Collagen type I-mediated activation of ERK/MAP Kinase is dependent on Ras, Raf-1 and protein phosphatase 2A in Jurkat T cells

Growing evidence indicates that interactions of T cells with extracellular matrix through beta1 integrins are important for the regulation of T cell-mediated immune responses and diseases. In this regard, we have recently demonstrated that collagen I (Coll I) through alpha2beta1 integrin inhibited Fas-induced apoptosis of T cells by activating a protein phosphatase 2A (PP2A)-dependent ERK/MAP Kinase pathway. As survival of T cells is critical for their functions, we further investigated the mechanisms underlying the activation of this pathway. Inhibition studies demonstrated that Coll I activates the ERK/MAP Kinase pathway in Jurkat T cells through the activation of Ras and Raf-1. Activation of PP2A was not necessary for the binding of Coll I to Jurkat T cells, but is required for the activation of Raf-1. In accordance, activation of Ras, Raf-1 and PP2A were also required for the ability of Coll I to protect Jurkat T cells from Fas-induced apoptosis. In contrast and despite its capacity to activate Ras, fibronectin (Fbn) failed to activate PP2A and Raf-1. These results might explain, at least in part, the weak ability of Fbn to activate ERK in T cells, supporting thus the differential signaling of beta1 integrin members in these cells. This study provides novel insights into the mechanisms by which beta1 integrins activate the ERK/MAP Kinase pathway in T cells, and is the first report to provide a role for PP2A in integrin-mediated ERK/MAP Kinase activation.     

Collagen type I signaling reduces the expression and the function of human receptor activator of nuclear factor-kappa B ligand (RANKL) in T lymphocytes

The mechanisms by which beta1 integrins modulate T cell functions are still poorly defined. We have previously reported that signaling via the collagen type I (Coll I) receptor, alpha2beta1 integrin, inhibited FasL expression and protected Jurkat T cells from activation-induced cell death (AICD). In this study, we examined whether Coll I signaling in T cells also modulates the expression of the human receptor activator of nuclear factor-kappaB ligand (RANKL), a recently identified TNF family member which has important functions in osteoclastogenesis, cell survival and apoptosis. Our results show that in both Jurkat T cells and human primary T cells, Coll I signaling significantly reduces activation-induced RANKL expression by 50-60%. We also found that RANKL is not involved in AICD but participates in doxorubicin-induced apoptosis of leukemia T cell lines including Jurkat, CEM and HSB-2. In this respect, Coll I protected leukemia T cell lines from doxorubicin-induced apoptosis by inhibiting doxorubicin-induced RANKL expression. Together, our results suggest that by limiting the production of RANKL, Coll I signaling may contribute to the resistance of leukemia T cells to chemotherapy. Our study also emphasizes the importance Coll I signaling may have in the control of RANKL-associated T cell functions.     

Complement regulatory protein Crry/p65-mediated signaling in T lymphocytes: role of its cytoplasmic domain and partitioning into lipid rafts

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I-related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR-dependent activation signals, including p56lck-, zeta-associated protein-70 (ZAP-70), Vav-1, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation but also costimulation-dependent mitogen-activated protein kinases (MAPK), such as the stress-activated c-Jun N-terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent-insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3epsilon, and p59-60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol-3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin-4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP-70, Akt, Vav-1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.     

CD46-mediated costimulation induces a Th1-biased response and enhances early TCR/CD3 signaling in human CD4+ T lymphocytes

The role of membrane cofactor protein (MCP, CD46) on human T cell activation has been analyzed. Coligation of CD3 and CD46 in the presence of PMA or CD28 costimuli enhanced IL-2, IFN-gamma, or IL-10 secretion by CD4+ T lymphocytes. The effect of CD46 on IL-10 secretion did not require additional costimuli like anti-CD28 antibodies or phorbol esters. CD46 also enhanced IL-2 or IFN-gamma secretion by CD4+ blasts. In contrast, IL-5 secretion was inhibited upon CD46-CD3 coligation, in all the cells analyzed. These effects were independent of IL-12 and suggest that CD46 costimulation promotes a Th1-biased response in human CD4+ T lymphocytes. CD46 enhanced TCR/CD3-induced tyrosine phosphorylation of CD3zeta and ZAP-70, as well as the activation of the ERK, JNK, and p38, but did not modify intracellular calcium. The effect of specific inhibitors shows that enhanced ERK activation contributes to augmented IFN-gamma and lower IL-5 secretion and, consequently, to the Th1 bias. Cross-linking CD46 alone induced weak tyrosine phosphorylation of CD3zeta and ZAP-70. However, CD46 cross-linking by itself did not induce cell proliferation or lymphokine secretion, and pretreatment of CD4+ T lymphocytes with anti-CD46 antibodies did not significantly alter TCR/CD3 activation.     

GADD45gamma mediates the activation of the p38 and JNK MAP kinase pathways and cytokine production in effector TH1 cells

The p38 and JNK stress-activated MAPK signal transduction pathways are activated by T cell receptor (TCR) signaling and are required for IFN-gamma production by TH1 effector cells. Here, we show that the expression of GADD45gamma is induced during T cell activation and that the level of expression is higher in TH1 cells than in TH2 cells. TH1 cells from GADD45gamma(-/-) mice are severely compromised in their abilities to activate p38 and JNK in response to TCR signaling, produce much less IFN-gamma upon restimulation, and are deficient in activation-induced cell death (AICD). Additionally, GADD45gamma deficiencies caused reduced contact hypersensitivity in mice. Thus, GADD45gamma mediates activation of the p38 and JNK pathways and effector function of TH1 cells.     

Notch 1 signaling regulates peripheral T cell activation

Notch signaling has been identified as an important regulator of leukocyte differentiation and thymic maturation. Less is known about the role of Notch signaling in regulating mature T cells. We examined the role of Notch 1 in regulating peripheral T cell activity in vitro and in vivo. Coligation of Notch 1 together with TCR and CD28 resulted in a dramatic inhibition of T cell activation, proliferation, and cytokine production. This effect was dependent on presenilin activity and induced the expression of HES-1, suggestive of Notch 1 signaling. Biochemical analysis demonstrated an inhibition of AKT and GSK3beta phosphorylation following Notch 1 engagement while other biochemical signals such as TCR and ERK phosphorylation remained intact. Similar effects were observed in vivo in an adoptive transfer model. Therefore, Notch 1 signaling may play an important role in regulating naive T cell activation and homeostasis.     

Activation of the extracellular signal-regulated kinase pathway is differentially required for TCR-stimulated production of six cytokines in primary T lymphocytes

The extracellular signal-regulated kinase (ERK) signaling pathway is strongly activated in response to TCR stimulation in normal T cells. However, the extent to which activation of the ERK pathway is necessary for TCR-stimulated cytokine production is not clear. We have addressed this question by use of two separate methods to interfere with TCR activation of the ERK cascade. The first approach utilized transient expression of a catalytically inactive form of mitogen-activated/ERK 1 (CI-MEK1), while the second involved using the MEK1- and MEK2-specific inhibitor PD98059 to block ERK activation by the TCR. In order to assess the requirement for ERK activation in T cell cytokine production, we have measured the effect of ERK inhibition upon the production of six cytokines, IL-3, IL-4, IL-5, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IFN-gamma, by newly activated normal mouse T cells in response to TCR stimulation. The results of experiments using both methods to block ERK activation have revealed a requirement for intact ERK signaling for the full elicitation of TCR-stimulated cytokine production. Dose-response analyses using the MEK inhibitor PD98059 showed that the TCR-stimulated production of all cytokines measured was affected by this treatment. However, the production of IL-3 and IL-4 was only partially dependent upon ERK activation, whereas IL-5, IL-10, IFN-gamma and GM-CSF production was severely affected by diminished ERK activation. We conclude that the ERK pathway is differentially involved in the activation of different cytokine genes in normal T cells.      

T cell costimulation via the integrin VLA-4 inhibits the actin-dependent centralization of signaling microclusters containing the adaptor SLP-76

Antigen-dependent T cell activation drives the formation of signaling microclusters containing the adaptor SLP-76. Costimulatory integrins regulate SLP-76 phosphorylation and could influence SLP-76 microclusters in the integrin-rich periphery of the immune synapse. We report that costimulation by the integrin VLA-4 (alpha4beta1) required SLP-76 domains implicated in microcluster assembly. Pro-adhesive ligands enlarged the contact and increased the number of SLP-76 microclusters regardless of their costimulatory potential. Costimulatory VLA-4 ligands also prevented the centralization of SLP-76, promoted microcluster persistence, prolonged lateral interactions between SLP-76 and its upstream kinase, ZAP-70, and retained SLP-76 in tyrosine-phosphorylated peripheral structures. SLP-76 centralization was driven by dynamic actin polymerization and was correlated with inward actin flows. VLA-4 ligation retarded these flows, even in the absence of SLP-76. These data suggest a widely applicable model of costimulation, in which integrins promote sustained signaling by attenuating cytoskeletal movements that drive the centralization and inactivation of SLP-76 microclusters.     

Beta 1/beta 3 integrin ligation is uncoupled from ERK1/ERK2 activation in cytotoxic T lymphocytes

beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize with very low levels of CD3 stimulation to activate the extracellular signal-regulated kinase (ERK)1 and ERK2 MAP kinases but that fibronectin alone induces no detectable MAP kinase activation in CTL. Surprisingly, antibodies to beta1 or beta 3 integrins were also unable to stimulate MAP kinase activation, suggesting that although beta 1 integrins are capable of stimulating MAP kinase activation in other cells, they cannot do so in CTL. In CTL, phosphorylation of proline-rich tyrosine kinase 2 downstream of integrin stimulation did not result in recruitment of the adaptor protein Grb2. Additionally, we examined the role of MAP kinases in regulating integrin-mediated adhesion. Anti-CD3-triggered adhesion to fibronectin was largely insensitive to the MAP kinase kinase inhibitor PD98059. Triggered cell-spreading on fibronectin was inhibited by PD98059 but not by U0126. In summary, ligation of beta 3 integrin by antibodies or fibronectin or of beta1 integrin by monoclonal antibodies fails to activate ERK MAP kinases, but integrin ligation synergizes with T cell receptor stimulation upstream of MAP kinases.     

Pyk2 is differentially regulated by β 1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

β₁ integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by β₁ integrin signaling in human T cells. Stimulation of Jurkat T cells with the α₄β₁ integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and α₄β₁ integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4⁺ T cells confirmed the ability of CD3-derived signals to synergize with β₁ integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell costimulation mediated specifically by β₁ integrins.     

Importance of MAPK pathways for microglial pro-inflammatory cytokine IL-1 beta production

In Alzheimer's disease (AD), chronically activated glia contribute to neuronal dysfunction through production of neuroinflammatory molecules like interleukin (IL)-1beta. As a first step to address the signaling pathways important for pro-inflammatory cytokine induction, and whether different activators use distinct pathways, we tested the involvement of mitogen-activated protein kinase (MAPK) pathways in microglial IL-1beta production. Microglial cultures stimulated with lipopolysaccharide, S100B, or beta-amyloid showed rapid activation of three different MAPKs (p38, ERK1/2, and JNK) and a later increase in IL-1beta levels, consistent with a possible mechanistic relationship between MAPK and IL-1beta. To more directly test this possibility, we stimulated microglia in the presence of selective MAPK inhibitors, and found that inhibition of each of the three MAPK pathways inhibited IL-1beta production in a concentration-dependent manner. In addition, the relative importance of each MAPK to IL-1beta production depended on the activating stimulus. These data demonstrate that MAPK pathways are important for microglial IL-1beta production, and suggest that different glial activators use distinct sets of signaling pathways to induce the same disease-relevant end-point in microglia.     

p38 MAPK is a critical regulator of the constitutive and the beta4 integrin-regulated expression of IL-6 in human normal thymic epithelial cells

Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor alpha6beta4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-kappaB and NF-IL6 transactivators. Here we show that alpha6beta4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, beta4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase 1 (PAK1) and MAPK kinases (MKK) 3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and beta4 integrin-induced production of IL-6 preventing NF-kappaB and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-kappaB activation. Overall, our results indicate that p38 MAPK and alpha6beta4 integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses.     

Increased expression of collagen receptors: α1β1 and α2β1 integrins on blood eosinophils in bronchial asthma

BACKGROUND: Eosinophils are one of the major effector cells in bronchial asthma. Their infiltration of airways correlates with the asthma severity. Recruitment and activation of eosinophils are partially mediated by integrins alpha4beta1 and alpha4beta7. Collagens type I and IV constitute important components of extracellular matrix and vascular basement membrane, respectively. Therefore, collagen-binding integrins (alpha1beta1 and alpha2beta1) may also play a role in eosinophil lung infiltration. OBJECTIVE: To evaluate the possible presence of alpha1beta1 and alpha2beta1 integrins on peripheral blood eosinophils from asthmatic subjects. METHODS: Collagen receptors were studied on eosinophils separated by immunomagnetic CD16-negative method from healthy donors (n=13) and patients with moderate persistent atopic bronchial asthma (n=15). Surface receptor identification was performed by flow cytometry and cell adhesion assay. RESULTS: Eosinophils isolated from the patients showed increased expression of both alpha1beta1 and alpha2beta1 integrins as compared with healthy controls. Moreover, adhesive function of eosinophils to collagen type IV was inhibited by snake venom disintegrins: viperistatin and obtustatin. These disintegrins contain KTS active motif and are specific inhibitors of alpha1beta1 integrin. CONCLUSION: We demonstrated for the first time that collagen receptors: alpha1beta1 and alpha2beta1 integrins are overexpressed on the surface of peripheral blood eosinophils of asthmatic subjects. Further studies may reveal potential application of KTS-disintegrins or their structural analogs for therapy of bronchial asthma.     

Rap1A positively regulates T cells via integrin activation rather than inhibiting lymphocyte signaling

T cell receptor (TCR) stimulation activates the small GTPase Rap1A, which is reported to antagonize Ras signaling and induces T cell anergy. To address its role in vivo, we generated transgenic mice that constitutively expressed active Rap1A within the T cell lineage. We found that active Rap1A did not interfere with the Ras signaling pathway or antagonize T cell activation. Instead of anergy, the T lymphocytes that constitutively expressed active Rap1A showed enhanced TCR-mediated responses, both in thymocytes and mature T cells. In addition, Rap1A activation was sufficient to induce strong activation of the beta1 and beta2 integrins via an avidity-modulation mechanism. This shows that, far from playing an inhibitory role during T cell activation, Rap1A positively influences T cells by augmenting lymphocyte responses and directing integrin activation.     

Intracellular analysis of interleukin-2 induction provides direct evidence at the single cell level of differential coactivation requirements for CD45RA+ and CD45RO+ T cell subsets.

Through measurements of intracellular cytokine production, evidence is provided at the single cell level that triggering different cell surface molecules preferentially activates discrete human peripheral blood (PB) T cell subsets. T cell costimulation due to cross-linking a variety of individual molecules (beta1, beta2, and beta7 integrins, CD26, CD43, or CD44), in conjunction with the CD3/TCR complex, preferentially activated CD45RO+ PB T lymphocytes. CD28, however, costimulated interleukin-2 (IL-2) production in both CD45RO+ and CD45RA+ subpopulations. The amount of soluble IL-2 produced by CD28 coactivation was 15-30-fold higher than that due to integrin or CD26-dependent coactivation, although even the lowest amount of soluble IL-2 produced was in the range of the high-affinity IL-2 receptor. The overall proliferative responses were similar among all costimulatory settings. This was in part due to the uniform upregulation of IL-2 receptor-alpha (IL-2R alpha) (CD25) expression on the entire T cell population activated under each of the different costimulatory conditions. The data provide direct evidence on a single cell level that activation of human CD45RA+ (naive) T cells is stringently controlled and, in these studies, limited to CD28 costimulation for induction of IL-2 production. In contrast, coactivation of CD45RO+ (memory) T lymphocytes can proceed by a variety of different PB T cell surface molecules.