Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

Cell death in the Schwann cell lineage and its regulation by neuregulin.

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.

Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.

Expression of corticotropin-releasing factor in inflamed tissue is required for intrinsic peripheral opioid analgesia.

Immune cell-derived opioid peptides can activate opioid receptors on peripheral sensory nerves to inhibit inflammatory pain. The intrinsic mechanisms triggering this neuroimmune interaction are unknown. This study investigates the involvement of endogenous corticotropin-releasing factor (CRF) and interleukin-1beta (IL-1). A specific stress paradigm, cold water swim (CWS), produces potent opioid receptor-specific antinociception in inflamed paws of rats. This effect is dose-dependently attenuated by intraplantar but not by intravenous alpha-helical CRF. IL-1 receptor antagonist is ineffective. Similarly, local injection of antiserum against CRF, but not to IL-1, dose-dependently reverses this effect. Intravenous anti-CRF is only inhibitory at 10(4)-fold higher concentrations and intravenous CRF does not produce analgesia. Pretreatment of inflamed paws with an 18-mer 3'-3'-end inverted CRF-antisense oligodeoxynucleotide abolishes CWS-induced antinociception. The same treatment significantly reduces the amount of CRF extracted from inflamed paws and the number of CRF-immunostained cells without affecting gross inflammatory signs. A mismatch oligodeoxynucleotide alters neither the CWS effect nor CRF immunoreactivity. These findings identify locally expressed CRF as the predominant agent to trigger opioid release within inflamed tissue. Endogenous IL-1, circulating CRF or antiinflammatory effects, are not involved. Thus, an intact immune system plays an essential role in pain control, which is important for the understanding of pain in immunosuppressed patients with cancer or AIDS.

Differential effects of staphylococcal toxic shock syndrome toxin-1 on B cell apoptosis.

Superantigens, such as toxic shock syndrome toxin 1 (TSST-1), have been implicated in the pathogenesis of several autoimmune and allergic diseases associated with polyclonal B cell activation. In this report, we studied the in vitro effects of TSST-1 on B cell activation. We show herein that TSST-1 produced antagonistic effects on Ig synthesis by peripheral blood mononuclear cells (PBMC) from normal subjects, depending on the concentration used; Ig production was inhibited at 1000 pg/ml (P < 0.01) and enhanced at 1 and 0.01 pg/ml (P < 0.01) of toxin. Cultures of PBMC were then examined for morphologic features and DNA fragmentation characteristic for apoptosis. B cells exhibited a significantly higher (P < 0.01) incidence of apoptosis after stimulation with 1000 pg/ml of TSST-1 compared with 1 or 0.01 pg/ml of toxin or medium alone. Abundant expression of Fas, a cell surface protein that mediates apoptosis, was detected on B cells after stimulation with 1000 pg/ml of TSST-1 and was significantly higher on B cells undergoing apoptosis than on live cells (P = 0.01). Additionally, increased Fas expression and B cell death occurred at concentrations of TSST-1 inducing the production of high amounts of gamma interferon (IFN-gamma), and both events could be blocked by neutralizing anti-IFN-gamma antibody. These findings suggest that high concentrations of TSST-1 can induce IFN-gamma-dependent B cell apoptosis, whereas at low concentrations it stimulates Ig synthesis by PBMC from normal subjects. These findings support the concept that staphylococcal toxins have a role in B cell hyperactivity in autoimmunity and allergy.

Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.

Human V delta 2+ gamma delta T-cell tolerance to foreign antigens of Toxoplasma gondii.

Little is known about the mechanisms involved in human gammadelta T-cell tolerance to self or to foreign antigens. Patients with congenital toxoplasmosis offer a unique opportunity to examine Vdelta2+ gammadelta T-cell tolerance. Analysis of gammadelta T cells in patients with congenital toxoplasmosis revealed evidence for anergy of these cells with or without clonal Vdelta2+ gammadelta T-cell expansion in the acute phase of the Toxoplasma infection. T cells in general were unresponsive and did not proliferate upon exposure to mitogens or to Toxoplasma lysate antigens or in response to live Toxoplasma-infected cells when the congenitally infected infants were 1 month of age, and they exhibited selective anergy to Toxoplasma lysate antigens and live Toxoplasma-infected cells when the infants were aged 5 months. During the chronic phase of congenital toxoplasmosis in the patients who were more than I year of age, the repertoires of the gammadelta T-cell receptors were found to be within normal ranges. In addition, in the chronic phase, the gammadelta T cells proliferated and secreted gamma-interferon in response to exposure to live Toxoplasmia-infected cells. By contrast, alphabeta T cells remained anergic. Vdelta2+ gammadelta T cells have been considered to undergo extrathymic maturation and thus to be subject to development of peripheral tolerance. Our findings indicate that Vdelta2+ gammadelta T-cell tolerance was lost in these infected infants earlier than alphabeta T-cell tolerance. These findings suggest that gammadelta T cells play a role in protection against Toxoplasma gondii in the chronic phase when congenitally infected children are more than 1 year of age, especially in those in whom alphabeta T cells continue to exhibit deficits in specific immune responses to Toxoplasma antigens.

Prolactin increases CD4/CD8 cell ratio in thymus-grafted congenitally athymic nude mice.

One distinctive effect on T-cell development was analyzed by selectively increasing serum prolactin (PRL) concentration in thymus-grafted congenitally athymic nude mice and by neutralizing PRL in suspension cultures of thymus from 1-day-old neonatal mice. Flow cytometric analysis of single-positive CD4+ and CD8+ cells derived from inguinal lymph nodes revealed a CD4/CD8 cell ratio of 2.2 +/- 0.18 (mean +/- SEM) in thymus-grafted nude mice that is similar to the ratio for immune-competent BALB/c mice (2.0 +/- 0.06). Addition of the pituitary to thymus-grafted nude mice significantly elevated serum PRL (P < 0.005) and increased the CD4/CD8 cell ratio (2.8 +/- 0.12; P < 0.005), demonstrating preferential stimulation of CD4+ cell development. T cells in nude mice receiving sham (submandibular salivary gland) or pituitary grafts alone were below detectable levels. Suspension cultures of neonatal thymus treated with anti-mouse PRL antiserum resulted in 20% and 30% decreases in double-positive CD4+8+ thymocytes and thymocyte viability, respectively. A 10-fold increase in double-negative CD4-8- thymocytes expressing the interleukin 2 receptor alpha chain, CD25, was also observed concurrently. Our findings illustrate an important way in which PRL may participate in two interrelated mechanisms: the regulation of peripheral single-positive cells and the maintenance of thymocyte viability during the double-positive stage of intrathymic differentiation.

Intravenous injection of soluble antigen induces thymic and peripheral T-cells apoptosis.

The mechanism by which tolerance is induced via systemic administration of high doses of aqueous antigen has been analyzed by using mice transgenic for a T-cell receptor specific for the influenza virus hemagglutinin (HA) peptide comprising amino acids 126-138. After intravenous injection of 750 (but not 75) micrograms of HA peptide, a state of hyporesponsiveness was rapidly induced. In the thymus, in situ apoptosis in the cortex and at the corticomedullary junction was responsible for a synchronous and massive deletion of CD4+ CD8+ thymocytes. In secondary lymphoid organs, HA-reactive T cells were initially activated but were hyporesponsive at the single cell level. After 3 days, however, those cells were rapidly deleted, at least partially, through an apoptotic process. Therefore, both thymic and peripheral apoptosis, in addition to T-cell receptor desensitization, contribute to high-dose tolerance.

Olfactory neuroblastoma is a peripheral primitive neuroectodermal tumor related to Ewing sarcoma.

Olfactory neuroblastoma (ONB) is a malignant tumor of the nasal mucosa whose histogenesis is unclear. A relationship to neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is based on morphologic similarities and the expression of similar neural antigens. However, the clinical presentation of ONB differs from that of NB, and MYCN amplification characteristic of NB is not observed. We have therefore examined the relationship of this malignancy to other classes of neural tumors. In previous studies, two ONB cell lines demonstrated cytogenetic features and patterns of protooncogene expression suggestive of a relationship to the Ewing sarcoma family of childhood peripheral primitive neuroectodermal tumors (pPNETs). The pPNETs show t(11;22)(q24;q12) or t(21;22)(q22;q12) chromosomal translocations fusing the EWS gene from 22q12 with either the FL11 gene on 11q24 or the ERG gene on 21q22. We therefore analyzed ONBs for the presence of pPNET-associated gene fusions. Both cell lines showed rearrangement of the EWS gene, and fluorescence in situ hybridization (FISH) of each case demonstrated fusion of EWS and FL11 genomic sequences. Moreover, both lines expressed EWS/FL11 fusion transcripts with in-frame junctions between exon 7 of EWS and exon 6 of FL11 as described for pPNETs. We identified similar gene fusions in four of six primary ONB cases. None of the cases expressed tyrosine hydroxylase, a catecholamine biosynthetic enzyme widely expressed in NB. Our studies indicate that ONB is not a NB but is a member of the pPNET family.

T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.

Localization of choline acetyltransferase in rat peripheral sympathetic neurons and its coexistence with nitric oxide synthase and neuropeptides.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1.

Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression.

The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4+ T cells by CD8+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8+ T cells. However, IL-2 induces CD8+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability to IL-2 to induce HIV expression. Five to 25 times fewer CD8+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25+) CD8+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies.