The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis.

Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.

Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation.

Immature double-positive (DP) thymocytes mature into CD4(+)CD8(-) cells in response to coengagement of TCR with any of a variety of cell surface "coinducer" receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.

Inhibition of interleukin-1-stimulated NF-kappaB RelA/p65 phosphorylation by mesalamine is accompanied by decreased transcriptional activity.

Nuclear factor kappaB (NF-kappaB) is an inducible transcription factor that regulates genes important in immunity and inflammation. The activity of NF-kappaB is highly regulated: transcriptionally active NF-kappaB proteins are sequestered in the cytoplasm by inhibitory proteins, IkappaB. A variety of extracellular signals, including interleukin-1 (IL-1), activate NF-kappaB by inducing phosphorylation and degradation of IkappaB, allowing nuclear translocation and DNA binding of NF-kappaB. Many of the stimuli that activate NF-kappaB by inducing IkappaB degradation also cause phosphorylation of the NF-kappaB RelA (p65) polypeptide. The transactivating capacity of RelA is positively regulated by phosphorylation, suggesting that in addition to cytosolic sequestration by IkappaB, phosphorylation represents another mechanism for control of NF-kappaB activity. In this report, we demonstrate that mesalamine, an anti-inflammatory aminosalicylate, dose-dependently inhibits IL-1-stimulated NF-kappaB-dependent transcription without preventing IkappaB degradation or nuclear translocation and DNA binding of the transcriptionally active NF-kappaB proteins, RelA, c-Rel, or RelB. Mesalamine was found to inhibit IL-1-stimulated RelA phosphorylation. These data suggest that pharmacologic modulation of the phosphorylation status of RelA regulates the transcriptional activity of NF-kappaB, independent of nuclear translocation and DNA binding. These findings highlight the importance of inducible phosphorylation of RelA in the control of NF-kappaB activity.

Gi proteins use a novel beta gamma- and Ras-independent pathway to activate extracellular signal-regulated kinase and mobilize AP-1 transcription factors in Jurkat T lymphocytes.

Receptors coupled to pertussis toxin (PTX)-sensitive Gi proteins regulate T lymphocyte cytokine secretion, proliferation, and chemotaxis, yet little is known about the molecular mechanisms of Gi protein signaling in mammalian lymphocytes. Using the Jurkat T lymphocyte cell line, we found that a stably expressed Gi protein-coupled receptor (the delta-opioid receptor (DOR1)) stimulates MEK-1 and extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) and transcriptional activity by an ERK target, Elk-1, via a mechanism requiring a PTX-sensitive Gi protein. Levels of beta-adrenergic receptor kinase-1 C-terminal fragment that inhibited signaling by Gi protein beta gamma subunits in these cells had no effect on DOR1 stimulation of either MEK-1- or Elk-1-dependent transcription, indicating that this pathway is independent of beta gamma. Analysis of this betagamma-independent pathway indicates a role for a herbimycin A-sensitive tyrosine kinase. Unlike beta gamma-mediated pathways, the beta gamma-independent pathway was insensitive to RasN17, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), and constitutive PI 3-kinase activity. The beta gamma-independent pathway regulates downstream events, since blocking it abrogated both Elk-1-dependent transcription and mobilization of the mitogenic transcription factor, AP-1, in response to DOR1 signaling. These results characterize a novel, Ras- and PI 3kinase-independent pathway for ERK activation by Gi protein signaling that is distinct from ERK activation by beta gamma and may therefore be mediated by the alphai subunit.

Delta-opioid receptors expressed by Jurkat T cells enhance IL-2 secretion by increasing AP-1 complexes and activity of the NF-AT/AP-1-binding promoter element.

Recent molecular evidence points to transient and/or stage-specific expression of delta- and kappa-opioid receptors by thymic and peripheral T lymphocytes. Since medical treatments or stress commonly increase opioid levels, it is important to understand the mechanisms by which opioids affect T lymphocyte functions. We therefore created and studied a T cell line expressing the cloned delta-opioid receptor (DOR1). DOR1 ligation by a specific DOR1 agonist, deltorphin, augmented IL-2 secretion by synergizing with signals from TCR-CD3 and CD28. Reporter gene constructs were used to map this effect of deltorphin to the AP-1- and NF-AT/AP-1-binding sites of the IL-2 promoter. Although DOR1 signaling increased [Ca2+]i, deltorphin enhanced transcriptional activity of the NF-AT/AP-1-binding site via a mechanism independent of calcineurin and distinct from the effects of elevated [Ca2+]i. Deltorphin also increased accumulation of AP-1 transcription factor complexes, suggesting that DOR1 augments IL-2 secretion by increasing the AP-1 component of the NF-AT/AP-1 transcription factor. These results advance the molecular understanding of opioid effects on lymphocytes, and in addition, demonstrate regulation of IL-2 synthesis and secretion by the novel mechanism of receptor-mediated AP-1 induction.

Class II histocompatibility molecules associate with calnexin during assembly in the endoplasmic reticulum.

Class II histocompatibility antigens are composed of polymorphic alpha and beta polypeptides which associate in the endoplasmic reticulum (ER) with a third, non-polymorphic invariant polypeptide (Ii). The alpha beta Ii complexes are subsequently transported through the Golgi to the endosomes, where the Ii chain dissociates before the alpha beta complex is transported to the cell surface. Results from transport-defective class II expression variant studies suggest that class II intracellular transport is regulated in multiple intracellular compartments. Consistent with this, a large number of studies have demonstrated that protein folding and/or oligomerization is facilitated in the ER by a class of proteins collectively known as molecular chaperones. In this report, we show that the ER-resident protein calnexin associates with human and murine class II antigens. Specifically, calnexin associates in the ER with free Ii polypeptides and partially assembled wild-type class II complexes, including A alpha and/or A alpha Ii complexes, as well as with alpha beta dimers isolated from class II transport-defective cells. Calnexin also physically associates with alpha beta Ii complexes, but not with mature alpha beta dimers. These results suggest that calnexin may regulate class II intracellular transport by facilitating the production of transport competent molecules out of the ER. In addition, we report that the nucleotide sequence of the gene encoding murine calnexin shows a high degree of homology to human IP90 and dog calnexin at both the nucleotide and deduced amino acid sequence level. The isolation of cDNA fragments encoding murine calnexin will allow us to further evaluate the functional consequences of calnexin-class II interaction.

Murine T helper cell-2 lymphocytes express type I and type II IL-1 receptors, but only the type I receptor mediates costimulatory activity.

The role of IL-1 in augmenting the Ag receptor-initiated activation program was evaluated in IL-4-producing (Th2) CD4+ murine T lymphocytes. Northern blot and 125I-labeled IL-1 alpha cross-linking analyses demonstrated that Th2 lymphocytes express both type I and type II IL-1R. The expression of both IL-1R isoforms on the surface of the Th2 cells is coordinately up-regulated in response to anti-CD3 cross-linking in the absence of detectable accessory cells. Analyses of the kinetics of IL-1R acquisition demonstrated that the peak level of type I and type II IL-1R mRNA expression occurs after the peak expression of mRNA encoding IL-2R alpha and IL-4, which are two IL-1-responsive events in the Th2 activation program. Type I IL-1R ligand-binding antagonists, IL-1R antagonist and anti-type I mAb, were used to evaluate the functional significance of Th2 cell expression of two IL-1R isoforms. The addition of either IL-1R antagonist or anti-type I mAb completely inhibited the IL-1 alpha-augmented component of the proliferative response stimulated by anti-CD3 plus exogenous IL-1 alpha. Together, these studies indicate that, although Th2 clones express inducible levels of both type I and type II IL-1R isoforms, the IL-1-induced intracellular signals involved in augmenting an anti-CD3-stimulated proliferative response are mediated solely through the type I IL-1R.

Recovery of viruses from three transport media incorporated into culturettes.

A double-blind prospective study was carried out to compare the rates of recovery of viruses from the upper respiratory tracts of children, using three different transport media. Stuart's, Hanks', and Leibovitz-Emory (LEM) media were packaged in the ampules of Culturettes and labeled in code by the manufacturer. Three swabs (one each from culturettes containing different media) were inserted simultaneously into the oropharynx or pressed onto unroofed dermal lesions, transported to the laboratory, and inoculated into MRC (Medical Research Council)-5 and primary monkey kidney-cell cultures. Eighty isolates were obtained from 200 specimens (40%) collected during all four seasons of the year. Parainfluenza virus, enterovirus, adenovirus, and herpes simplex virus accounted for 79% of the isolates. Of 80 isolates, 72 (90%) were recovered in Hanks', 64 (80%) in Stuart's, and 63 (79%) in LEM. The differences were not significant.