Requirement for distinct Janus kinases and STAT proteins in T cell proliferation versus IFN-gamma production following IL-12 stimulation

While IL-12 is known to activate JAK2 and TYK2 and induce the phosphorylation of STAT4 and STAT3, little is known regarding how the activation of these signaling molecules is related to the biologic effects of IL-12. Using an IL-12-responsive T cell clone (2D6), we investigated their requirements for proliferation and IFN-gamma production of 2D6 cells. 2D6 cells could be maintained with either IL-12 or IL-2. 2D6 lines maintained with IL-12 (2D6(IL-12)) or IL-2 (2D6(IL-2)) exhibited comparable levels of proliferation, but produced large or only small amounts of IFN-gamma, respectively, when restimulated with IL-12 after starvation of either cytokine. 2D6(IL-12) induced TYK2 and STAT4 phosphorylation. In contrast, their phosphorylation was marginally induced in 2D6(IL-2). The reduced STAT4 phosphorylation was due to a progressive decrease in the amount of STAT4 protein along with the passages in IL-2-containing medium. 2D6(IL-12) and 2D6(IL-2) similarly proliferating in response to IL-12 induced comparable levels of JAK2 activation and STAT5 phosphorylation. JAK2 was associated with STAT5, and IL-12-induced STAT5 phosphorylation was elicited in the absence of JAK3 activation. These results indicate that IL-12 has the capacity to induce/maintain STAT4 and STAT5 proteins, and that TYK2 and JAK2 activation correlate with STAT4 phosphorylation/IFN-gamma induction and STAT5 phosphorylation/cellular proliferation, respectively.     

Selective neural cell adhesion molecule signaling by Src family tyrosine kinases and tyrosine phosphatases

Nerve growth cone guidance is a highly complex feat, involving coordination of cell adhesion molecules, trophic factor gradients, and extracellular matrix proteins. While navigating through the developing nervous system, the growth cone must integrate diverse environmental signals into a singular response. The repertoire of growth cone responses to these extracellular cues includes axonal growth, fasciculation, and synaptic stabilization, which are achieved through dynamic changes in the cytoskeleton and modulation of gene expression. It has become evident that interactions between cell adhesion molecules can activate intracellular signaling pathways in neurons. Such signaling pathways are just beginning to be defined for the axonal growth promoting molecules L1 and NCAM which are members of the immunoglobulin (Ig) superfamily. Recent findings have revealed that L1 and NCAM induce neurite outgrowth by activating intracellular signaling pathways in the growth cone mediated by two different members of the src family of nonreceptor protein tyrosine kinases (PTKs), pp60(c-src) and p59(fyn5,6). Growth cones display diverse morphologies and variable motility on these different cell adhesion molecules, which are likely to be generated by src kinases. In this review we will address novel features of nonreceptor PTKs of the src family which dictate their distinctive molecular interactions with cell adhesion molecules and signaling components.     

Cooperative effects of interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor: a new myeloid cell line inducible to eosinophils

The cytokines interleukin-3 (IL-3); IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to contribute to the proliferation and differentiation of eosinophil progenitors. Recently, it was determined that the cellular receptors for these three cytokines share a common beta-chain while having unique alpha-chains. Thus, there is considerable interest in how these cytokines and their receptors interact in promoting production of eosinophils. We have established a cell line (AML14) from a patient with acute myelogenous leukemia that will consistently exhibit eosinophilic differentiation in suspension in response to IL-3, IL-5, and GM-CSF. Proliferation with only modest differentiative effects was observed in response to a single cytokine. Combinations of two cytokines gave variable results, with GM-CSF + IL-3 and IL-3 + IL-5 causing more proliferation than a single cytokine but little more differentiation. The combination of GM-CSF + IL-5 caused marked enhancement of eosinophilic differentiation with only modest augmentation of proliferation. The combination of all three cytokines was most effective in stimulating both proliferation and eosinophilic differentiation (up to 70% of cells) of AML14 cells. Specific binding of GM-CSF and IL-5 to AML14 cells can be conveniently studied by flow cytometric methods, and cross-competition of these two cytokines for their respective receptors was demonstrated. IL-3 was shown to partially compete for IL-5 binding on AML14 cells. Although specific IL-3 binding could not be demonstrated by flow cytometry, mRNA for the alpha-chains of the IL-3, IL-5, and GM-CSF receptors and the beta-chain common to all three receptors was detected in AML14 cells. The AML14 cell line may be a useful model for the study of cooperative interactions of IL-3, IL-5, GM-CSF, and their respective receptors in the promotion of eosinophil progenitor growth and differentiation.     

Lipopolysaccharide and IL-1 alpha activate CNS pathways as measured by NK cell activity

An outward current (Iout) was produced by stereoisomers of beta-hydroxy-L-glutamic acid (L-BHGA), an L-glutamic acid (L-Glu) derivative, applied by brief pneumatic pressure ejection on an identifiable neurone type, v-LCDN (ventral-left cerebral distinct neurone), of Achatina fulica Férussac. However, L- and D-Glu were almost ineffective on this neurone type. The pharmacological features of this Iout caused by L-BHGA were elucidated in the present study. According to the dose (pressure duration)-response studies on the L-BHGA stereoisomers that produced the Iout, the effective potency of threo-L-BHGA was approximately similar to that of erythro-L-BHGA. The dose (pressure duration)-response curve of quisqualic acid was shifted towards the left direction from those of threo-and erythro-L-BHGA, suggesting that the binding activity of quisqualic acid to the receptors would be stronger than those of the L-BHGA stereoisomers. GABA, glycine and L-homocysteic acid showed an inward current (Iin) on this neurone type, in contrast to the Iout caused by L-BHGA. beta-Alanine and taurine had absolutely no effect. Therefore, no amino acid inhibitory neurotransmitter candidate was found for this neurone type except for L-BHGA. It was assumed that L-BHGA, in either threo-or erythro-configuration, would be an inhibitory neurotransmitter for this neurone type. Mammalian L-Glu receptor antagonists. D(-)-AP-5, (+/-)-CPP, CNQX and L(+)-AP-3, applied by perfusion, showed no effect on the Iout of v-LCDN caused by threo-L-BHGA, indicating that the features of the inhibitory receptor activated by L-BHGA were much different from those of any type of the mammalian L-Glu receptors. Among the inhibitors of ATP-sensitive K+ channel, glipizide significantly inhibited the Iout caused by threo-L-BHGA, whereas tolbutamide did not. Inhibitors of intracellular signal transduction systems, H-7, H-8, H-9, staurosporine, calphostin C, KT5823 and W-7, had no effect on the Iout caused by threo-L-BHGA, suggesting that the receptors activated by threo-L-BHGA would be ionotropic.     

Fluoroaluminate induces activation and association of Src and Pyk2 tyrosine kinases in osteoblastic MC3T3-E1 cells

Fluoride is known to increase bone mass in vivo, probably through stimulation of osteoblast proliferation; however, the mechanisms of fluoroaluminate action in osteoblasts have not yet been elucidated. We have previously shown that in osteoblastic MC3T3-E1 cells, fluoroaluminate stimulates G protein-mediated protein tyrosine phosphorylation (Scaronuscarona, M., Standke, G. J. R., Jeschke, M., and Rohner, D. (1997) Biochem. Biophys. Res. Commun. 235, 680-684). Although the Ser/Thr kinases Erk1, Erk2, and p70(S6K) were activated in response to fluoroaluminate, the identity of fluoroaluminate-activated tyrosine kinase(s) remained elusive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate induces a 110-kDa tyrosine-phosphorylated protein that we identify as Pyk2, a cytoplasmic tyrosine kinase related to Fak (focal adhesion kinase). The tyrosine phosphorylation of Pyk2 increased in a dose- and time-dependent manner. The autophosphorylation activity of Pyk2 increased 3-fold and reached its maximum within 10 min of fluoroaluminate treatment. Fluoroaluminate also induced activation of Src and the association of Pyk2 with Src. The phosphorylation of Src-associated Pyk2 increased >20-fold in in vitro kinase assays, suggesting that Pyk2 is phosphorylated by Src. Although MC3T3-E1 cells express much more Fak than Pyk2, Src preferentially associated with Pyk2. In vitro, Pyk2 bound to the Src SH2 domain, suggesting that this interaction mediates the Src-Pyk2 association in cells. These data indicate that osteoblastic cells express Pyk2, which is tyrosine-phosphorylated and activated in response to G protein activation by fluoroaluminate, and that the mechanism of Pyk2 activation most likely involves Src. Thus, Src and Pyk2 are tyrosine kinases involved in G protein-mediated tyrosine phosphorylation in osteoblastic cells and may be important for the osteogenic action of fluoroaluminate.     

Tyrosine phosphorylation-dependent and -independent associations of protein kinase C-delta with Src family kinases in the RBL-2H3 mast cell line: regulation of Src family kinase activity by protein kinase C-delta

Src kinases and protein kinase C (PKC) have been well studied for their role in oncogenic and normal cellular processes. Herein we report on a novel regulatory pathway mediated by the interaction of PKC-delta with p53/56Lsy (Lyn) and with p60Src (Src) that results in the phosphorylation and increased activity of Lyn and Src. In the RBL-2H3 mast cell line, the interaction of PKC-delta with Lyn required the activation of the high affinity receptor for IgE (FcsigmaRI) while the interaction with Src was constitutive. Increased complex formation of PKC-delta with Lyn or Src led to increased serine phosphorylation and activity of the Src family kinases. Conversely, Lyn was found to phosphorylate Lyn-associated and recombinant PKC-delta in vitro and the tyrosine 52 phosphorylated PKC-delta was recruited to associate with the Lyn SH2 domain. The constitutive association of PKC-delta with Src did not result in the tyrosine phosphorylation of PKC-delta prior to or after FsigmaRI engagement. However in cells over-expressing PKC-delta, FsigmaRI engagement resulted in the dramatic inhibition of Src activity and some inhibition of Lyn activity. Thus, the interaction and cross-talk of PKC-delta with Src family kinases suggests a novel and inter-dependent mechanism for regulation of enzymatic activity that may serve an important role in cellular responses.     

A role for JNK/SAPK in proliferation, but not apoptosis, of IL-3-dependent cells

Activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) has been implicated in the induction of apoptosis in a variety of systems [1] [2] [3] [4] [5] [6] [7] [8]. BAF3 cells are pre-B cells that undergo apoptosis following IL-3 withdrawal or ceramide treatment [9] [10]. JNK/SAPK in BAF3 cells is stimulated by ceramide and also during cell proliferation in response to IL-3 [11], but its role in the apoptotic response is not clear. We have devised a method of selectively inhibiting JNK/SAPK activity using a dual-specificity threonine/tyrosine phosphatase, M3/6. Expression of this phosphatase in BAF3 cells prevented ceramide stimulation of JNK/SAPK activity but did not affect apoptosis following IL-3 withdrawal or ceramide treatment. IL-3-stimulated proliferation of BAF3 cells expressing the phosphatase was, however, inhibited. Hence JNK/SAPK activation is likely to be involved in the proliferative response of these cells but is not required for apoptosis. Selective ablation by dual-specificity phosphatases should be a general method for determining the functions of specific mitogen-activated kinase pathways.     

Abrogation of IL-3 requirements and stimulation of hematopoietic cell proliferation in vitro and in vivo by carboxylic acids

Short-chain fatty acids, such as butyrate and propionate, induce fetal globin gene expression and are under clinical investigation in the beta-hemoglobinopathies. Limitations of the short-chain fatty acids as therapeutics include their rapid metabolism and a tendency to induce cell growth arrest if administered for prolonged periods. In studies described here, the cellular effects of other inducers of fetal globin, phenoxyacetic acid and derivatives of short-chain fatty acids and cinnamic acids, were investigated in the human erythroid cell line K562, the IL-3 dependent multi-lineage cell line (32D), and in mice and primates. Several test compounds supported 32D cell proliferation despite a 50-fold depletion of IL-3, which resulted in growth arrest and apoptotic death in control cells. The degree of proliferation induced by certain test compounds was similar to the degree of proliferation induced by Erythropoietin and G-CSF in the cells. Eight of ten compounds induced gamma globin mRNA in K562 cells. A 2.5 to 6-fold increase in reticulocytosis was observed in vivo in mice treated with two prototype compounds. Pharmacokinetic studies of three prototype compounds demonstrated millimolar plasma concentrations after single oral doses for many hours in primates. These findings identify orally bioavailable compounds which induce gamma globin gene expression and hematopoietic cell proliferation through an activity which partially abrogates requirements for IL-3. Such compounds provide potential for oral therapeutics which stimulate proliferation of hematopoietic cells of multiple lineages, as well as inducing fetal globin.     

Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation

The receptor tyrosine kinase Kit plays critical roles in hematopoiesis, gametogenesis and melanogenesis. In mast cells, Kit receptor activation mediates several cellular responses including cell proliferation and suppression of apoptosis induced by growth factor deprivation and gamma-irradiation. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules. We have investigated the role of phosphatidylinositol 3'-kinase (PI 3-kinase) and Src kinases in Kit-mediated cell proliferation and suppression of apoptosis induced both by factor deprivation and irradiation in bone marrow-derived mast cells (BMMC). Analysis of Kit-/- BMMC expressing mutant Kit receptors and the use of pharmacological inhibitors revealed that both signaling pathways contribute to these Kit-mediated responses and that elimination of both pathways abolishes them. We demonstrate that the PI 3-kinase and Src kinase signaling pathways converge to activate Rac1 and JNK. Analysis of BMMC expressing wild-type and dominant-negative mutant forms of Rac1 and JNK revealed that the Rac1/JNK pathway is critical for Kit ligand (KL)-induced proliferation of mast cells but not for suppression of apoptosis. In addition, KL was shown to inhibit sustained activation of JNK induced by gamma-irradiation and concomitant irradiation-induced apoptosis.     

Evaluation of apoptosis and cell proliferation in experimentally induced renal cysts

Cell proliferation and apoptosis in renal cysts induced by streptozotocin, alloxan and ferric-nitrilotriacetate were investigated in rats. In the kidneys of all treated animals dilated tubules at the cortico-medullary region, large cysts, glomerular cysts and tubular dilation in the medullary area were found. Both cell proliferation and apoptosis were increased in the epithelium of the non-dilated tubules, in the mesangial and interstitial cells. Cells lining the dilated tubules or cysts demonstrated apoptosis but their proliferating activity was low. By calculating the proliferation-apoptosis ratio we found that alloxan did not change the balance between the two mechanisms. Meanwhile streptozotocin resulted in an increased apoptosis and ferric-nitrilotriacetate in an increased cell proliferation. p53 expression might be responsible for the uncontrolled proliferation in rats treated with ferric-nitrilotriacetate as this oncoprotein was diffusely present in tubular cell nuclei. The observed apoptosis seemed to be independent of bcl-2 oncoprotein expression. We assume that the initial factor in such cystogenesis should be a cellular injury due to direct toxic or to the diabetogenic effect of the drugs. The latter is more likely as all the animals were hyperglycemic and insulin treatment following administration of streptozotocin prevented the morphologic changes.     

Regulation of proliferation, differentiation and survival by the IL-3/IL-5/GM-CSF receptor family

The receptors for the I1-3/IL-5/GM-CSF cytokine family are composed of a heterodimeric complex of a cytokine-specific alpha chain and a common beta chain (betac). Binding of IL-3/IL-5/GM-CSF to their respective receptors rapidly induces activation of multiple intracellular signalling pathways, including the Ras-Raf-ERK, the JAK/STAT, the phosphatidylinositol 3-kinase PKB, and the JNK/SAPK and p38 signalling pathways. This review focuses on recent advancements in understanding how these different signalling pathways are activated by IL-3/IL-5/GM-CSF receptors, and how the individual pathways contribute to the pleiotropic effects of IL-3/IL-5/GM-CSF on their target cells, including proliferation, differentiation, survival, and effector functions.     

Costimulation via TCR and IL-1 receptor reveals a novel IL-1alpha-mediated autocrine pathway of Th2 cell proliferation

Previous studies have shown that triggering of Th2 cells via the TCR is sufficient for production of IL-4 but not for proliferation of these cells. Proliferation of Th2 cells occurs only in the additional presence of a costimulatory signal delivered by IL-1. For the majority of Th2 cell clones, this type of proliferation was found to be independent of IL-4. Here, we further investigated the mechanism of IL-4-independent proliferation. We demonstrate that, after costimulation via TCR and IL-1R, but not via either receptor alone, Th2 cells are triggered to produce cell-associated IL-1alpha, as detected at the level of function, protein, and mRNA expression. In the presence of the TCR signal, autocrine IL-1alpha is then able to costimulate IL-4-independent proliferation of Th2 cells and to further enhance its own production. Thus, our results point to a novel, IL-4-independent, self-amplifying autocrine pathway of Th2 cell proliferation that requires a signal via the TCR and a costimulatory signal via IL-1R. This pathway may explain frustrating results in experimental models that attempted to treat established Th2-mediated diseases in vivo with IL-4-neutralizing agents alone.     

Transfected glucocorticoid receptor and certain GR fragments evoke cell death in malignant lymphoid, not myeloid cell lines

We compared the data from four growth hormone (GH) immunoassays for analyzing 24-h GH profiles in four apparently normal subjects and four obese subjects (508 serum samples). The detection limit was 0.02 microgram/L for one immunochemiluminometric assay (ICMA), 0.1 microgram/L for two IRMAs, and 0.4 microgram/L for one RIA. All GH pulses with a peak ICMA value > 1 microgram/L were detected by each of the other methods. Overall, the correlation coefficient between the values obtained with all four assays exceeded 0.90. However, for GH concentrations < or = 0.25 microgram/L, acceptable concordance (r2 > or = 0.80) was reached only between the ICMA and one IRMA; between the ICMA and the RIA, concordance was acceptable only for GH concentrations > or = 10 micrograms/L. In the normal subjects, the percentage of undetectable values was 0% with the ICMA but 29% with one of the IRMAs; in obese subjects, the corresponding values were 12% and 38%.     

Cytotoxicity of the anticancer agents cisplatin and taxol during cell proliferation and the cell cycle

The cerebral extraction and retention of three radioiodinatéd SPECT perfusion tracers were measured using residue detection in a baboon. A permeability-surface area product PS' with special relevance to SPECT was calculated from the retention of tracer in the brain after 10 min. PS' differs from the traditional PS value, which is calculated from the tracer clearance curve at 2 min. The PS' values ranged from 50 to 95 mL/min/100 g, decreased in the order [123I]IMP > [123I]iodoperidol approximately [123I]HIPDM, and did not differ for specific activities of 10 MBq/mmol to 74 TBq/mmol. These radioiodinated compounds exhibited extraction characteristics superior to those of [99mTc]HMPAO but underestimated cerebral blood flow when flows were above 20-30 mL/min/100 g, underscoring the need for development of a more ideal SPECT perfusion tracer.     

Phospholipase C-mediated signaling is altered during HaCaT cell proliferation and differentiation

The extracellular investment of oocytes in a number of species contains species-specific receptors to which spermatozoa bind as a prelude to fertilization; however, little is known about the nature and distribution of sperm receptors in avian oocytes. In order to elucidate the early step of fertilization in birds, we observed the binding of spermatozoa to the perivitelline layer (PL) of quail ova. When the PL obtained from the largest follicles were incubated in vitro with spermatozoa, perforations were observed. The presence of trypsin inhibitors during incubation inhibited the sperm-induced perforations of the PL and binding of spermatozoa to the PL could be observed. The number of spermatozoa bound to the PL increased in the ovum from more mature follicles, and concentrated binding of spermatozoa to the PL overlying the germinal disc region was observed in the largest follicle. The number of spermatozoa bound to the PL overlying the germinal disc region decreased in the oviposited eggs. These results demonstrate that sperm receptors exist in the PL over the germinal disc in the mature follicle.     

The interference effects of hexadecylphosphocholine on proliferation and membrane phospholipid metabolism in human myeloid leukemia cell lines

Membrane phospholipids are important regulators of cellular function. The phospholipid activities, such as lipid composition and transportation, contribute to cellular homeostasis in the lifespan of cells. Alterations in phospholipids result in the movement of bilayer lipids and the initiation of coagulation, recognition and internalization. Hexadecylphosphocholine (HePC) exerts antitumor potencies and represents a new class of antitumor agents targeted to the cellular membrane. Human myeloid leukemia cell lines HL-60 and K562 employed in this study were inhibited by HePC in vitro. The results indicate that the HL-60 cell line was sensitive, while K562 was resistant to HePC. Synthetic HePC is an alkyllysophospholipid analog which interacted with the cell membrane, thereby altering lipid composition and metabolism of membrane phospholipids and modulating intracellular calcium in human myeloid leukemia HL-60 and K562 cell lines. The contents of membrane phospholipids, including phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), were determined quantitatively with high performance liquid chromatography. The sensitivity of myeloid leukemia HL-60 and K562 cell lines to HePC probably depends on the different distribution of these four phospholipids in the cellular membrane, or on the response of these phospholipids to HePC. The cytosolic free calcium ([Ca++]i) concentration increased by HePC confirmed that [Ca++]i was released from the intracellular calcium pool and is associated with cell differentiation and apoptosis. We investigated the hypothesis that the antiproliferative effect of HePC was mediated through the interference with cellular membrane phospholipids, including choline-containing phospholipids (PC), aminophospholipids (PE and PS) and PI, in eukaryotic cells.     

CD40-mediated stimulation contributes to lymphocyte proliferation, antibody production, eosinophilia, and mastocytosis during an in vivo type 2 response, but is not required for T cell IL-4 production

CD40/CD40 ligand interactions are required for the development of T cell-dependent Ab responses in vivo. The role of these cell surface molecules in contributing to T cell cytokine production and the development of effector populations other than B cells and T cells is, however, less well defined. We have examined the in vivo effects of blocking CD40/CD40 ligand interactions on the type 2 mucosal immune response that follows oral inoculation of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of anti-gp39 (CD40L) mAb (MR1) blocked H. polygyrus-induced elevations in serum IgG1 levels and inhibited elevations in blood eosinophils and mucosal mast cells at day 14 after inoculation. Anti-gp39 mAb markedly inhibited B cell blastogenesis 8 days after H. polygyrus inoculation but did not inhibit elevations in B cell class II MHC expression. Maximal elevations in B7-2 expression required signaling through both CD40 and the IL-4R. Elevations in T cell cytokine gene expression and elevations in the number of IL-4-secreting cells were unaffected by treatment with anti-gp39 mAb, although IL-4 production was inhibited by anti-IL-4R mAb. These results suggest that CD40/CD40L interactions are not required to activate T cells to produce cytokines but are required for the activation and proliferation of other effector cells associated with the type 2 response.