IL-2 receptor signaling through the Shb adapter protein in T and NK cells

We have investigated the effect of hypoxia on the excitatory synaptic transmission in the substantia gelatinosa neurons using perforated-patch-clamp configuration. Brief periods of hypoxia induced a depression in the evoked excitatory postsynaptic current (eEPSC) amplitude. The hypoxia-induced depression of eEPSC was not observed in the presence of theophylline, a nonselective adenosine receptor antagonist, and DPCPX, a selective adenosine receptor A1 antagonist. Application of adenosine (100 microM) also depressed eEPSC in a similar way as with hypoxia. This adenosine-induced depression of eEPSC was inhibited by DPCPX. Hypoxia and exogenous adenosine decreased the frequency of the spontaneous excitatory postsynaptic current (sEPSC) but not the amplitude of sEPSC and increased the paired-pulse ratio. From these results, it is suggested that acute hypoxia depresses the excitatory synaptic transmission by activating the presynaptic adenosine A1 receptor.     

Multiple cytokines regulate the NK gene complex-encoded receptor repertoire of mature NK cells and T cells

Mature NK cells comprise a highly diverse population of lymphocytes that express different permutations of receptors to facilitate recognition of diseased cells and perhaps pathogens themselves. Many of these receptors, such as those belonging to the NKRP1, NKG2, and Ly49 families are encoded in the NK gene complex (NKC). It is generally thought that these NKC-encoded receptors are acquired by a poorly understood stochastic mechanism, which operates exclusively during NK cell development, and that following maturation the repertoire is fixed. However, we report a series of observations that demonstrates that the mature NK cell repertoire in mice can in fact be radically remodeled by multiple cytokines. Thus, both IL-2 and IL-15 selectively induce the de novo expression of Ly49E on the majority of mature NK cells. By contrast, IL-4 not only blocks this IL-2-induced acquisition of Ly49E, but reduces the proportion of mature NK cells that expresses pre-existing Ly49 receptors and abrogates the expression of NKG2 receptors while leaving the expression of several NKRP1 receptors unaltered. IL-21 also abrogates NKG2 expression on mature NK cells and selectively down-regulates Ly49F. IL-4 and IL-21 additionally cause dramatic and selective alterations in the NKC-encoded receptor repertoire of IL-2-activated T cells but these are quite different to the changes induced on NK cells. Collectively these findings reveal an unexpected aspect of NKC receptor expression that has important implications for our understanding of the function of these receptors and of the genetic mechanisms that control their expression.     

Unique signaling properties of B cell antigen receptor in mature and immature B cells: implications for tolerance and activation

Immature B cells display increased sensitivity to tolerance induction compared with their mature counterparts. The molecular mechanisms underlying these differences are poorly defined. In this study, we demonstrate unique maturation stage-dependent differences in B cell Ag receptor (BCR) signaling, including BCR-mediated calcium mobilization responses. Immature B cells display greater increases in intracellular calcium concentrations following Ag stimulation. This has consequences for the induction of biologically relevant responses: immature B cells require lower Ag concentrations for activation than mature B cells, as measured by induction of receptor editing and CD86 expression, respectively. BCR-induced tyrosine phosphorylation of CD79a, Lyn, B cell linker protein, and phospholipase Cgamma2 is enhanced in immature B cells and they exhibit greater capacitative calcium entry in response to Ag. Moreover, B cell linker protein, Bruton's tyrosine kinase, and phospholipase Cgamma2, which are crucial for the induction of calcium mobilization responses, are present at approximately 3-fold higher levels in immature B cells, potentially contributing to increased mobilization of calcium. Consistent with this possibility, we found that the previously reported lack of inositol-1,4,5-triphosphate production in immature B cells may be explained by enhanced inositol-1,4,5-triphosphate breakdown. These data demonstrate that multiple mechanisms guarantee increased Ag-induced mobilization of calcium in immature B cells and presumably ensure elimination of autoreactive B cells from the repertoire.     

Activation of NK cells by an endocytosed receptor for soluble HLA-G

Signaling from endosomes is emerging as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. The activity of natural killer (NK) cells, which are important effectors of innate immunity and regulators of adaptive immunity, is controlled primarily by receptors that are at the cell surface. Here we show that cytokine secretion by resting human NK cells is induced by soluble, but not solid-phase, antibodies to the killer cell immunoglobulin-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen (HLA)-G. KIR2DL4 was constitutively internalized into Rab5-positive compartments via a dynamin-dependent process. Soluble HLA-G was endocytosed into KIR2DL4–containing compartments in NK cells and in 293T cells transfected with KIR2DL4. Chemokine secretion induced by KIR2DL4 transfection into 293T cells occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. The profile of genes up-regulated by KIR2DL4 engagement on resting NK cells revealed a proinflammatory/proangiogenic response. Soluble HLA-G induced secretion of a similar set of cytokines and chemokines. This unique stimulation of resting NK cells by soluble HLA-G, which is endocytosed by KIR2DL4, implies that NK cells may provide useful functions at sites of HLA-G expression, such as promotion of vascularization in maternal decidua during early pregnancy.     

Maintenance of vascular integrity in the embryo requires signaling through the fibroblast growth factor receptor

Basic fibroblast growth factor (FGF)-2 is important for vessel formation and/or maintenance of vascular integrity in the embryo. FGF signaling may be mediated through transmembrane tyrosine kinase receptors or directly through intracellular pathways that do not involve receptor activation. To determine the role of receptor-mediated signaling in endothelial cells, an adenovirus encoding truncated FGF receptor (FGFR)-1, under the control of the cytomegalovirus promoter, was expressed in endothelial cells. FGF signaling was impaired, as indicated by inhibition of MAPK phosphorylation. Functional consequences included inhibition of endothelial cell migration and induction of apoptosis. To address the role of endothelial FGFR signaling in vascular development, recombinant adenovirus encoding a dominant-negative FGFR was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Previous studies demonstrated that transgenes delivered via adenovirus, under the control of the cytomegalovirus promoter, are expressed selectively in the developing vasculature. Embryos expressing a control adenovirus developed normally, whereas those expressing the FGFR-1 mutant exhibited abnormal embryonic and extra-embryonic vascular development. These data demonstrate that FGF, by signaling through the FGFR, plays a pivotal role in the development and maintenance of a mature vascular network in the embryo.     

Gs protein-coupled adenosine receptor signaling and lytic function of activated NK cells

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.     

Acquisition of viral receptor by NK cells through immunological synapse

Occasional EBV infection of human NK cells may lead to malignant diseases such as naso-pharyngeal NK lymphoma although NK cells do not express CD21, the primary receptor for EBV. Here we show that during early EBV infection in patients, NK cells attacked EBV-infected autologous B cells. In vitro, NK cells activated by conjugation to CD21(+) B-EBV cell targets transiently acquired a weak CD21(+) phenotype by synaptic transfer of few receptor molecules onto their own membrane. In the presence of viral particles, these ectopic receptors allowed EBV binding to the novel NK cell host. Hence, trans-synaptic acquisition of viral receptor from target cells might constitute an unsuspected mode of infection for otherwise unreachable lymphoid hosts.     

Development of the thymus requires signaling through the fibroblast growth factor receptor R2-IIIb

Mice deficient for fibroblast growth factor (Fgf)R2-IIIb show a block in thymic growth after embryonic day 12.5, a stage that just precedes its detection in thymic epithelial cells. Fgf7 and Fgf10, the main ligands for FgfR2-IIIb, are expressed in the mesenchyme surrounding the thymic epithelial primordium, and Fgf10-deficient mice also exhibit impaired thymic growth. Hence, Fgf signaling is essential for thymic epithelial proliferation. In addition to the proliferative block, most thymic epithelial cells fail to progress from an immature cytokeratin 5-positive to a cytokeratin 5-negative phenotype. Nevertheless, sufficient epithelial cell differentiation occurs in the severely hypoplastic thymus to allow the development of CD4/CD8-double-positive thymocytes and a very small number of single-positive thymocytes expressing TCRs.     

Perforin-dependent cryptococcal microbicidal activity in NK cells requires PI3K-dependent ERK1/2 signaling

Previously, NK cells have been reported to kill the opportunistic fungal pathogen Cryptococcus neoformans through a perforin-dependent mechanism; however, the receptor and signaling involved are unknown. In this report we sought to identify the signaling pathways activated and required for direct perforin-mediated killing of microbes. In this study, using the NK-like cell line YT and primary peripheral blood NK cells, it is demonstrated that YT cells kill C. neoformans and that the killing is accompanied by the activation of PI3K. We demonstrate that inhibition of either the catalytic subunit (using a pharmacological inhibitor) or the alpha-regulatory subunit (using small interfering RNA knockdown) of PI3K significantly inhibited the killing of C. neoformans. Downstream of PI3K, ERK1/2 was activated in a PI3K-dependent fashion and was required for cryptococcal killing. Furthermore, we demonstrate that perforin release from YT cells can be detected by 4 h after contact of the YT cells with C. neoformans and that the release of perforin is blocked by pharmacological inhibition of either PI3K or ERK1/2. Defective degranulation is rooted in the inability to polarize perforin-containing granules toward the target. Finally, we demonstrate that PI3K-ERK1/2-dependent signaling is activated and required for the killing of C. neoformans by primary NK cells. Taken together, these data identify a conserved PI3K-ERK1/2 pathway that is used by NK cells during the direct killing of C. neoformans and demonstrate that the pathway is essential in the formation and activation of the microbicidal mechanism.     

Activation of an alternative death receptor-induced signaling pathway in human hepatocytes under caspase arrest

Caspases are thought to be essential in execution of death receptor-induced apoptosis. However, recent findings suggest the existence of alternative pathways independent of caspases. We provide further evidence for such signaling in hepatocytes. RESULTS: Death receptor-induced activation of caspases and apoptosis in primary murine hepatocytes was completely blocked in presence of 1.5 microM N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk). Whereas the same concentration of the inhibitor was sufficient to block TNF receptor 1-, CD95- or TRAIL receptor 1/-2-induced activation of caspases in primary human hepatocytes or HepG2 cells, complete prevention apoptotic cell death needed almost 100 microM zVAD-fmk. Under caspase-inhibitory but non-protective conditions, i.e. at 1.5 microM zVAD-fmk, various serine protease inhibitors prevented apoptosis-like cell death. Neither sole arrest of caspases nor inhibition of serine proteases alone protected human hepatocytes. CONCLUSION: Human but not murine hepatocytes bear the potential to activate a permissive, serine protease inhibitor-sensitive alternative death signaling pathway under caspase-inhibitory conditions.     

Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway

Neurite outgrowth is an important process in neural regeneration and plasticity, especially after neural injury, and recent evidence indicates that several G_αi/o protein-coupled receptors play an important role in neurite outgrowth. The neuropeptide (NP)FF system contains two G_αi/o protein-coupled receptors, NPFF₁ and NPFF₂ receptors, which are mainly distributed in the central nervous system. The aim of the present study was to determine whether the NPFF system is involved in neurite outgrowth in Neuro 2A cells. We showed that Neuro 2A cells endogenously expressed NPFF₂ receptor, and the NPFF₂ receptor agonist dNPA inhibited cyclic adenosine monophosphate (cAMP) production stimulated by forskolin in Neuro 2A cells. We also demonstrated that NPFF and dNPA dose-dependently induced neurite outgrowth in Neuro 2A cells, which was completely abolished by the NPFF receptor antagonist RF9. Pretreatment with mitogen-activated protein kinase inhibitors PD98059 and U0126 decreased dNPA-induced neurite outgrowth. In addition, dNPA increased phosphorylation of extracellular signal-regulated kinase (ERK) in Neuro 2A cells, which was completely antagonized by pretreatment with U0126. Our results suggest that activation of NPFF₂ receptor stimulates neurite outgrowth in Neuro 2A cells through activation of the ERK signaling pathway. Moreover, NPFF₂ receptor may be a potential therapeutic target for neural injury and degeneration in the future.     

Flagellin-induced tolerance of the Toll-like receptor 5 signaling pathway in polarized intestinal epithelial cells

Salmonella typhimurium is a gram-negative enteric pathogen that invades the mucosal epithelium and is associated with diarrheal illness in humans. Flagellin from S. typhimurium and other gram-negative bacteria has been shown to be the predominant proinflammatory mediator through activation of the basolateral Toll-like receptor 5 (TLR5). Recent evidence has shown that prior exposure can render immune cells tolerant to subsequent challenges by TLR ligands. Accordingly, we examined whether prior exposure to purified flagellin would render human intestinal epithelial cells insensitive to future contact. We found that flagellin-induced tolerance is common to polarized epithelial cells and prevents further activation of proinflammatory signaling cascades by both purified flagellin and Salmonella bacteria but does not affect TNF-alpha stimulation of the same pathways. Flagellin tolerance is a rapid process that does not require protein synthesis, and that occurs within 1 to 2 h of flagellin exposure. Prolonged flagellin exposure blocks activation of the NF-kappaB, MAPK, and phosphoinositol 3-kinase signaling pathways and results in the internalization of a fraction of the basolateral TLR5 without affecting the polarity or total expression of TLR5. After removal of flagellin, cells require more than 24 h to fully recover their ability to mount a normal proinflammatory response. We have found that activation of phosphoinositol 3-kinase and Akt by flagellin has a small damping effect in the early stages of flagellin signaling but is not responsible for tolerance. Our study indicates that inhibition of TLR5-associated IL-1 receptor-associated kinase-4 activity occurs during the development of flagellin tolerance and is likely to be the cause of tolerance.     

Activation of natural killer (NK) cells in vivo with H-2 and non-H-2 alloantigens

Intraperitoneal inoculation of allogeneic lymphoid cells rapidly activates cytotoxic cells in the peritoneum which are nonadherent and express the NK-1, asialo-GM₁, and Thy-1 antigens. Allogeneic spleen cells were very efficient at activating these natural killer (NK) cells, while allogeneic thymocytes were much less effective. Heat-killed allogeneic cells or sonicates also could augment NK activity. — Incompatibility atH-2K, H-2I-A, orH- 2D readily evoked NK cell activity, whileH-2S- andH-2I-E/C-associated disparities did not. Non-H- 2 differences also stimulated NK activity and augmentation was particularly evident inMls-disparate combinations. Thus, the same alloantigens which efficiently activate T cells also activate NK cells.     

BMPR2 expression is suppressed by signaling through the estrogen receptor

Background

Low endogenous testosterone levels have been shown to be a risk factor for the development of cardiovascular disease and cardiovascular benefits associated with testosterone replacement therapy are being advocated; however, the effects of endogenous testosterone levels on acute coronary vasomotor responses to androgen administration are not clear. The objective of this study was to compare the effects of acute androgen administration on in vivo coronary conductance and in vitro coronary microvascular diameter in intact and castrated male swine.

Methods

Pigs received intracoronary infusions of physiologic levels (1?C100 nM) of testosterone, the metabolite 5??-dihydrotestosterone, and the epimer epitestosterone while left anterior descending coronary blood flow and mean arterial pressure were continuously monitored. Following sacrifice, coronary arterioles were isolated, cannulated, and exposed to physiologic concentrations (1?C100 nM) of testosterone, 5??-dihydrotestosterone, and epitestosterone. To evaluate effects of the androgen receptor on acute androgen dilation responses, real-time PCR and immunohistochemistry for androgen receptor were performed on conduit and resistance coronary vessels.

Results

In vivo, testosterone and 5??-dihydrotestosterone produced greater increases in coronary conductance in the intact compared to the castrated males. In vitro, percent maximal dilation of microvessels was similar between intact and castrated males for testosterone and 5??-dihydrotestosterone. In both studies epitestosterone produced significant increases in conductance and microvessel diameter from baseline in the intact males. Androgen receptor mRNA expression and immunohistochemical staining were similar in intact and castrated males.

Conclusions

Acute coronary vascular responses to exogenous androgen administration are increased by endogenous testosterone, an effect unrelated to changes in androgen receptor expression.     

Leukemic priming of resting NK cells is killer Ig-like receptor independent but requires CD15-mediated CD2 ligation and natural cytotoxicity receptors

Resting human NK cells require a two-stage activation process that we have previously described as "priming" and "triggering." NK-sensitive tumor cells provide both priming and triggering signals. NK-resistant tumors evade lysis, mostly by failure to prime; however, we recently reported a tumor cell line (CTV-1) that primes resting NK cells but fails to trigger lysis. In this article, we report two additional leukemia cell lines that prime NK cells but are resistant to lysis. Tumor-mediated NK priming is via CD2 binding to a ligand within CD15 on the tumor cell. NK-resistant RAJI cells became susceptible to NK lysis following transfection and expression of CD15. Blockade of CD15 on K562 cells or on CD15(+) RAJI cells significantly inhibited lysis, as did blockade of CD2 on resting NK cells. NK priming via CD2 induced CD16 shedding, releasing CD3ζ to the CD2, leading to its phosphorylation and the subsequent phosphorylation of linker for activation of T cells and STAT-5 and synthesis of IFN-γ. Blockade of C-type lectin receptors significantly suppressed the tumor-mediated priming of NK cells, whereas blockade of Ig-superfamily-like receptors had no effect at the NK-priming stage. Tumor priming of resting NK cells was irrespective of HLA expression, and blockade of HLA-killer Ig-like receptor interactions did not influence the incidence or degree of priming. However, CD15-CD2 interactions were critical for NK priming and were required, even in the absence of HLA-mediated NK inhibition. Tumor-mediated priming led to a sustained primed state, and the activated NK cells retained the ability to lyse NK-resistant tumors, even after cryopreservation.     

Long-term retention of mature NK1.1+ NKT cells in the thymus

The NKT cell pool in the thymus contains immature (NK1.1(-)) and mature (NK1.1(+)) subsets that represent distinct linear stages of a linear developmental pathway. An unexplained paradox is why immature NK1.1(-) NKT cells are mainly exported to the periphery instead of the more mature and more abundant NK1.1(+) NKT cells. In this study we have determined that mature NK1.1(+) NKT cells are retained by the thymus to form an extremely long-lived resident population capable of rapid and prolonged production of IFN-gamma and IL-4. The retention of mature NKT cells provides an explanation for why the periphery is mainly seeded by immature NK1.1(-) cells despite mature NK1.1(+) NKT cells being more abundant in the thymus. This is the first study to identify a mature T cell subset retained within the thymus and is additional evidence of the distinct developmental pathways of mainstream T cells and NKT cells.     

Chemorepellent signaling through the PACAP/lysozyme receptor is mediated through cAMP and PKC in Tetrahymena thermophila

Pituitary adenylate cyclase-activating peptide and lysozyme are potent chemorepellents which act through the same receptor in Tetrahymena. Using in vivo behavioral studies, we have found that the pituitary adenylate cyclase-activating peptide/lysozyme receptor appears to signal through a G-protein pathway which is mediated through both adenosine 3'5'monophosphate and protein kinase C. Avoidance to pituitary adenylate cyclase-activating peptide and lysozyme is inhibited by the G-protein inhibitor, guanosine 5'-O-(2thiodiphosphate), the adenosine 3'5'monophate analog, Rp-adenosine-3', 5' cyclic monophosphorothioate, and the protein kinase C inhibitors, calphostin C and bisindolylmaleimide IV. A proposed model for signaling through the pituitary adenylate cyclase-activating peptide/lysozyme receptor is briefly outlined.