An instructive component in T helper cell type 2 (Th2) development mediated by GATA-3.

Although interleukin (IL)-12 and IL-4 polarize naive CD4(+) T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

Role of the Stat4 N domain in receptor proximal tyrosine phosphorylation.

Stat4 is activated by the cytokines interleukin 12 and alpha interferon (IFN-alpha) and plays a significant role in directing development of naïve CD4(+) T cells to the Th1 phenotype. Signal transducers and activators of transcription (STAT) proteins undergo phosphorylation on a conserved tyrosine residue, resulting in homo- and heterodimerization, nuclear translocation, and DNA binding. Stat4 can bind to single IFN-gamma-activated sites (GASs) as a dimer or bind two tandem GASs as a pair of STAT dimers, or tetramer, stabilized through N-terminal domain (N domain) interactions between dimers. We uncovered an unexpected effect of the Stat4 N domain in controlling the proximal activation of Stat4 by tyrosine phosphorylation at activated receptor complexes. Mutation of the N domain at tryptophan residue W37, predicted to interrupt N domain dimer formation, unexpectedly prevented IFN-alpha-induced tyrosine phosphorylation of the Stat4 monomer, blocking dimer formation and nuclear translocation. Furthermore, N domains appear to exert private STAT functions, since interchanging the N domains between Stat1 and Stat4 prevented receptor-mediated tyrosine phosphorylation in one case and interrupted STAT-specific gene activation in another. Finally, replacement of the N domain of Stat1 with that of Stat4 abrogated the normal Stat2 dependence of Stat1 phosphorylation, again suggesting the domains are not equivalent. Thus, in addition to its role in STAT tetramerization, the conserved STAT N domain appears to participate in very proximal steps of receptor-mediated ligand-induced tyrosine phosphorylation.

A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules.

A synthetic DNA construct has been developed as a standard molecule whereby murine cytokine mRNA molecules can be quantified by the reverse transcription-polymerase chain reaction (RT-PCR). The construct, designated Cytoquant 1, allows the quantification of murine IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha, TGF-beta, GM-CSF, CD4, CD8, HPRT and beta-actin mRNA levels. This technique is based on the amplification of a transcribed RNA molecule from Cytoquant 1 as an internal standard control in both the RT and PCR reactions. The quantification data from these analyses are expressed in absolute values, i.e. molecules/cell, which allows the data derived from separate experiments to be compared. In this study, mRNAs encoding beta-actin, IL-10, IFN-gamma and GM-CSF have been quantitated in both Th1 and Th2 cell clones with, and without, stimulation. The quantitative analysis data are highly reproducible and cytokine mRNA concentrations are reflective of restricted cytokine secretion patterns. Furthermore, constitutive cytokine mRNA levels are detectable in resting cells, eliminating the need for exogenous stimulation. The high degree of sensitivity and accuracy make this methodology uniquely suited for the study of T-cell subset cytokine expression in both in vivo and in vitro biological models.

Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides.

Developing mammals are more sensitive than adults to a variety of organophosphorothioate insecticides (OPs), compounds which act in vivo by inhibition of cholinesterase (ChE). Little is known, however, regarding age-related differences in biochemical responses to these toxicants. The time course of ChE inhibition and recovery in whole brain was compared in neonatal (7 days of age) and adult (80-100 days of age) rats after treatment with maximal tolerated doses (MTDs) of either methyl parathion (MPS), parathion (PS) or chlorpyrifos (CPF). Neonatal rats were more sensitive than adults in all cases (MTDs for MPS, PS and CPF; neonates = 7.8, 2.1 and 45 mg/kg, s.c.; adults = 18, 18, and 279 mg/kg, s.c., respectively). In general, maximal brain ChE inhibition was similar (greater than 78%) in both age groups but ChE activity recovered faster in neonates. Plasma and erythrocyte ChE activities correlated relatively well (r = 0.794-0.943) with brain ChE activity in neonatal rats at all time points between 4 h and 7 days after treatment but similar correlations between circulating and brain ChE activities in adults were more variable (r = 0.211-0.917). The results indicate that neonatal rats are more sensitive to acute lethality from these compounds and that MTD exposures produce extensive brain ChE inhibition in both age groups. Significant inhibitor-related and age-related differences in the duration of ChE inhibition can ensue, however, following such OP exposures.

Comparative neurochemical and neurobehavioral effects of repeated chlorpyrifos exposures in young and adult rats.

Neonatal (7 days old) rats are markedly more sensitive than adults (3 months old) to the acute toxic effects of the insecticide, chlorpyrifos (CPF). In the present study, we have compared the effects of subacute CPF exposures in these same age groups. Repeated doses of CPF (40 mg/kg, SC, every 4 days, total of 4 doses) caused extensive inhibition of cortical, hippocampal, and striatal cholinesterase (ChE) activity in adult rats at 4 (90-92%) and 14 (71-78%) days after the last treatment. Rats treated similarly during postnatal maturation (beginning on day 7) showed a much lower degree of ChE inhibition (21-60%) at these time points. Muscarinic ([3H]quinuclidinyl benzilate, QNB) receptor binding in cortex, hippocampus, and striatum was reduced in adult brain at 4 (30-43%) and 14 (22-32%) days after the final treatment, whereas receptor densities were only marginally affected (5-11% reduction) in young rats. Basal motor activity levels were not affected in either young or adult rats as a function of CPF exposure. CPF-treated adult rats exhibited higher activity levels after challenge with scopolamine (1 mg/kg, IP) at 2, 4, 6, and 8 weeks after treatment, whereas CPF exposure did not affect the motoric response to scopolamine in rats treated during postnatal maturation. These data suggest that although neonatal rats are more sensitive to acute lethal effects from high doses of CPF, adult rats exhibit more persistent neurochemical and neurobehavioral alterations following repeated, lower-level exposures.

Long-term neurochemical and behavioral effects induced by acute chlorpyrifos treatment.

A single dose of the organophosphate insecticide O,O'-diethyl-O-3,5,6- trichloro-2-pyridylphosphorothioate [chlorpyrifos (CPF), 279 mg/kg, SC] caused extensive inhibition of cortical and striatal cholinesterase (ChE) activity in adult rats at 2 (94-96%), 4 (82-83%), and 6 (58-60%) weeks after treatment. These persistent changes in ChE activity were concomitant with reductions in muscarinic receptor binding sites in cortex (34, 33, and 18% reduction in Bmax) and striatum (48, 40, and 23% reduction in Bmax) at 2, 4, and 6 weeks after exposure. Neither ChE activities nor muscarinic receptor densities were different from control levels at 12 weeks after exposure. CPF treatment caused a reduction in locomotor activity for the first 2 days after treatment, after which basal activity levels were not different from controls. CPF-treated rats showed higher activity relative to controls, however, following challenge with scopolamine (1 mg/kg, IP) at 2, 4, 6, 8, and 12 weeks after treatment. These data indicate that acute exposure to CPF in adult rats can cause long-term neurobehavioral changes that may persist following the recovery of neurochemical parameters associated with exposure and tolerance to cholinesterase inhibitors.

Toxicity of sediment-associated nitroaromatic and cyclonitramine compounds to benthic invertebrates.

The toxicity of nitroaromatic (2,4-diaminonitrotoluene [2,4-DANT] and 1,3,5-trinitrobenzene [TNB]) and 14C-labeled cyclonitramine compounds (hexahydro-1,3,5-trinitro-1,3,5-triazine [RDX] and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine [HMX]) to the marine polychaete Neanthes arenaceodentata and the estuarine amphipod Leptocheirus plumulosus following 10- or 28-d exposures to spiked sediments was investigated. Organismal-level effects on survival, growth, and reproduction and cellular-level effects on apoptosis (programmed cell death) were evaluated. Because cyclonitramines have low affinity for sediment, overlying water was not exchanged in the RDX and HMX exposures. Nitroaromatics sorbed strongly to sediment, resulting in near complete resistance to solvent extraction. Cyclonitramines sorbed weakly to sediment, as more 14C-activity was found in the overlying water than in the sediment at exposure termination. No significant decrease in survival or growth was observed with cyclonitramines at initial sediment concentrations as high as 1,000 microg/g. Survival was significantly affected by nitroaromatics at nominal sediment concentrations as low as 200 microg/g, with L. plumulosus being more sensitive than N. arenaceodentata. Growth was significantly decreased at sublethal concentrations of 2,4-DANT for N. arenaceodentata. Reproduction, measured only with L. plumulosus, was significantly decreased only in the highest RDX treatment and also in the lower TNB treatment. However, no decrease was observed in higher concentrations of TNB. Body burden at exposure termination was below detection limit (1 microg/kg) for all compounds. Significant inhibition of apoptosis was not accompanied by significant decreases in growth or reproduction. Because of its critical function in many biological processes. alterations in this endpoint may result in adverse effects on the organism and could be used as an early indicator of toxicity.

Differential temporal and spatial expression of mRNA encoding extracellular matrix components in decidua during the peri-implantation period.

Changes in the temporal and spatial patterns of expression of mRNA encoding uterine extracellular matrix (ECM) proteins were determined during the peri-implantation period. Northern blot hybridization of cDNAs corresponding to laminin (LM) B1, LM B2, entactin, fibronectin, collagen (CL) type IV alpha 1, and CL IV alpha 2 was performed on RNA extracted from either whole mouse uteri or endometrial explants between Day 4, i.e., the day of implantation, and Day 7 of pregnancy, when the decidual response is well established. These analyses revealed a dramatic increase in LM B2, CL IV alpha 1, and CL IV alpha 2 mRNA expression by Day 7 of pregnancy. Relative levels of the mRNA encoding other ECM components, including LM B1, were not altered when compared to changes in the relative level of expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. The differential expression of the B chains of LM appeared to be limited to the stromal cells of the endometrium. In situ hybridization of uterine sections with cRNA probes corresponding to LM B1, LM B2, and CL IV alpha 1 demonstrated that LM B1 was expressed temporally in high amounts in the primary decidual zones (PDZ) and persisted throughout PDZ degeneration. LM B2 mRNA was expressed in both primary and secondary decidual zones and persisted through Day 8 of pregnancy. CL IV alpha 1 mRNA expression mimicked that of LM B2. Oviduct ligation on Day 2 of pregnancy was used to prevent embryo transport to one uterine horn, whereas decidualization and embryo implantation were permitted in the contralateral horn. This experiment demonstrated that the increases in uterine ECM mRNA expression were not due solely to the changing hormonal milieu of the uterus. ECM components, including CL IV, have been shown to bind growth factors such as transforming growth factor-beta (TGF-beta) in an insoluble but biologically active form. The remarkable similarity between the pattern of CL IV and LM B2 expression and previously reported TGF-beta deposition (Tamada et al., Mol Endocrinol 1990; 4:965-972) prompted examination of the effects of this growth factor on blastocyst development in vitro. TGF-beta 1 was tested for its ability to alter embryo outgrowth on LM-coated tissue culture surfaces; however, significant differences in the rate or extent of outgrowth in the presence of TGF-beta were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective mannosylation of glycosylphosphatidylinositol in Lec35 Chinese hamster ovary cells.

The Lec35 mutation (previously designated PIR) of Chinese hamster ovary cells is a recessive mutation that affects the participation of mannose-P-dolichol (MPD) in dolichol-P-P-oligosaccharide biosynthesis in vivo, even though MPD and the respective MPD-dependent mannosyltransferases are present. The Lec35 phenotype can be partially corrected by disrupting Lec35 cells and performing the transferase reactions in vitro, suggesting that the defect may be related to mislocalization of MPD. In this study, we examined the effect of the Lec35 mutation on glycosylphosphatidylinositol (GPI) lipid biosynthesis, another pathway that requires MPD. Our data indicate that the first mannosylation reaction of GPI lipid biosynthesis is defective in Lec35 cells, with the accumulation of glucosaminylphosphatidylinositol having a fatty acyl group on inositol and a base-resistant alkyl group attached to glycerol. The same intermediate accumulates in Lec15 (MPD synthase-defective) cells. The defective mannosylation reaction of Lec35 cells was corrected in vitro and shown to require MPD. These results demonstrate that the Lec35 gene governs a general aspect of MPD metabolism affecting both GPI lipid and dolichol-P-P-oligosaccharide biosynthesis. To provide additional insight into the role of the Lec35 gene, we give evidence for an inefficient pool of MPD in Lec35 membranes.

Recruitment of Stat4 to the human interferon-alpha/beta receptor requires activated Stat2.

Stat4 activation is involved in differentiation of type 1 helper (Th1) T cells. Although Stat4 is activated by interleukin (IL)-12 in both human and murine T cells, Stat4 is activated by interferon (IFN)-alpha only in human, but not murine, CD4(+) T cells. This species-specific difference in cytokine activation of Stat4 underlies critical differences in Th1 development in response to cytokines and is important to the interpretation of murine models of immunopathogenesis. Here, we sought to determine the mechanism of Stat4 recruitment and activation by the human IFN-alpha receptor. Analysis of phosphopeptide binding analysis suggests that Stat4 does not interact directly with tyrosine-phosphorylated amino acid residues within the cytoplasmic domains of either of the subunits of the IFN-alpha receptor complex. Expression of murine Stat4 in the Stat1-deficient U3A and the Stat2-deficient U6A cell lines shows that IFN-alpha-induced Stat4 phosphorylation requires the presence of activated Stat2 but not Stat1. Thus, in contrast to the direct recruitment of Stat4 by the IL-12 receptor, Stat4 activation by the human IFN-alpha receptor occurs through indirect recruitment by intermediates involving Stat2.

Selective activation of the N-glycosylation apparatus in uteri by estrogen.

Estrogen rapidly, preferentially and markedly enhances the rate of N-linked glycoprotein synthesis in mouse uteri. In contrast, the rate of glycoprotein turnover is unaffected by the hormone. Estrogen's effect on the expression of mRNA coding for glycoproteins was studied using an in vitro translation-glycosylation system as well as by Northern/slot blot analyses. Both approaches indicated that estrogen did not have a preferential stimulatory effect on the general expression of glycoprotein mRNA. Neither was there a significant change in the relative levels of specific mRNA coding for several N-linked glycoproteins, i.e. laminin B1 and B2, fibronectin, and uvomorulin, as a function of estrogen treatment. Immunoprecipitation studies also demonstrated no change in the relative rates of synthesis of the corresponding core proteins for laminin or fibronectin. Taken together, these results suggested that estrogen primarily stimulated glycoprotein synthesis by stimulating the glycosylation apparatus, and not by increasing synthesis of protein acceptors. Previous studies have indicated that of a variety of potential regulatory points in the pathway of N-linked glycoprotein assembly, only expression of mannosylphosphoryldolichol synthase (MPDS) increases sufficiently to account for the increase in glycoprotein expression observed in response to estrogen. Consistent with these observations, it was found that injection of uterine poly(A+) RNA from estrogen-treated uteri into Xenopus oocytes markedly stimulated MPDS activity in the oocytes. In contrast, injection of RNA from non-estrogen-treated uteri did not stimulate MPDS activity in oocytes. Collectively, these results indicate that steroid hormones can modulate glycoprotein expression by preferentially stimulating the glycosylation apparatus. Nonetheless, one of estrogen's effects on the glycosylation apparatus, induction of MPDS activity, appears to occur at a transcriptional level.

DDT toxicity and critical body residue in the amphipod Leptocheirus plumulosus in exposures to spiked sediment.

The lethal and sublethal toxicity of dichlorodiphenyltrichloroethane (DDT) to the estuarine amphipod Leptocheirus plumulosus was determined using sediment spiked with (14)C-labeled compound. Juvenile amphipods were exposed to concentrations up to 9.9 nmol/g dry weight (3.5 microg/g). Acute effects on survival were determined in a 10-day experiment. Chronic effects on survival, growth, and reproduction were assessed in a 28-day experiment. The DDT in the sediments transformed to dichlorodiphenyldichloroethane (DDD), dichlorodiphenyldichloroethylene (DDE), and polar metabolites during the 14-day sediment storage prior to exposing the amphipods. The mixture of DDT and its breakdown products (tDDT) was comprised mostly of DDT at the beginning of the exposures. DDD was the prevalent compound at termination of the 28-day exposure. Complete mortality occurred at sediment concentrations of tDDT as low as 7 nmol/g (2.3 microg/g) in both acute and chronic experiments. Most of the mortality appeared to have occurred within the first 4 days of exposure. No sublethal reductions in growth or reproduction were observed in the 28-day experiment. In the 10-day experiment, where amphipods did not receive supplemental food, growth was significantly increased in DDT treatments where survival was not affected. The concentration of tDDT in amphipod tissues was determined at exposure termination. In the 10-day experiment, a mean body residue of 14 nmol/g wet weight was associated with significant mortality (30%). Lower critical body residues were observed in the 28-day experiment, where the median lethal tissue residue (LR(50)) was 7.6 (6.8--8.4, 95% confidence interval) nmol/g wet weight. Based on previous studies, the lethal critical body residue for L. plumulosus is similar to those determined for freshwater amphipods and substantially lower than those determined for cladocerans and polychaetes.

Selective loss of type I interferon-induced STAT4 activation caused by a minisatellite insertion in mouse Stat2.

The use of murine systems to model pathogen-induced human diseases presumes that general immune mechanisms between these species are conserved. One important immunoregulatory mechanism involves linkage of innate and adaptive immunity to direct the development of T helper subsets, for example toward subset 1 (TH1) development through STAT4 activation. In analyzing type I interferon signaling, we uncovered a difference between murine and human cells which may affect how these two species control linkage between innate and adaptive immunity. We show that in humans, type I interferons induce TH1 development and can activate STAT4 by recruitment to the IFN-alpha receptor complex specifically via the carboxy-terminus of STAT2. However, the mouse Stat2 gene harbors a minisatellite insertion that has altered the carboxy-terminus and selectively disrupted its capacity to activate STAT4, but not other STATs. This defect in murine Stat2 suggests that the signals leading to STAT4 activation and TH1 development in CD4+ T cells are different between mice and humans.

Signaling and transcription in T helper development.

The recognition of polarized T cell subsets defined by cytokine production was followed by a search to define the factors controlling this phenomenon. Suitable in vitro systems allowed the development of cytokine "recipes" that induced rapid polarization of naïve T cells into Th1 or Th2 populations. The next phase of work over the past several years has begun to define the intracellular processes set into motion during Th1/Th2 development, particularly by the strongly polarizing cytokines IL-12 and IL-4. Although somewhat incomplete, what has emerged is a richly detailed tapestry of signaling and transcription, controlling an important T cell developmental switch. In addition several new mediators of control have emerged, including IL-18, the intriguing Th2-selective T1/ST2 product, and heterogeneity in dendritic cells capable of directing cytokine-independent Th development.

Cancer dormancy. VII. A regulatory role for CD8+ T cells and IFN-gamma in establishing and maintaining the tumor-dormant state.

Dormant tumor cells resistant to ablative cancer therapy represent a significant clinical obstacle due to later relapse. Experimentally, the murine B cell lymphoma (BCL1) is used as a model of tumor dormancy in mice vaccinated with the BCL1 Ig. Here, we used this model to explore the cellular mechanisms underlying dormancy. Our previous studies have demonstrated that T cell-mediated immunity is an important component in the regulation of tumor dormancy because Id-immune T cells adoptively transferred into passively immunized SCID mice challenged with BCL1 cells significantly increased the incidence and duration of the dormant state. We have extended these observations and demonstrate that CD8+, but not CD4+, T cells are required for the maintenance of dormancy in BCL1 Ig-immunized BALB/c mice. In parallel studies, the transfer of Id-immune CD8+ cells, but not Id-immune CD4+ cells, conferred significant protection to SCID mice passively immunized with nonprotective levels of polyclonal anti-Id and then challenged with BCL1 cells. Furthermore, the ability of CD8+ T cells to induce a state of dormancy in passively immunized SCID mice was completely abrogated by treatment with neutralizing alpha-IFN-gamma mAbs in vivo. In vitro studies demonstrated that IFN-gamma alone or in combination with reagents to cross-link the surface Ig induced both cell cycle arrest and apoptosis in a BCL1 cell line. Collectively, these data demonstrate a role for CD8+ T cells via endogenous production of IFN-gamma in collaboration with humoral immunity to both induce and maintain a state of tumor dormancy.