A role for the Tec family kinase ITK in regulating SEB induced Interleukin-2 production in vivo via c-jun phosphorylation
© Ragin et al; licensee BioMed Central Ltd. 2005
Received: 20 December 2004
Accepted: 22 July 2005
Published: 22 July 2005
Exposure to Staphylococcal Enterotoxin B (SEB), a bacterial superantigen secreted by the Gram-positive bacteria Staphyloccocus aureus, results in the expansion and eventual clonal deletion and anergy of Vβ8+ T cells, as well as massive cytokine release, including Interleukin-2 (IL-2). This IL-2 is rapidly secreted following exposure to SEB and may contribute to the symptoms seen following exposure to this bacterial toxin. The Tec family kinase ITK has been shown to be important for the production of IL-2 by T cells stimulated in vitro and may represent a good target for blocking the production of this cytokine in vivo. In order to determine if ITK represents such a target, mice lacking ITK were analyzed for their response to SEB exposure.
It was found that T cells from mice lacking ITK exhibited significantly reduced proliferative responses to SEB exposure in vitro, as well as in vivo. Examination of IL-2 production revealed that ITK null mice produced reduced levels of this cytokine in vitro, and more dramatically, in vivo. In vivo analysis of c-jun phosphorylation, previously shown to be critical for regulating IL-2 production, revealed that this pathway was specifically activated in SEB reactive Vβ8+ (but not non-reactive Vβ6+) T cells from WT mice, but not in Vβ8+ T cells from ITK null mice. However, toxicity analysis indicated that both WT and ITK null animals were similarly affected by SEB exposure.
These data show that ITK is required for IL-2 production induced by SEB in vivo, and may regulate signals leading IL-2 production, in part by regulating phosphorylation of c-jun. The data also suggest that perturbing T cell activation pathways leading to IL-2 does not necessarily lead to improved responses to SEB toxicity.
Superantigens (SAGs) are microbial toxins of bacterial and viral origin with the ability to activate 5–20% of the T cell population, causing T cell activation, cytokine release and systemic shock [1, 2]. Most SAGs share the ability to simultaneously bind the class II major histocompatibility complex molecules and the variable region of the T cell receptor β-chain, without the need to be processed by antigen presenting cells [1, 2]. Thus SEB can interact directly with MHC class II molecules on APCs and activate T cells bearing the proper TcR Vβ chains. The result of this interaction is large-scale stimulation of any T cell that expresses the proper TCR Vβ chain. A number of studies have shown that when mice are challenged with a SAG such as Staphylococcal Enterotoxin B (SEB), toxicity results from massive induction of cytokines derived from T-helper-type-1 (TH1) type cells such as IL-2, IFN-γ, TNF-α and TNF-β [1, 3]. This cytokine production is accompanied by expansion of the numbers of SEB reactive T cells, followed by cell death and the induction of functional "anergy" [4–6].
In order for a T cell to be activated antigen presenting cells (APC) must present antigen, either an antigenic peptide or SAG. In vitro, this interaction with the TcR has been shown to lead to the activation of a number of tyrosine kinases, including the Src family kinase Lck, the Syk family kinase Zap-70 and the Tec family kinase ITK (for review see [7–10]). This then results in the activation of a number of signaling pathways including members of the MAPK family of kinases, ERK, JNK and p38, followed by transcription factor activation . In vivo, activation of these T cells lead to the induction of cytokine secretion within hours of SEB exposure [1, 3].
ITK is expressed primarily in T cells, NK cells and mast cells [11–13]. In T cells, it is rapidly activated following TcR crosslinking in vitro [14, 15]. Mice lacking ITK exhibit reduced proliferation and IL-2 production in vitro, and reduced T cell differentiation in vitro and in vivo, with TH2 cell differentiation preferentially affected [15–19]. The observed reduced proliferation of ITK null T cells in vitro is IL-2 dependent as it could be rescued by the addition of exogenous IL-2 . However, these animals are not entirely immunocompromised, with residual responses against LCM, Vaccinia and VS viruses . We have tested whether ITK null mice are susceptible to SEB induced IL-2 secretion. We show here that mice lacking ITK have much reduced IL-2 production and T cell expansion in response to SEB in vitro and in vivo. We also show that SEB induced the activation of the JNK MAPK pathway in responding T cells in vivo, and that ITK null T cells were defective in the activation of this pathway in vivo. However, toxicity analysis indicated that both WT and ITK null animals were similarly affected by SEB exposure. Our data suggest that ITK is required for full IL-2 secretion following SEB exposure, and that this may be due to the regulation of the JNK pathway by ITK in vivo. However, reducing T cell signals does not necessarily lead to better physiological responses to SEB exposure.
ITK deficient T cells proliferate less efficiently than WT T cells in response to varying concentrations of SEB in vitro
ITK null T cells secrete less IL-2 in response to SEB in vitro
Reduced expansion of Vβ8+CD4+ population in ITK null animals in response to SEB exposure in vivo
Defective SEB induced IL-2 secretion in vivo in ITK null mice
Defective phosphorylation of c-jun induced by SEB in ITK null T cells
Similar toxicity of SEB on WT and ITK null mice
Effect of SEB on health and survival of WT and ITK null mice. Mice (WT or ITK null) were injected with the indicated amount of LPS, followed 4 hours later by 50 μg SEB, both delivered Ip. Alternatively, mice were injected with 20 mg. D-Gal and 50 μg SEB at the same time. All injections were delivered intraperitoneally. Mice were then monitored for the presence of ruffled fur, mucous in feces and lethargy. *mice had ruffled fur, mucous in feces and were lethargic. **mouse died over the course of the experiment. ***mouse had to be euthanized due to severity of sickness.
50 μg SEB + 150 μg LPS
50 μg SEB + 150 μg LPS
50 μg SEB + 20 mg D-Gal
50 μg SEB + 20 mg D-Gal
It is well established that exposure to SEB results in large scale cytokine production, which in part is responsible for symptoms of exposure to this toxin [1, 2, 35]. T cells have been shown to be largely responsible for this excessive cytokine production [35, 36]. Current suggestions for pharmacologically blocking the symptoms of SEB exposure include T cell signal transduction inhibitors such as CsA and Perfenidone, however, these agents have significant side effects [37, 38]. Here we present a promising target for inhibition IL-2 production induced by SEB exposure, the tyrosine kinase ITK. We show that mice lacking ITK have significantly reduced T cell expansion and IL-2 secretion upon exposure to SEB in vivo. Furthermore, we show that the SEB induced signaling pathway leading to c-jun phosphorylation, an indication of JNK pathway activation, is significantly reduced in ITK null T cells, although these T cells could respond to SEB activation by upregulating CD69, and there was no difference in the overall toxicity of SEB/LPS in these mice compared to WT mice.
Previous work performed in vitro suggests that ITK is required for the anti-TcR/CD3 antibody mediated activation of the transcription factor NFAT, via regulation of calcium influx into TcR stimulated T cells [19, 39]. ITK null T cells also exhibit reduced AP-1 DNA binding activity when stimulated in vitro with anti-CD3 antibodies . It has also been shown that JNK activation lies in part downstream of ITK following TcR crosslinking in vitro . However, these experiments were performed in vitro and it is not clear that effects seen in vitro would reflect what happens in vivo, since other cell-cell interactions may allow for activation of pathways that are not seen using in vitro activation with antibodies. Our data however, suggest that indeed, ITK lies downstream of the TcR and is required for IL-2 secretion and c-jun phosphorylation in vivo. Corroborating a role for c-jun activation in IL-2 production, mice carrying a dominant negative c-jun have much reduced IL-2 secretion in vitro  (mice lacking c-jun die during embryogenesis ). Similarly, JNK has been implicated in IL-2 secretion by T cells in vitro [25–27]. Our data lend support to this idea that the JNK-c-jun pathway is involved in the production of IL-2 by T cells in vivo.
Curiously, the immunosuppresant CsA has been shown to inhibit the effects of SEB exposure in mice if delivered prior to SEB exposure , however it does not inhibit SEB induced effects in monkeys if delivered at the same time as SEB . The effects of CsA on SEB induced symptoms has been suggested to be due to its effects on inhibiting IL-2 and other cytokine production due to inhibition of activation of the transcription factor NFAT [36, 41], however, CsA also inhibits activation of the JNK pathway following TcR/CD3 and CD28 stimulation [29, 30], and so CsA pretreatment may act to prevent early T cell activation of these pathways, thus blocking cytokine production and protecting mice from the effects of subsequent SEB exposure.
Mice lacking ITK exhibit reduced T cell responses in vitro, but also have reduced percentages of CD4+ T cells (approximately 60–70% of WT, ). While this would be expected to reduced the overall T cell response in vivo to SEB exposure, if these cells were able to respond to SEB, we would expect an equivalent reduction in the amount of IL-2 secreted in vivo in response to SEB. We would also expect to see an equivalent reduction in proliferation in vitro. However, in vivo, we observed much reduced IL-2 secretion, and reduced expansion of Vβ8+CD4+ T cells, although this was not as dramatic as the reduction in IL-2 secretion. Indeed, it has been observed that SEB induced T cell expansion in IL-2 null mice, indicating that IL-2 is not necessary for expansion of T cells in vivo following SEB exposure. However, even with the reduced T cell responses observed in ITK null mice, they still suffer from SEB/LPS induced toxicity similar to that seen in the WT mice. It should be noted however, that C57BL/6 mice are much less sensitive to SEB induce lethal shock than Balb/c mice . Thus we observed very few deaths in our experiments, and ITK may protect Balb/c mice from actual death. We are currently crossing these mice onto the Balb/c background to determine if this is the case.
In conclusion, we have show that ITK is required for IL-2 production induced by SEB in vivo and in vitro. We have also shown that ITK may regulate signals leading IL-2 production in part by regulating phosphorylation of c-jun. However, mice lacking ITK exhibit similar responses to SEB toxicity. The data suggest that the inability of T cell lacking ITK to produce IL-2 cannot be overcome by SAG stimulation, and that perturbing T cell activation pathways leading to IL-2 production does not necessarily lead to improved responses to SEB toxicity.
Wild type mice and ITK-deficient mice (C57BL/6 background, ) between 8 and 10 weeks were used in all experiments. RAG1-/- mice were a kind gift of Dr. Eric Harvill (Penn State University) and were similarly used between 6–10 weeks of age. The Institutional Animal Care and Use Committee of The Pennsylvania State University approved all experiments.
In vivo expansion assays
WT and ITK deficient mice were injected intraperitoneally (i.p.) with 50 μg SEB in PBS (Sigma-Aldrich, St. Louis, MO) and after 2 days, were sacrificed and splenocytes stained with antibodies specific for Vβ8 (SEB reactive T cells) or Vβ6 (non-reactive T cells) and CD4 directly conjugated to FITC and PE respectively (BDPharmingen, San Diego, CA). Alternatively, mice were injected i.p. with 50 μg SEB and eye-bled every 2 days for 5 days. Following lysis of red blood cells, the remaining cells were stained as described above for the splenocytes. Lymphocytes were identified by flow cytometry by their forward and side scatter characteristics.
We used a protocol reported by Zell et al to determine the phosphorylation status of c-jun, as a measure of activation of the JNK-c-jun pathway in SEB responding T cells. WT and ITK deficient mice were injected i.p. or intravenously (i.v.) with 50 μg SEB and allowed to survive for an 1 hr. Similar results were found with both routes of administration. Animals were sacrificed and spleens, lymph nodes, and blood rapidly isolated, and dounced in 2% paraformaldehyde to rapidly fix the cells as previously described . Cells were stained with antibodies to Vβ8 or Vβ6 directly conjugated to PE (BDPharmingen, San Diego, CA), then permeabilized with saponin. Cells were then stained for intracellular phosphorylated-c-jun with a monoclonal antibody against phosphorylated c-jun (IgG1, Cell Signaling, Beverly, MA), followed by biotinylated rabbit anti-mouse IgG1 and streptavidin conjugated to FITC. Controls included secondary reagents alone. Upregulation of CD69 by SEB in vivo was performed using similar approaches, except that T cells were not fixed or permeabilized prior to staining with PE-conjugated anti-CD69 monoclonal antibody (BDPharmingen, San Diego, CA). This was followed by analysis by flow cytometry, with post analysis performed using the WinMDI program 2.8 (The Scripps Research Institute, La Jolla, CA).
In vitro proliferation
Lymph nodes and spleens were isolated from WT and ITK deficient mice and pooled. Red blood cells were removed using ACK lysis buffer, and cells resuspended in complete RPMI. Cells were then left unstimulated or stimulated with varying concentrations of SEB (5 μg/ml, 0.5 μg/ml, 0.05 μg/ml, 0.005 μg/ml, and 0.0005 μg/ml) at a concentration of 2 × 106 cells/ml. These cells were incubated at 37°C for 5 days and pulsed once a day for 5 days with 0.5 μCi tritiated thymidine per well for 12 hrs. before harvest. T cells were purified from WT and ITK null mice using MiniMACS separation columns (Miltenyi Biotec Inc, Auburn, CA), and plated at 2 × 106 cells/ml and stimulated as above, in the presence of splenocytes from RAG null mice pre-treated with mitomycin C. All experiments were done in triplicate.
In vitro cytokine analysis
Pooled lymphocytes and splenocytes from WT and ITK deficient mice were incubated with varying concentrations of SEB (5 μg/ml, 0.5 μg/ml, 0.05 μg/ml, 0.005 μg/ml, and 0.0005 μg/ml) for 5 days as described above for in vitro proliferation. Supernatants were sampled once a day over this period and analyzed for IL-2 using ELISAs according to the manufacturer's recommendations (BDPharmingen, San Diego, CA).
In vivo cytokine analysis
WT and ITK deficient mice were injected with 50 μg SEB i.v., and serum isolated from cardiac blood from mice 1, 2, 4, 8, 12, and 24 hrs. post SEB exposure and used to perform an ELISA to determine IL-2 levels. PBS injected mice served as controls, and there was no change in IL-2 secretion in these animals.
Values were compared using student's t test and considered significant if p < 0.05.
List of abbreviations used
Interleukin-2 inducible T cell Kinase
Staphylococcal Enterotoxin B
T-helper 1 cells
T-helper 2 cells
Spleen tyrosine kinase
Zeta chain Associated protein-70
Lstra cell kinase
Mitogen Activated Protein Kinase
Extracellular signal Regulated Kinase
c-jun N-terminal kinase
Natural Killer cells
Phorbol Myristic Acid
Phosphate Buffered Saline
Nuclear Factor of Activated T cells
Activator protein 1
We thank members of the August lab and the Center for Molecular Immunology & Infectious Disease at Penn State for helpful comments and suggestions. We also thank Elaine Kunze and Susan Magargee in the Center for Quantitative Cell Analysis at Penn State for excellent technical help. We also thank Dr. Dan Littman (New York University Medical School, NY, NY) for kindly providing us with the ITK null mice.
This work was supported in part by a Johnson & Johnson Focused Giving Grant (to A.A.), the American Heart Association (0330036N), and Public Health Service Grants AI-51626 (to A.A.) and AI-46261 (to A.J.H.). MJR is a Ford Foundation Scholar.
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