (Fig. 1 ). Data from one representative experiment of at least three independent experiments are shown. The data indicate that CD28 plus jacalin induced phosphorylation of LAT (Fig. 1A) and Tec (Fig. 1B) compared with stimulation with jacalin alone or CD28 alone.


Figure 1
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  		Figure 1. (A) CD28XL induces phosphorylation of LAT and Tec. Lane 1, Medium control; lane 2, PMA + ionomycin; lane 3, CD28; lane 4, CD28 + jacalin; lane 5, jacalin. Upper panel was probed with antiphosphotyrosine antibody (Anti-Pty); lower panel with LAT antibody. Cells in medium alone were used as negative control (lane 1), and cells stimulated with PMA plus ionomycin (lane 2) were used as positive controls. CD4+ T cells (10x106) were incubated with anti-CD28 antibody (5 µg/106 cells) for 30 min at 4°C on a rotator, cells were brought to room temperature, jacalin (50 µg/ml) and cells were incubated at 37°C, washed, and lysed, and lysates were immunoprecipitated with anti-LAT antibodies and immunoblotted as indicated (lane 4). Data from one representative experiment of at least three independent experiments are shown. The data indicate that CD28 plus jacalin induced phosphorylation of LAT when compared with stimulation with CD28 (lane 3) or with jacalin alone (lane 5). (B) CD28XL induces phosphorylation of Tec. Lane 1, Medium control; lane 2, PMA + ionomycin; lane 3, jacalin; lane 4, jacalin; lane 5, CD28 + jacalin. Upper panel was probed with antiphosphotyrosine antibody; lower panel with Tec antibody. Cells in medium alone were used as negative control (lane 1), and cells stimulated with PMA plus ionomycin (lane 2) were used as positive controls. CD4+ T cells (10x106) were incubated with anti-CD28 antibody (5 µg/106 cells) for 30 min at 4°C on a rotator, cells were brought to room temperature, jacalin (50 µg/ml) and cells were incubated at 37°C, washed, and lysed, and lysates were immunoprecipitated with anti-Tec antibodies and immunoblotted as indicated (lane 5). Data from one representative experiment of at least three independent experiments are shown. The data indicate that CD28 plus jacalin induced phosphorylation of Tec when compared with stimulation with jacalin (lane 3) or with CD28 alone (lane 4).

 
CD28XL induces phosphorylation of p38
Cells in medium alone were used as negative control, and cells stimulated with PMA plus ionomycin were used as positive controls. CD4+ T cells (10x106) were incubated with anti-CD28 antibody (5 µg/106 cells) for 30 min at 4°C on a rotator, cells were brought to room temperature, jacalin (50 µg/ml) was added, and cells were incubated at 37°C for different times (Fig. 2A ). Cells were transferred quickly to ice and washed with ice-cold washing buffer, cell pellets were lysed for 30 min on ice with lysis buffer, lysed samples were microcentrifuged for 20 min at 14,000 RPM at 4°C, and supernatants were separated by PAGE and probed with phospho-p38 antibody or p38 antibody (Fig. 2A) . Data are shown from one representative experiment of at least three independent experiments. The data indicate that phosphorylation of p38 peaked at 10 min (Fig. 2A) , and CD28 plus jacalin induced phosphorylation of p38 (Fig. 2B) compared with stimulation with jacalin alone or CD28 alone. Incubation of cells with p38 MAPK inhibitor (SB203580, 400 nM) prior to stimulation inhibited phosphorylation of p38 (Fig. 2B , lane 7).


Figure 2
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  		Figure 2. CD28XL induces phosphorylation of p38. CD4+ T cells (10x106) were incubated with anti-CD28 antibody (5 µg/106 cells) for 30 min at 4°C on a rotator, cells were brought to room temperature, and jacalin (50 µg/ml) and cells were incubated at 37°C for different times: 0, 10, 30, 45, and 60 min (A). Upper panel, Probing with control p38; lower panel, probing with phosphor-p38 antibody. Data are from one representative experiment of at least three independent experiments. The data indicate that phosphorylation of p38 peaked at 10 min (A), and CD28 plus jacalin induced phosphorylation of p38 (B, lane 5) compared with stimulation with jacalin (B, lane 6) alone or CD28 alone (B, lane 4). Incubation of cells with p38 MAPK inhibitor (SB203580, 400 nM) prior to stimulation inhibited phosphorylation of p38 (B, lane 7).

 
CD28XL induces IL-4 mRNA synthesis
Cells were stimulated for 24 h. Total cellular RNA extraction and RT-PCR were performed according to the manufacturer’s instructions (Ambion). RNA was isolated and subsequently subjected to RT-PCR analysis using primers specific for the human IL-4 and the human-actin cDNA as an internal control. cDNA samples were isolated on agarose gels visualized by SyberGreen and photographed (Fig. 3 ). CD28XL induced transcription of IL-4 as assessed by RT-PCR and is comparable with that induced by PMA plus ionomycin. Actinomycin D as well as p38 MAPK inhibitor SB203580 inhibited CD28 plus a jacalin-induced increase of IL-4 mRNA completely (data not shown).


Figure 3
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  		Figure 3. CD28XL induces IL-4 mRNA synthesis. Cells were stimulated for 24 h. Total cellular RNA extraction and RT-PCR were performed according to the manufacturer’s instructions (Ambion). RNA was isolated and subsequently subjected to RT-PCR analysis using primers specific for the human IL-4 and the human-actin cDNA as an internal control. cDNA samples were isolated on agarose gels visualized by SyberGreen and photographed. CD28XL induced transcription of IL-4 as assessed by RT-PCR and is comparable with that induced by PMA plus ionomycin.

 
CD28XL induces IL-4 secretion
Cells treated as described above were cultured for 48 h, and culture supernatants were harvested and analyzed for IL-4 using a commercial kit from Biosource International, according to the manufacturer’s instructions. There is increased IL-4 secretion following stimulation with CD28 plus jacalin when compared with treatment with jacalin alone (Fig. 4 ). Incubation of cells with p38 MAPK inhibitor (SB203580, 400 nM) prior to stimulation was performed to assess the role of p38 in IL-4 production. The data suggest that prior treatment with inhibitor decreased IL-4 secretion (Fig. 4) .


Figure 4
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  		Figure 4. CD28XL induces IL-4 secretion. Cells treated as described above were cultured for 48 h, and culture supernatants were harvested and analyzed for IL-4 using a commercial kit from Biosource International, according to the manufacturer’s instructions. There is increased IL-4 secretion following stimulation with CD28 plus jacalin when compared with treatment with jacalin alone. Incubation of cells with p38 MAPK inhibitor (SB203580, 400 nM) prior to stimulation was performed to assess the role of p38 in IL-4 production. The data suggest that prior treatment with inhibitor decreased IL-4 secretion.

 
CD28XL induces decreased levels of TNF-{alpha} secretion
Cells treated as described above were cultured for 48 h, and culture supernatants were harvested and analyzed for TNF-{alpha} using a commercial kit from Biosource International, according to the manufacturer’s instructions. There is decreased TNF-{alpha} secretion following stimulation with CD28 plus jacalin when compared with treatment with PMA plus ionomycin (Fig. 5 ).


Figure 5
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  		Figure 5. CD28XL induces decreased levels of TNF-{alpha} secretion. Cells treated as described above were cultured for 48 h, and culture supernatants were harvested and analyzed for TNF-{alpha} using a commercial kit from Biosource International, according to the manufacturer’s instructions. There is decreased TNF-{alpha} secretion following stimulation with CD28 plus jacalin when compared with treatment with PMA plus ionomycin.

 
Stimulation was consistent to analyze p38 phosphorylation. Culture conditions to analyze IL-4 and TNF-{alpha} secretions were also similar. The data show a positive relationship between enhanced phosphorylation of p38 (Fig. 2) , increased IL-4 gene transcription (Fig. 3) , and increased production of IL-4 (Fig. 4) . However, we did not observe a positive relationship between p38 phosphorylation and TNF-{alpha} secretion. IL-4 secretion and TNF-{alpha} secretion in jacalin plus CD28 costimulation were compared with the positive control treatment of PMA plus ionomycin, where we see an increased secretion of IL-4 and TNF-{alpha} by PMA + ionomycin treatment. Our culture system appears to favor IL-4 production.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In recent years, experimental evidence showed that signal transduction through CD28 not only augmented TCR-mediated proliferation, IL-2 production, and T cell survival but also played an important role in differentiation of Th cells into type 1 or type 2 cells [18 , 10 ]. CD28 costimulation has been shown to promote production of Th2 cytokines including IL-4 [11 ]. Blocking the CD28-B7 interaction blocks the induction of IL-4 synthesis, indicating that CD28 costimulation is important in regulation of IL-4 synthesis [12 , 13 , 22 ]. In the current study, we have shown that jacalin treatment alone or CD28 treatment alone failed to induce IL-4 synthesis in CD4+ T cells. However, when cells were stimulated with jacalin in the presence of anti-CD28 antibody, there is enhanced phosphorylation of MAPK family member p38 and IL-4 secretion. In the current study, we have also analyzed phosphorylation of LAT and Tec following stimulation with CD28 plus jacalin and found that there is enhanced phosphorylation. Previously, it has been shown by others that CD3/CD28-induced IL-4 transcription was inhibited upon coexpression of dominant-negative forms of Vav, the adaptor proteins LAT and SLP76, and protein kianse C {theta} [23 ]. Yang et al. [20 ] have shown that CD28 activates Tec via Src family PTK, and transcription of the IL-4 promoter is enhanced by overexpression of wild-type Tec but inhibited by overexpression of a kinase-dead version of Tec following CD28 activation. These results imply that Tec can modulate transcription of Th1 and Th2 cytokines in a kinase-dependent manner. Consistent with the hypothesis postulated above that Lck can regulate Tec activation, overexpression of kinase-dead Lck can block Tec-induced cytokine expression following CD28 ligation [21 ]. Our findings are in agreement with previous observations by Zhang et al. [24 ] and Schafer et al. [25 ] that CD28 costimulation induces a shift toward Th2 phenotype. p38 can be activated by multiple stimuli. Ricon and co-workers [26 ] showed that "specific inhibitors of p38 MAP kinase block the production of IFN-{gamma} by Th1 cells without affecting the IL-4 production by Th2 cells." These authors also report that "activation of p38 MAP kinase kinase 6 in transgenic mice caused increased production of IFN-{gamma}." It is true that these authors found the effect of p38 on IFN-{gamma} production in contrast to our findings. These discrepancies could be related to experimental design and cell types among others. We used antigen-independent lectin stimulation in normal CD4+ T cells. As jacalin itself failed to induce IL-4 production, the goal of our study was to examine the role of CD28 as a costimulatory molecule in lectin-induced IL-4 production. Future studies may focus on analyzing other cytokines to understand the molecular mechanisms.

In summary, we have provided evidence that Th2 cell differentiation can be induced by signaling through CD28 in the absence of TCR ligation and hence, in an antigen-independent manner. Engagement of CD28 in the presence of jacalin initiated IL-4 gene transcription in T cells and activated the PI-3K, the JNK/stress-activated protein kinase (data not shown), and the p38 MAPK pathways. CD28-induced Th2 cell differentiation could be dependent on IL-4 and activation of the MAPK p38, as the p38 MAPK inhibitor down-modulated IL-4 secretion. As CD28, along with jacalin, was sufficient to provide the signals required for antigen-independent Th2 cell differentiation, CD28-mediated Th2 cell generation might provide a mechanism to understand cellular immunity by generating activated Th2 effector cells.


   ACKNOWLEDGEMENTS
 
A grant from C. W. Post Campus Research Committee of Long Island University (Brookville, NY) supported this work. We acknowledge Dr. Thoedora Grauer, Dean, and Ron Modesto, Chairman, Department of Biomedical Sciences, School of Health Professions and Nursing, C. W. Post Campus, for providing additional monetary support and Mr. Paul Dominguez, Laboratory Manager, Department of Biomedical Sciences, for helping with materials and equipment. Ms. Susan Bodak, Department of Biomedical Sciences, and Ms. Louise Miller, Nutrition Department, School of Health Professions and Nursing, C. W. Post Campus, also deserve recognition for their help.

Received September 12, 2005; revised November 16, 2005; accepted November 25, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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