Alternate signalling pathways from the interleukin-2 receptor.

Interleukin-2 (IL-2) plays a major role in the proliferation of cell populations during an immune reaction. The beta(c) and gamma(c) subunits of the IL-2 receptor (IL-2R) are sufficient and necessary for signal transduction. Despite lacking known catalytic domains, receptor engagement leads to the activation of a diverse array protein tyrosine kinases (PTKs). In resting or anergised T cells, Jak3 is not activated. Signals arising from the PROX domain of the gamma(c) subunit activate p56(lck) (lck) leading to the induction of anti-apoptotic mechanisms. When Jak3 is activated, in primed T cells, other PTKs predominantly mediate the induction of anti-apoptotic mechanisms and drive cellular proliferation. This review intends to suggest a role for these differences within the context of the immune system.     

A membrane-proximal region of the interleukin-2 receptor gamma c chain sufficient for Jak kinase activation and induction of proliferation in T cells.

The interleukin-2 (IL-2) receptor (IL-2R) consists of three distinct subunits (alpha, beta, and gamma c) and regulates proliferation of T lymphocytes. Intracellular signalling results from ligand-mediated heterodimerization of the cytoplasmic domains of the beta and gamma c chains. To identify the residues of gamma c critical to this process, mutations were introduced into the cytoplasmic domain, and the effects on signalling were analyzed in the IL-2-dependent T-cell line CTLL2 and T-helper clone D10, using chimeric IL-2R chains that bind and are activated by granulocyte-macrophage colony-stimulating factor. Whereas previous studies of fibroblasts and transformed T cells have suggested that signalling by gamma c requires both membrane-proximal and C-terminal subdomains, our results for IL-2-dependent T cells demonstrate that the membrane-proximal 52 amino acids are sufficient to mediate a normal proliferative response, including induction of the proto-oncogenes c-myc and c-fos. Although gamma c is phosphorylated on tyrosine upon receptor activation and could potentially interact with downstream molecules containing SH2 domains, cytoplasmic tyrosine residues were dispensable for mitogenic signalling. However, deletion of a membrane-proximal region conserved among other cytokine receptors (cytoplasmic residues 5 to 37) or an adjacent region unique to gamma c (residues 40 to 52) abrogated functional interaction of the receptor chain with the tyrosine kinase Jak3. This correlated with a loss of all signalling events analyzed, including phosphorylation of the IL-2R beta-associated kinase Jak1, expression of c-myc and c-fos, and induction of the proliferative response. Thus, it appears in T cells that Jak3 is a critical mediator of mitogenic signaling by the gamma c chain.     

Ryanodine receptor signaling is required for anti-CD3-induced T cell proliferation, interleukin-2 synthesis, and interleukin-2 receptor signaling

Ryanodine receptors (RyR) are involved in regulating intracellular Ca(++) mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR-selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor-mediated activation events and cytokine-dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)-2 by T lymphocytes responding to mitogenic anti-CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation-induced expression of the alpha chain (CD25) of the high affinity IL-2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL-2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL-2-dependent proliferation of CTLL-2 T cells. These data indicate that RyR are involved in regulating IL-2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR-associated Ca(++) signaling regulates T cell proliferation by promoting both IL-2 synthesis and IL-2-dependent cell cycle progression.     

The level of interleukin-2 and of soluble interleukin-2 receptor in patients with Balkan nephropathy

Balkan Nephropathy (BN) is defined as a clinical entity with unknown etiology. The involvement of immune system in pathogenesis of BN is not well defined yet. The aim of this study was to gain more insight into the cellular immune mechanisms in BN. We determined some factors implied in cellular immunity, such as the serum level of IL-2 and of IL-2 soluble receptor (sIL-2R), and the presence of IL-2 receptor alpha chain (CD25) on T cells membrane. The study was performed on 15 patients with BN, 15 patients with Chronic Pyelonephritis (CPN), and 10 healthy controls from a non-endemic area. Our study showed no significant differences between IL-2 level and CD25+ cells percentage in CPN compared to controls, but a significantly increased level of sIL-2R. The BN sIL-2R is significantly lower than sIL-2R in CPN, and associates an important T cell activation (high CD25+ presence, elevated IL-2 level) compared to CPN. Our conclusion is that while the high sIL-2R level could down modulate T cell activity in CPN, BN sIL-2R level is ineffective in limiting the activation effects of IL-2 on T cells. The results suggest that cellular immunity could have a role in the pathogenesis of N.     

Cytokine-independent Jak3 activation upon T cell receptor (TCR) stimulation through direct association of Jak3 and the TCR complex

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common gamma chain (gammac) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/gammac but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3zeta via its JH4 region, which is a different region from that for gammac association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.     

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor.

Murine interleukin-5 (IL-5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL-5 receptor (IL-5-R) is composed of at least two membrane protein subunits and is responsible for IL-5-mediated signal transduction. One subunit of the high affinity IL-5-R is a 60 kDa membrane protein (p60 IL-5-R) whose cDNA was isolated using the anti-IL-5-R monoclonal antibody (mAb), H7. This subunit alone binds IL-5 with low affinity. The second subunit does not bind IL-5 by itself, and is expressed not only on IL-5-dependent cell lines but also on an IL-3-dependent cell line, FDC-P1. Expression of the p60 IL-5-R cDNA in FDC-P1 cells, which do not bind IL-5, reconstituted the high affinity IL-5-R. We have characterized the second subunit of the IL-5-R by using another anti-IL-5-R mAb, R52.120, and the anti-IL-3-R mAb, anti-Aic-2. The anti-Aic-2 mAb down-regulated binding of IL-5 to an IL-5-dependent cell line, Y16. Both R52.120 and anti-Aic-2 mAbs recognized membrane proteins of 130-140 kDa expressed on FDC-P1 and Y16 cells. The R52.120 mAb recognized both murine IL-3-R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL-5-R was reconstituted on an L cell transfectant co-expressing AIC2B and p60 IL-5-R, whereas only the low affinity IL-5-R was detected on a transfectant co-expressing AIC2A and p60 IL-5-R.(ABSTRACT TRUNCATED AT 250 WORDS)     

A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit,IL-23R

IL-23 is a heterodimeric cytokine composed of the IL-12p40 "soluble receptor" subunit and a novel cytokine-like subunit related to IL-12p35, termed p19. Human and mouse IL-23 exhibit some activities similar to IL-12, but differ in their capacities to stimulate particular populations of memory T cells. Like IL-12, IL-23 binds to the IL-12R subunit IL-12Rbeta1. However, it does not use IL-12Rbeta2. In this study, we identify a novel member of the hemopoietin receptor family as a subunit of the receptor for IL-23, "IL-23R." IL-23R pairs with IL-12Rbeta1 to confer IL-23 responsiveness on cells expressing both subunits. Human IL-23, but not IL-12, exhibits detectable affinity for human IL-23R. Anti-IL-12Rbeta1 and anti-IL-23R Abs block IL-23 responses of an NK cell line and Ba/F3 cells expressing the two receptor chains. IL-23 activates the same Jak-stat signaling molecules as IL-12: Jak2, Tyk2, and stat1, -3, -4, and -5, but stat4 activation is substantially weaker and different DNA-binding stat complexes form in response to IL-23 compared with IL-12. IL-23R associates constitutively with Jak2 and in a ligand-dependent manner with stat3. The ability of cells to respond to IL-23 or IL-12 correlates with expression of IL-23R or IL-12Rbeta2, respectively. The human IL-23R gene is on human chromosome 1 within 150 kb of IL-12Rbeta2.     

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling.     

Responses to T cell receptor/CD3 and interleukin-2 receptor stimulation are altered in T cells from B cell non-hodgkin's lymphomas

T cells infiltrating (T-TIL) B cell non-Hodgkin's lymphomas (NHL) are thought to represent a local host response to the tumor. However, tumor progression in the presence of this T cell infiltrate suggests that the T-TIL may be functionally impaired. To address this issue we determined whether response to stimulation of T-TIL from 25 patients with NHL through the T cell receptor (TCR/CD3) and the interleukin-2 (IL-2) receptor (IL-2R) was intact, since activation of these receptors is important for proliferation and cytokine production. Our results demonstrate defects in response to stimulation via TCR/CD3 and the IL-2R in T-TIL cells from patients with NHL that were not observed with T cells from the peripheral blood. T-TIL showed minimal proliferation to anti-CD3 and only modest proliferation to IL-2 alone or when combined with anti-CD3. Moreover, cytokine production in T-TIL was impaired since stimulation through the TCR/CD3 complex did not induce mRNA for interferon (IFN), IL-2, IL-4 or IL-10. The functional unresponsiveness of these cells may be linked to altered signalling through the TCR/CD3 since an abnormal tyrosine phosphorylation pattern was detected in T-TIL after stimulation with anti-CD3.     

Interleukin 2 receptor expression by T cells in human aging

Aged individuals have depressed cell-mediated immunity and diminished T cell proliferation to mitogenic and antigenic stimuli. Because T cell responses depend on the surface expression and normal function of interleukin 2 receptors, we measured the quantities and affinities of cell surface IL-2R and the amount of soluble IL-2R alpha chain (p55) release in vitro in PHA-stimulated mononuclear cells from healthy aged (greater than or equal to 65 years old) and young (less than or equal to 39 years old) donors. At the peak of the PHA response, the fraction of cells expressing IL-2R alpha chain (CD25+) was lower in the aged (43% vs 56%, P = 0.033). Relative to the lower proliferation and CD25 expression, old donor cells released unexpectedly high quantities of soluble alpha chain into culture supernatants. However, the average affinities and the mean numbers of high- and low-affinity surface receptors per CD25+ cell were equivalent in cells from eight pairs of aged and young donors (1850 vs 1586 high affinity, and 20,655 vs 23,466 low affinity, P greater than 0.2 for both). The soluble IL-2R released by stimulated cells had no effect on proliferative responses, because addition of saturating doses of exogenous recombinant IL-2 did not increase cellular proliferation, and addition of soluble anchor-minus recombinant IL-2R alpha chain did not suppress it. These results indicate that in healthy older individuals, diminished numbers of T cells can be induced to express cell surface IL-2R following mitogenic stimulation, although aged CD25+ can express a normal complement of IL-2R molecules. In the aged, either CD25+ cells release excessive quantities or a subset of cells synthesizes and releases soluble IL-2R alpha chain into the extracellular environment without expressing it on the cell surface.     

Streptavidin suppresses T cell activation and inhibits IL-2 production and CD25 expression

Streptavidin is widely used as a detection tool in biology research because of its high affinity and specificity binding to biotin. Biotin-streptavidin system has also been explored for detection of infection and tumor in clinical medicine. Here, we show immunosuppressive property of streptavidin on T cell activation and proliferation. Upon CD3 and CD28 stimulation, CD4(+) T cells produce interleukin 2 (IL-2) and express IL-2 receptor α chain (CD25). Addition of streptavidin in T cell culture suppressed IL-2 synthesis and CD25 expression with no cytotoxicity. The immunosuppressive effect of streptavidin was reversed by excessive biotin. Conjugated to a single chain anti-CD7 variable fragment (scFvCD7), streptavidin was directly delivered to T cells and showed substantially more profound suppressive effect on T cell activation. These results suggest that streptavidin could potentially be used as a novel immunomodulator.     

Efficient internalization of IL-2 depends on the distal portion of the cytoplasmic tail of the IL-2R common gamma-chain and a lymphoid cell environment

The common gamma-chain (gammac), a subunit of the IL-2R, is essential for high affinity ligand binding and signal transduction due to Jak3 association to gammac. Another consequence of IL-2/IL-2R interaction is rapid receptor-mediated endocytosis of the receptor-ligand complex. In the present study, we establish that this rapid endocytosis of IL-2 in a T cell tumor line is dependent upon the cytoplasmic tail of gammac. Deletion mutants of the cytoplasmic tail mapped this activity to 9 aa of gammac, 45-54 aa distal to the transmembrane region. In contrast, ligand-independent constitutive endocytosis of gammac occurred more slowly and was dependent upon a PEST sequence in a more membrane-proximal region of the cytoplasmic tail of gammac. Thus, this receptor subunit may use distinct sorting signals for its constitutive regulation and ligand-induced endocytosis. Rapid endocytosis of IL-2 was inhibited by the tyrosine kinase inhibitor genistein, implicating a role for a signal transduction pathway in IL-2 internalization. However, one T cell line bearing a mutant gammac exhibited impaired endocytosis of IL-2, despite normal IL-2-induced Jak/STAT activation. Furthermore, inefficient endocytosis of IL-2 was noted after transfection of the COS7 epithelial cell line with the IL-2R, and further reconstitution of these cells with Jak/STAT proteins did not enhance this internalization. Collectively, these latter findings indicate that rapid endocytosis of IL-2 is dependent upon cellular signaling in lymphoid cell environment that is not solely a consequence of the presence of the Jak/STAT pathway.     

Smad-dependent cooperative regulation of interleukin 2 receptor alpha chain gene expression by T cell receptor and transforming growth factor-beta

The interleukin 2 receptor alpha chain (IL-2Ralpha) is a component of high affinity IL-2 receptors and thus critically regulates T cell growth and other lymphoid functions. Five positive regulatory regions together control lineage-restricted and activation-dependent IL-2Ralpha induction in response to antigen and IL-2. We now show that TGF-beta cooperates with T cell receptor (TCR) signaling to increase IL-2Ralpha gene expression. Moreover, we identify a sixth positive regulatory region that regulates IL-2Ralpha expression in cells treated with anti-CD3 + anti-CD28 as well as TGF-beta and show that this region contains binding sites for Smad3, AP-1, and cAMP-responsive element-binding protein/ATF proteins. The importance of Smad complexes is indicated by impaired IL-2Ralpha induction by TGF-beta in CD4+ T cells from both Smad3-/- and Smad4-/- mice. Thus, we have identified a novel positive regulatory region in the IL-2Ralpha gene that mediates TGF-beta-dependent induction of the gene. These findings have implications related to IL-2Ralpha expression on activated T cells and regulatory T cells.     

Immunoregulatory functions of paf-acether. III. Down-regulation of CD4+ T cells high-affinity IL-2 receptor expression

In the present report, we further explored the mechanisms by which 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), a phospholipid mediator of inflammation inhibited PHA-induced CD4+ cell proliferation. Evidence was obtained that CD4+ cells stimulated with either PHA or immobilized OKT3 in the presence of paf at concentrations that block CD4+ cell proliferation, exhibited a marked decrease in high affinity IL-2R expression. Importantly, paf did not prevent the binding of IL-2 to its receptor. Scatchard analysis of the binding data indicated that paf caused more than 50% decrease in the number of IL-2 high affinity sites per cell, whereas the receptor ligand affinity remained essentially constant. Moreover, the down-regulation of high affinity IL-2R was also accompanied by a loss of IL-2-dependent proliferative capacity. Together these data suggest that decreased expression of high affinity IL-2R may contribute to the diminished proliferative activity observed in CD4+ cells stimulated with PHA or immobilized OKT3 in the presence of paf. They further emphasize the potential role of lipid proinflammatory mediators such as paf in the regulation of T cell activation.     

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

PUFAs have been shown to mediate immune re-sponse especially the functions of T cells[1]. Recent researches have demonstrated that PUFAs can in-crease membrane fluidity and modify the functions of membrane receptors and enzymes in T cell membra-ne[2,3]. M…     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.