bluuebunny/crossref_metadata_2025 huggingface dataset:
A role for calcium influx in setting the threshold for CD4+CD8+ thymocyte negative selection.
Authors: Kane L P , Hedrick S M
Published: December 2022

We have applied an in vitro system that mimics thymic negative selection to investigate signaling pathways that may be important for the removal of autoreactive cells from the thymus. We sought to more precisely determine the contribution of calcium-dependent pathways to CD4+CD8+ thymocyte deletion that is mediated by either an antigenic peptide or a peptide analogue. We show that the requirement for external calcium influx is dependent upon the strength of the deleting ligand. Furthermore, these results correlate well with a requirement, under certain circumstances, for signaling through the calcium/calmodulin-dependent phosphatase calcineurin. The use of suboptimal stimuli may, therefore, be useful in revealing biochemical pathways important for CD4+CD8+ thymocyte negative selection.
In vivo T cell response to viral superantigen. Selective migration rather than proliferation.
Authors: Le Bon A , Lucas B , Vasseur F , Penit C , Papiernik M
Published: December 2022

Superantigens induce T cell activation and proliferation in vitro, and some also induce cell activation in vivo. MMTV(SW) is an infectious mouse mammary tumor virus (MMTV) encoding a superantigen with the same Vbeta specificity as MIs-1a (Mtv-7), which induces a strong local response in vivo. injection of MMTV(SW) into mouse footpads leads to accumulation of superantigen-reactive T cells (Vbeta6+CD4+) and B cells in the draining lymph nodes (LN). We investigated the kinetics of this cell accumulation by measuring cell activation (blastogenesis, CD25 and CD69 expression), cell migration (using syngenic FITC-labeled CD4+ cells and L-selectin detection), and cell proliferation (using in vivo labeling with bromodeoxyuridine). Specific T cells selectively migrated to the draining LN. Accumulating Vbeta6+CD4+ T cells were large CD69+ cells, but remained CD25 negative and showed down-regulated L-selectin expression. Their DNA synthesis rate, studied by pulse labeling and continuous administration of bromodeoxyuridine, was increased, but remained too low to explain the draining LN hyperplasia. These data show that the local T cell response to MMTV(SW) mainly consists of selective migration followed by local activation of reactive T cells, and that cell proliferation is only a minor component of the response. By contrast, the optimal dose of staphylococcal enterotoxin B that, nevertheless, leads to a lower reactive T cell accumulation in the draining LN induces a very high proliferation rate.
Characterization of molecular interactions between intercellular adhesion molecule-1 and leukocyte function- associated antigen-1.
Authors: Woska J R , Morelock M M , Jeanfavre D D , Bormann B J
Published: December 2022

The ability of soluble ICAM-1 (sICAM-1) to inhibit ICAM-1/LFA-1 adhesion events has been reported previously by numerous investigators. sICAM-1 has been demonstrated to inhibit various in vitro assays at concentrations ranging from 2 nM to greater than 40 microM. Given the hypothesis that circulating ICAM-1 modulates immune functions, the ability of sICAM-1 to inhibit cellular functions may have significant ramifications. Considering the potential clinical importance of the interaction between ICAM-1 and its receptor, LFA-1, it is necessary to understand this receptor-ligand interaction at a molecular level. In this study, direct binding experiments were utilized to determine the affinity between biotinylated monomeric sICAM-1 and immobilized LFA-1 (approximately 130 nM). Competitive binding experiments with unlabeled sICAM-1 and a truncated form of sICAM-1 (D1D2) yielded similar affinities. The specificity of this interaction was characterized using mAbs directed against sICAM-1 or LFA-1. This assay system was extended to include multimeric species using nonblocking mAbs directed against domains D4 and D5 of sICAM-1. Dimerizing sICAM-1 with a mAb alphaD4 or alphaD5 increased the affinity for immobilized LFA-1 by two orders of magnitude (approximately 4 nM), an effect presumably due to avidity. These results indicate that while the monomeric sICAM-1/LFA-1 interaction may involve only a moderate binding affinity, multimeric ICAM-1 present on a cell surface may bind cell surface-immobilized LFA-1 with very high avidity. These sICAM-1/LFA-1 molecular assays should be useful in defining the efficacy of potential antagonists.
Murine IL-12 is involved in Calmette-Guérin bacillus-induced sensitization and is by itself sufficient to sensitize mice to the lethal effects of human TNF1.
Authors: Cauwels A , Fiers W , Brouckaert P
Published: December 2022

Human TNF, a specific agonist of murine (m)TNF-R55, is fairly harmless in healthy mice, but readily induces lethality in animals sensitized by a malignant tumor or a bacterial infection. We here report that IL-12 is both a necessary and sufficient mediator in Calmette-Guérin bacillus-induced sensitization to the lethal effects of hTNF. Evidence for this involvement is provided by the protection observed after anti-mIL-12 Ab treatment and by the fact that Calmette-Guérin bacillus-induced sensitization can be mimicked by repetitive mIL-12 administration. IL-12 exerts this effect by inducing IFN-gamma in NK cells. Tumor-induced sensitization, however, does not seem to involve mIL-12.
NK cell infiltration into lung, liver, and subcutaneous B16 melanoma is mediated by VCAM-1/VLA-4 interaction.
Authors: Fogler W E , Volker K , McCormick K L , Watanabe M , Ortaldo J R , Wiltrout R H
Published: December 2022

The mechanisms that regulate the adhesion and migration of NK cells to and across endothelium have been studied under nonflow conditions; however, the involvement of these processes in vivo is poorly understood. The present studies investigated the potential vascular adhesion ligand interactions that determine the in vivo recruitment of NK cells to pulmonary and hepatic parenchyma, and s.c. tumor after treatment of mice with biologic response modifiers. Seventy-two hours after a single injection of the cytokine-inducing agent poly-L-lysine stabilized in carboxylmethyl cellulose (poly-ICLC), pulmonary NK cell lytic activity and N-alpha-carbobenzoxy-L-lysine thiobenzyl ester (BLT)-esterase were augmented 29- and 14-fold, respectively, and the number of lung-associated NK cells was increased from 2.3 x 10(5) to 7.4 x 10(5). Similar fold increases in NK cell number and activity were observed in the liver and s.c. B16 melanoma after poly-ICLC injection or in the lungs and liver of mice treated with IL-2. Concomitant treatment of mice with alpha-VCAM-1 or alpha-VLA-4 mAb, but not alpha-ICAM-1 or alpha-LFA-1, abrogated the poly-ICLC and IL-2-induced increase in organ-associated NK activity and percentage of tumor-associated NK cells, resulted in a 61 to 76% decrease in pulmonary and hepatic NK cell number, and was independent of T and/or B cells. The decrease in NK cell number in organ parenchyma and tumor lesions was correlated to an increase in the number of NK cells in peripheral blood, but not bone marrow. These results demonstrate that VCAM-1/VLA-4 interaction is critically involved in the infiltration of newly recruited NK cells in to lung, liver, and progressively growing tumor after mobilization from the bone marrow.
Respiratory syncytial virus infection enhances neutrophil and eosinophil adhesion to cultured respiratory epithelial cells. Roles of CD18 and intercellular adhesion molecule-1.
Authors: Stark J M , Godding V , Sedgwick J B , Busse W W
Published: December 2022

Respiratory syncytial virus (RSV) infections in children precipitate acute episodes of respiratory obstruction that are associated with influx of inflammatory cells into the airway. Since RSV can induce the expression of adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), by the respiratory epithelium, the hypothesis has been proposed that ICAM-1 expression contributes to airway inflammation by supporting adhesion and retention of infiltrating inflammatory leukocytes. To test this hypothesis, A549 cells (an immortalized human alveolar epithelial type II cell-like fine) were infected with RSV, and the ability of these infected monolayers to support adhesion by human neutrophils (NEUT) and eosinophils (EOS) was measured. RSV infection significantly increased ICAM-1 expression by A549 monolayers (p < 0.001). Although NEUT adhesion to A549 monolayers was significantly enhanced following RSV infection (p < 0.001), infection alone resulted in little change in EOS adherence. However, if EOS were first activated with phorbol ester (PMA), adhesion to both control and RSV-infected A549 cells was enhanced, with greater levels of adhesion supported by RSV-infected cultures (p < 0.001). The requirement for EOS activation (but not for NEUT activation) before adhesion remained when NEU and EOS were prepared and compared from the same donor. Despite this difference, NEUT and EOS adhesion was reduced by blocking Abs to epithelial ICAM-1 or granulocyte CD18 adhesion proteins (p < 0.01). However, only NEUT adhesion was blocked by Ab to CD11a. Our results show that RSV infections of respiratory epithelial monolayers can promote inflammatory cell adherence which could, in turn, potentially contribute to the airway injury and obstruction that accompanies bronchiolitis.
IL-4 and IFN-gamma synergistically increase total polymeric IgA receptor levels in human intestinal epithelial cells. Role of protein tyrosine kinases.
Authors: Denning G M
Published: December 2022

IL-4 and IFN-gamma increase release of secretory component (SC), the polymeric IgA (plgA)-binding segment of the plgA receptor (plgAR), by the human intestinal epithelial cell line HT29. Moreover, these two cytokines synergistically increase plgA binding and cell surface staining for the receptor. To understand better the mechanism by which these cytokines regulate plgAR, we did quantitative immunoblotting using Abs against secretory component. We found that synergy occurs at the level of total cellular plgAR. Additionally, time course studies indicated that maximal receptor levels required >24-h incubation, that reaching maximal levels required at least 18 h of cytokine treatment, and that receptor levels remained elevated as long as cytokines were present. Conversely, if cytokines were removed, then cellular plgAR levels decreased with an approximate t1/2 of 20 h. Finally, synergy required the simultaneous presence of both cytokines throughout the treatment period. Direct measurement of second messengers and inhibitor studies suggest that Ca2+, cAMP, protein kinase A, and protein kinase C do not play major roles in regulating cellular plgAR levels by either cytokine, and do not contribute to the mechanism of synergy. In contrast, protein tyrosine kinase inhibitors potently inhibited all cytokine-dependent increases in total cellular plgAR. These results suggest that IL-4 and IFN-gamma increase cellular plgAR levels in HT29 cells predominantly by activating protein tyrosine kinase-dependent signaling pathways.
Evolution of hemopoietic ligands and their receptors. Influence of positive selection on correlated replacements throughout ligand and receptor proteins.
Authors: Shields D C , Harmon D L , Whitehead A S
Published: December 2022

The rates of amino acid replacement in cytokines and their receptors are high and vary considerably. To determine whether this reflects the action of positive selection, rates of nonsynonymous DNA substitution were examined and found to exceed the synonymous substitution rate in certain exons of rodent IL-3, granulocyte-macrophage stimulating factor, and IL-4. To determine the extent to which positive selection could account for correlations between the amino acid replacement rates of hemopoietins and their receptors, rates were examined in various domains: the correlation with ligand rate was not confined to the ligand-binding domain of the receptor, but extended into the transmembrane and cytoplasmic domains and even to leader peptide domains of both ligand and receptor. As the majority of these replacements are unlikely to be strongly advantageous, different levels of both positive and purifying selection contribute to the extensive variation in hemopoietin/receptor evolutionary rates. Changes in a few residues critical for ligand-receptor interaction may be followed by changes of lesser selective importance in both molecules: replacements of growth hormone residues that form hydrogen or salt bridges with the receptor occur in lineages in which there are many concurrent replacements. A ligand/receptor rate correlation is not found between the seven-transmembrane receptors and their ligands, whose mature forms are often short and completely conserved. This study predicts that a minority of concurrent evolutionary changes in hemopoietins and their receptors reflect directly compensatory changes.
The antitumoral effect of the immunomodulator AS101 and paclitaxel (Taxol) in a murine model of lung adenocarcinoma.
Authors: Kalechman Y , Shani A , Dovrat S , Whisnant J K , Mettinger K , Albeck M , Sredni B
Published: December 2022

The immunomodulator ammonium trichloro(dioxyethylene-0-0')tellurate (AS101) has been shown to possess antitumoral properties in several murine models. In the present study, we demonstrate a synergistic in vivo antitumor effect of AS101 and Taxol against early stage Madison 109 lung adenocarcinoma. Treatment with optimal doses of Taxol (25 and 17 mg/kg) and AS101 (0.5 mg/kg) resulted in 66.6 and 43.3% cures. We propose that the antitumor effect is the result of both a direct and indirect effect of the drugs on tumor cells. AS101 and Taxol directly inhibited clonogenicity of M109 cells in a synergistic dose-dependent manner. Exposure of M109 cells to clinically achievable concentrations of Taxol and AS101 produced a synergistic internucleosomal DNA fragmentation associated with programmed cell death. We suggest that AS101 renders tumor cells more susceptible to chemotherapy in general and to Taxol in particular, partly by increasing the wild-type p53 protein expression that is required for efficient execution of the death program. Moreover, we demonstrate a synergistic effect of AS101 and Taxol in increasing the tumoricidal activity of macrophages. This activity is produced by nitric oxide secretion. The synergistic antitumoral effects of AS101 and Taxol were partly ablated both in vitro and in vivo by inhibition of nitric oxide synthase. These findings indicate that AS101 in combination with Taxol may be a promising antitumor drug, and illustrate the mechanism of action of both drugs when acting synergistically. Phase II clinical trials have been initiated using AS101 in combination with Taxol.
Herpes simplex virus type 1-mediated up-regulation of IL-12 (p40) mRNA expression. Implications in immunopathogenesis and protection.
Authors: Kanangat S , Thomas J , Gangappa S , Babu J S , Rouse B T
Published: December 2022

In this report we investigated whether induction of IL-12 occurs in response to herpes simplex virus (HSV) infection of the mouse eye, which may serve to regulate the nature of the subsequent immune response. The data show early induction and continued maintenance of IL-12 (p40) mRNA in the cornea and draining lymph node upon ocular infection with HSV. Using a very sensitive radioimmunoassay technique, IL-12 (p40) protein also was detected in the cornea upon ocular HSV infection. Unfractionated splenocytes and enriched populations of dendritic cells, macrophages, and neutrophils all responded to HSV infection in vitro by up-regulating the expression of IL-12 (p40) mRNA. However, cultured corneal cells failed to generate IL-12 (p40) upon exposure to HSV. The data indicate that inflammatory cells that infiltrate the cornea in response to HSV-1 infection are the main source of IL-12 in the eye. Our results are consistent with the hypothesis that IL-12 induction in the eye acts as a triggering event that biases HSV-specific immunity to a type 1 T cell response, which, in the environment of the eye, culminates in an immunopathologic disease.
B7-1 is superior to B7-2 costimulation in the induction and maintenance of T cell-mediated antileukemia immunity. Further evidence that B7-1 and B7-2 are functionally distinct.
Authors: Matulonis U , Dosiou C , Freeman G , Lamont C , Mauch P , Nadler L M , Griffin J D
Published: December 2022

Although intact, viable tumor cells rarely induce a clinically significant immune response in vivo, immunogenicity can be elicited by irradiated tumor cells that protect against subsequent challenge with wild-type intact viable tumor cells. Genetic modification of murine tumor cells, by transfection of cDNAs encoding either cytokines, MHC molecules, or costimulatory molecules, has been capable of inducing antitumor immunity. We and others have previously demonstrated that expression of the B7-1 costimulatory molecule, in either immunogenic or nonimmunogenic tumors, can protect against subsequent challenge with wild-type tumor cells. In this work, using a murine model of acute myeloid leukemia, we demonstrate that the B7-1 costimulatory molecule is superior to the B7-2 molecule in its capacity to protect against wild-type tumor challenge and eradicate minimal residual disease. These results provide compelling evidence that the B7-1 and B7-2 costimulatory signals are functionally distinct, thus resulting in clinically significant differences in the induction of antitumor immunity in vivo.
Bacterial lipopolysaccharide induces the association and coordinate activation of p53/56lyn and phosphatidylinositol 3-kinase in human monocytes.
Authors: Herrera-Velit P , Reiner N E
Published: December 2022

p53/56lyn and other src family tyrosine kinases become activated in monocytes treated with LPS. In a variety of systems, phosphatidylinositol 3-kinase (PI 3-kinase) is believed to be a downstream effector of tyrosine kinases, and activation of PI 3-kinase results in increased levels of D3-phosphorylated metabolites of phosphatidylinositol (PtdIns). To examine whether LPS activates PI 3-kinase, freshly isolated human, peripheral blood monocytes were labeled in vitro with [32P]orthophosphate, and inositol phospholipids were detected after extraction and separation of lipids by TLC. Levels of PtdIns 3,4,5-trisphosphate (PtdIns 3,4,5-P3) were elevated within minutes of exposure of cells to LPS. Analysis of 32P-labeled lipid extracts of U937 cells by HPLC confirmed that levels of PtdIns 3,4,5-P3 increased rapidly following LPS treatment. Increased levels of PtdIns 3,4,5-P3 in LPS-treated cells resulted from an increase in the specific activity of PI 3-kinase. Thus, anti-PI 3-kinase immunoprecipitates prepared from unlabeled monocytes and assayed in an in vitro phosphorylation assay, using PtdIns as substrate, showed higher enzymatic activity when these were prepared from lysates of LPS-treated cells as compared with control cells. PI 3-kinase activity in immunoprecipitates was elevated as early as 2 min after LPS exposure and was dose dependent, with increased activity being observed at LPS concentrations as low as 10 pg/ml. Activation of PI 3-kinase involved signaling through the monocyte cell surface molecule CD14, since pretreatment of cells with Abs to CD14 abrogated LPS-induced increases in PtdIns 3,4,5-P3. Immunoprecipitates of p53/56lyn from LPS-treated cells showed a time-dependent and transient increase in PI 3-kinase activity assayed in vitro, coordinate with activation of p53/56lyn, indicating that LPS induces the association and simultaneous activation of these two enzymes in vivo. These findings indicate that monocytes respond to LPS with the rapid activation of PI 3-kinase, resulting in transient increases in levels of PtdIns 3,4,5-P3. This process is CD14 dependent and involves the physical association of PI 3-kinase with activated p53/56lyn.
Study of complement-mediated anaphylaxis in humans. The role of IgG subclasses (IgG1 and/or IgG4) in the complement-activating capacity of immune complexes.
Authors: Bergamaschini L , Santangelo T , Faricciotti A , Ciavarella N , Mannucci P M , Agostoni A
Published: December 2022

The role of Ig classes and subclasses in complement activation has been investigated both in vitro and in experimental animals, but not in humans. This study was conducted to determine the immunologic events of post-transfusion anaphylaxis in humans, and the effects of immune complexes of different IgG subclass compositions on complement activation. The ability of immune complexes containing mixed IgG1 and IgG4 or IgG4 Ab only to activate complement was investigated in two patients with von Willebrand's disease (a congenital bleeding disorder). This disease was complicated by precipitating IgG alloantibodies to von Willebrand factor (vWF), which triggered complement-mediated anaphylaxis after infusion of vWF-containing preparations. Complement activation was greater in the presence of mixed IgG1- and IgG4-vWF complexes than with IgG4-vWF alone. In one patient, the generation of large amounts of C4a, C3a, and terminal complement complexes following the formation of mixed IgG1- and IgG4-vWF was associated with life-threatening anaphylaxis. In this patient, IgG4 appeared to have no inhibitory effect on antigen-bound IgG1-mediated complement activation. In the other patient, formation of IgG4-vWF led to a lesser degree of complement activation and was associated with moderately severe anaphylaxis. Since neither patient showed any biochemical alterations indicating the involvement of mast cells or the contact phase of coagulation at any time, it is probable that the pathogenetic mechanism of the clinical syndrome was a direct effect of complement anaphylatoxins on vascular permeability and smooth muscle contraction. In both patients, IgG-vWF bound C4 and C3 (IgG4-vWF to a lesser extent than mixed IgG1- and IgG4-vWF), and this probably prevented serum sickness as a complication.
HIV glycoprotein 120 enhances intercellular adhesion molecule-1 gene expression in glial cells. Involvement of Janus kinase/signal transducer and activator of transcription and protein kinase C signaling pathways.
Authors: Shrikant P , Benos D J , Tang L P , Benveniste E N
Published: December 2022

It is well established that the two major glial cells in the central nervous system (CNS), astrocytes and microglia, are key participants in mediating the neurologic dysfunction associated with HIV infection of the CNS. In this study, we investigated the ability of the major envelope glycoprotein of HIV, glycoprotein 120 (gp120), to regulate intercellular adhesion molecule-1 (ICAM-1) expression in glial cells, because ICAM-1 is important in mediating immune responsiveness in the CNS, facilitating entry of HIV-infected cells into the CNS, and promoting syncytia formation. Our results indicate that gp120 enhances ICAM-1 gene expression in primary rat astrocytes, primary human astrocytes, a human astroglioma cell line CRT, and primary rat microglia. The signal transduction events involved in gp120-mediated enhancement of ICAM-1 appear to involve activation of both protein kinase C and tyrosine kinase, because inhibitors of protein kinase C and tyrosine kinase abrogate gp120-mediated ICAM-1 expression in both astrocytes and microglia. Moreover, gp120 induces tyrosine phosphorylation of signal transducer and activator of transcription (STAT-1 alpha) as well as the Janus kinase (JAK2) in glial cells. We also demonstrate that gp120-mediated ICAM-1 expression has functional significance, as it enhances the ability of monocytic cells to bind to gp120-stimulated human astrocytes in an ICAM-1/beta 2 integrin-dependent fashion. These results provide new insights into how gp120 can influence the involvement of glial cells in the pathogenesis of AIDS dementia complex.
CD28-mediated up-regulation of beta 1-integrin adhesion involves phosphatidylinositol 3-kinase.
Authors: Zell T , Hunt S W , Mobley J L , Finkelstein L D , Shimizu Y
Published: December 2022

Cross-linking of the CD28 Ag on T cells results in increased beta 1-integrin-mediated adhesion to fibronectin. Chimeric contructs containing the CD28 cytoplasmic domain fused to the extracellular and transmembrane regions of CD2 were expressed in HL60 cells to investigate CD28-mediated regulation of adhesion. Ab cross-linking of the CD2/28 chimera resulted in increased beta 1-dependent adhesion of HL60 transfectants to fibronectin. Induced binding was completely inhibited by the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Cross-linking of the CD2/28 chimera also induced association of the p85 subunit of PI 3-K with the CD2/28 cytoplasmic domain. In contrast, cross-linking of a CD2/28 chimera containing a tyrosine to phenylalanine substitution in the YMNM motif did not result in increased adhesion to fibronectin and did not lead to association of the chimera with PI 3-K. These results directly implicate the YMNM motif and PI 3-K in the regulation of beta 1-integrin activity by the CD28 Ag.
Chemokines regulate T cell adherence to recombinant adhesion molecules and extracellular matrix proteins.
Authors: Lloyd A R , Oppenheim J J , Kelvin D J , Taub D D
Published: December 2022

Chemokines are a family of structurally related, low m.w. proteins that regulate leukocyte migration both in vitro and in vivo. By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites of inflammation during the genesis of an immune response. Chemokines have been shown to induce leukocyte adhesion to endothelium, to facilitate trans-endothelial passage, and to direct cell migration along a protein gradient (chemotaxis). The chemokines (macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, RANTES, and IFN-inducible protein-10) have recently been reported to be chemotactic for T cells. We have investigated the potential activity of these proteins in regulation of T cell adhesion. These chemokines induce T cell adhesion to purified, recombinant human adhesion molecules (rhICAM-1, rhVCAM-1) and to ECM proteins: fibronectin, collagen, and laminin. The chemokine-induced adhesion process occurs rapidly, is dose-dependent, and appears to be mediated via beta 1 and beta 2 integrins. The enhanced T cell adhesion is not associated with an increased surface expression of adhesion proteins, suggesting that chemokines stimulate the development of a high affinity state in the integrin molecules. Our findings provide in vitro evidence of a critical role for chemokines in T cell adhesion to endothelial adhesion molecules and ECM proteins, thereby promoting haptotactic migration of T cells to sites of inflammation in vivo.
Requirement for phosphatidylinositol 3'-kinase to protect hemopoietic progenitors against apoptosis depends upon the extracellular survival factor.
Authors: Minshall C , Arkins S , Freund G G , Kelley K W
Published: December 2022

Hemopoietic growth factors promote survival of progenitor cells by preventing their apoptotic death. Recently, phosphatidylinositol 3'-kinase (PI 3-kinase) has been shown to be integral in the pathway by which insulin and nerve growth factor prevent apoptosis. In this work, we show that IL-3-dependent FDCP-1/Mac-1 murine hemopoietic progenitors express receptors for another growth factor, insulin-like growth factor-I (IGF-I), and that both IL-3 and IGF-I stimulate PI 3-kinase activity. We then demonstrate that IGF-I shares with IL-3 the properties of significantly promoting proliferation and enhancing survival of myeloid progenitor cells at concentrations as low as 3 ng/ml. IL-3 and IGF-I efficiently promote cell survival in the presence of inhibitors of either RNA synthesis (actinomycin D) or mitosis (mitomycin C), suggesting that both ligands promote survival by a process that is largely independent of RNA synthesis. To determine whether PI 3-kinase mediates IL-3- and IGF-I-induced inhibition of apoptosis, FDCP-1/Mac-1 cells were incubated with the PI 3-kinase inhibitor, wortmannin. While wortmannin inhibited both basal and IGF-I- and IL-3-induced PI 3-kinase enzyme activity, it did not affect the ability of IL-3 to protect FDCP-1/Mac-1 cells from apoptosis, even though it abrogated the IGF-I-induced inhibition of apoptosis. These data demonstrate that even though activation of PI 3-kinase is a pleiotropic feature of both IL-3 and IGF-I receptors in myeloid progenitors, prevention of apoptosis by IL-3 but not IGF-I is independent of PI 3-kinase. Survival of hemopoietic progenitors is therefore maintained by at least two different intracellular signaling pathways, one requiring PI 3-kinase and one that does not.
Thymic stromal cells can support B cell differentiation from intrathymic precursors.
Authors: Montecino-Rodriguez E , Johnson A , Dorshkind K
Published: December 2022

T lineage cells are the predominant intrathymic population, but low numbers of thymic B cells are also present in the thymus. Recent studies describing the contribution of thymic B cells to the shaping of the T cell repertoire have raised the question as to whether they are produced intrathymically and the potential role of the thymic stroma in this process. During the course of studies aimed at testing the B cell developmental potential of CD4-CD8-CD3/TCR- triple-negative thymocytes present in a newly developed, stromal cell-dependent, long term culture system, the ability of selected thymic stromal cells to support distinct stages of B cell development was revealed. Some thymic stromal cell lines maintained cells with B cell developmental potential but could not support their differentiation, while other selected lines efficiently supported the development of surface IgM-expressing cells from triple-negative precursors. Thymic surface IgM+ B cells were generated most efficiently from thymocytes harvested from relatively young mice. Taken together, the results of this study demonstrate that the thymic microenvironment exhibits a functional heterogeneity in its ability to support different stages of B cell development.
IL-7 differentially modulates the expression of IFN-gamma and IL-4 in activated human T lymphocytes by transcriptional and post-transcriptional mechanisms.
Authors: Borger P , Kauffman H F , Postma D S , Vellenga E
Published: December 2022

We investigated the role of IL-7 on the expression of IFN-gamma and IL-4 in human T lymphocytes. IL-7 alone did not induce IFN-gamma or IL-4 mRNA. However, IL-7 dose-dependently up-regulates the anti-CD3- or anti-CD3/anti-CD28-induced IFN-gamma and IL-4 mRNA expression. Used at an optimal concentration, IL-7 (5 ng/ml) increased the accumulation of IFN-gamma (eightfold) and IL-4 (2.5-fold) mRNAs, which could not be blocked by anti-IL-12 treatment. The enhanced IFN-gamma mRNA accumulation was observed within 3 to 6 h, without altering the pattern of the kinetics. However, longer exposure (> 12 h) did not result in different IFN-gamma expression for anti-CD3/anti-CD28 vs anti-CD3/anti-CD28 plus IL-7-stimulated T lymphocytes. mRNA stability studies revealed that IL-7 stabilizes both IFN-gamma and IL-4 mRNA transcripts: 40 and 60 min in anti-CD3/anti-CD28-stimulated T cells vs 120 and 90 min in T cells costimulated with anti-CD3/anti-CD28 plus IL-7. Nuclear run-on assays revealed that the transcription rate of the IFN-gamma gene increased approximately twofold in the presence of IL-7, without affecting the transcription rate of the IL-4 gene. The IL-7-mediated IFN-gamma up-regulation could not be inhibited by cycloheximide treatment, in contrast to IL-4 gene expression. However, the promotive effect of IL-7 on IFN-gamma and IL-4 gene expression could be blocked by genistein and cyclosporin A. Finally, it was demonstrated that the effect of IL-7 on IFN-gamma mRNA accumulation was also reflected at the protein level. In summary, these data demonstrate that IL-7 preferentially up-regulates IFN-gamma expression in activated T lymphocytes, which is accomplished at transcriptional and post-transcriptional levels.
Role of the TCR binding region of the HLA class I alpha 2 domain in regulation of cell adhesion and proliferation.
Authors: Pettersen R D , Hestdal K , Lie S O , Gaudernack G
Published: December 2022

In addition to Ag presentation for T cell surveillance, MHC molecules have been implicated in mediating regulatory signals. We have assessed biologic responses following engagement of the TCR accessible region of the HLA class I alpha 2 domain. mAbs directed to this domain specifically induced cell aggregation of normal hematopoietic and leukemic cells. The functional consequences were unique since other mAbs reactive with HLA class I residues outside the TCR binding domain did not induce cell aggregation. The adhesion response required ATP, mRNA, protein, and actin synthesis and did not depend on LFA-1/ICAM interactions. Cell aggregation was also induced when all but four of the intracytoplasmic residues of the class I molecule were deleted, indicating that transduction of signals leading to cell adhesion does not require this portion of the molecule. mAbs directed to HLA class I alpha 2 amino acid residues within the TCR binding domain were also able to inhibit proliferation of normal mitogen-stimulated T cells. Growth inhibition correlated with down-regulated expression of CD25, CD28, and CD95, suggesting that reduced transduction of costimulatory signals is involved. Although HLA class I signals inducing cell aggregation required engagement of positions within the TCR binding region, growth inhibitory signals could be generated through positions both within and adjacent to this domain. Taken together, engagement of specific positions within the TCR binding domain of the class I alpha 2 helix results in active cellular responses. Thus, this region may be directly involved in signal transduction following CTL recognition of target cells.
Contribution of nitric oxide to the host parasite equilibrium in toxoplasmosis.
Authors: Hayashi S , Chan C C , Gazzinelli R , Roberge F G
Published: December 2022

We studied the effect of nitric oxide (NO) production on the evolution of toxoplasmosis in C57BL/6 mice. Infection was induced by i.p. injection of Toxoplasma gondii strain ME49. NO synthesis was inhibited by treatment with aminoguanidine, a structural analogue of L-arginine. The severity of infection was evaluated by histopathologic examination of the brain. In the infected mice treated for 2 wk with aminoguanidine, we observed an increase in the number of toxoplasma tachyzoites and intracellular cysts accompanied by an exacerbated inflammation of the brain tissue compared with that in controls. When spleen cells from infected mice were stimulated in culture with toxoplasma Ag, there was a marked cytotoxic effect on cells collected during the acute stage of infection and an inhibition of proliferation of the remaining viable lymphocytes. These effects were correlated with high levels of NO and PGE2 production. The suppression of NO synthesis prevented cell death and restored the lymphocyte proliferative response as well as lymphokine production. The neutralization of IFN-gamma or TNF-alpha had no effect on NO production in the cultures of infected mouse spleen cells. Cultures in which purified macrophages and lymphocytes from infected and naive mice were mixed indicated that the production of NO was dependent on lymphocyte activation. In the later stages of infection, when the production of NO was abating, preventing PGE2 secretion with indomethacin also increased the lymphocyte proliferative response. We conclude that the opposing effects of NO in toxoplasmosis, which protects against Toxoplasma gondii and at the same time limits the immune response, probably contribute to the establishment of the characteristic chronic state of host parasite equilibrium.
Acute phase levels of C-reactive protein enhance IL-1 beta and IL-1ra production by human blood monocytes but inhibit IL-1 beta and IL-1ra production by alveolar macrophages.
Authors: Pue C A , Mortensen R F , Marsh C B , Pope H A , Wewers M D
Published: December 2022

C-reactive protein (CRP), the major acute phase protein in humans, was purified free of endotoxin (LPS) (< 10 pg of LPS/mg of purified CRP) and evaluated for its ability to modulate LPS-induced production of IL-1 beta and IL-1 receptor antagonist (IL-1ra) from human PBMC and lung macrophages. PBMC (5 x 10(6)/ml) released low levels of IL-1 beta in response to either CRP (250 micrograms/ml) or LPS (100 ng/ml) for 18 h (0.3 +/- 0.1 and 1.5 +/- 0.7 ng/ml, respectively). However, when CRP (250 micrograms/ml) and LPS (100 ng/ml) were combined, PBMC released 9.7 +/- 2.9 ng/ml (p < 0.001 vs LPS alone). This synergy was removed by immunodepletion of CRP before stimulation. With respect to IL-1ra, although CRP induced IL-1ra production from PBMC (0.8 +/- 0.3 ng/ml control, 2.6 +/- 1.3 ng/ml with CRP), CRP did not synergize with LPS for IL-1ra production (15.0 +/- 0.7 ng/ml LPS alone vs 15.4 +/- 1.4 ng/ml LPS and CRP). In contrast, lung macrophages responded to CRP quite differently than PBMC. Macrophages (10(6)/ml) were not stimulated to produce IL-1 beta or IL-1ra by CRP alone. When combined with LPS, CRP inhibited IL-1 beta and IL-1ra release induced by LPS (for IL-1 beta release, LPS induced 3.0 +/- 1.7 ng/ml vs 1.1 +/- 0.4 for combined LPS and CRP; for IL-1ra release, LPS induced 12.9 +/- 2.3 ng/ml vs 7.6 +/- 2.3 ng/ml for combined LPS and CRP). These data suggest that acute phase levels of CRP may have divergent effects depending on the target population. CRP may be largely proinflammatory to blood monocytes responding to LPS since IL-1 beta production is augmented over IL-1ra production. However, in tissue compartments the effects of CRP may be largely immunosuppressive to LPS-induced tissue macrophage IL-1 beta production.
Ligand-independent activation of the glucocorticoid receptor by ursodeoxycholic acid. Repression of IFN-gamma-induced MHC class II gene expression via a glucocorticoid receptor-dependent pathway.
Authors: Tanaka H , Makino Y , Miura T , Hirano F , Okamoto K , Komura K , Sato Y , Makino I
Published: December 2022

The therapeutic effectiveness of ursodeoxycholic acid (UDCA) for various autoimmune liver diseases strongly indicates that UDCA possesses immunomodulatory activities. Experimental evidence also supports this notion, since, for example, UDCA has been shown to suppress secretion of IL-2, IL-4, and IFN-gamma from activated T lymphocytes, and Ig production from B lymphocytes. To investigate the mechanical background of UDCA-mediated immunomodulation, we asked whether UDCA interacts with the intracellular signal transduction pathway, especially whether it is involved in immunosuppressive glucocorticoid hormone action. For this purpose, we used a cloned Chinese hamster ovary cell line, CHOpMTGR, in which glucocorticoid receptor cDNA was stably integrated. In immunocytochemical analysis, we found that treatment with UDCA promoted the nuclear translocation of the glucocorticoid receptor in a ligand-independent fashion, which was further confirmed by immunoprecipitation assays. Moreover, the translocated glucocorticoid receptor demonstrated sequence-specific DNA binding activity. Transient transfection experiments revealed that treatment of the cells with UDCA marginally enhanced glucocorticoid-responsive gene expression. We also showed that UDCA suppressed IFN-gamma-mediated induction of MHC class II gene expression via the glucocorticoid receptor-mediated pathway. Together, UDCA-dependent promotion of translocation of the glucocorticoid receptor may be associated with, at least in part, its immunomodulatory action through glucocorticoid receptor-mediated gene regulation.
The main IgE-binding epitope of a major latex allergen, prohevein, is present in its N-terminal 43-amino acid fragment, hevein.
Authors: Alenius H , Kalkkinen N , Reunala T , Turjanmaa K , Palosuo T
Published: December 2022

Polypeptides of natural rubber latex (NRL) that elute from surgeon's gloves and other manufactured rubber products can sensitize exposed individuals and elicit severe hypersensitivity reactions. Previously, we showed that prohevein is a major allergen in NRL, the source material for rubber manufacturing. To analyze which region of the molecule carries the main IgE-binding epitope(s), we purified prohevein and its C-domain from NRL by gel filtration, reverse phase chromatography, and electroelution. In immunoblotting, prohevein bound IgE from 15 of 20 (75%), and the prohevein C-domain from 3 of 20 (15%) latex-allergic patient sera. In ELISA, 36 of 52 (69%) patient sera showed IgE binding to prohevein, whereas 11 of 52 (21%) sera had IgE to prohevein C-domain. We then purified from a brand of highly allergenic surgeon's gloves six hydrophilic peptides that revealed in amino-terminal sequencing 100% identity to the N-terminus of prohevein. In mass spectrometry, all purified peptides gave a molecular mass of 4719 +/- 1.9 daltons, which corresponds to the molecular mass of hevein (4719.1 daltons), a 43-amino acid N-terminal fragment of prohevein. Purified hevein inhibited 72% of IgE binding from pooled sera of NRL-allergic patients to solid phase glove extract and 45% of IgE binding to solid phase NRL. Of the 43 NRL-allergic patient sera tested, 56% showed IgE Abs to purified hevein in ELISA. In skin prick testing, purified hevein elicited positive reactions in three-quarters of the latex-allergic patients tested. These results indicate that the majority of prohevein's IgE-binding ability resides in its N-terminal fragment, known as hevein. In one highly allergenic latex glove examined, the majority of IgE-binding ability was attributable to hevein molecules, suggesting that these peptides can be significant sensitizers in NRL allergy.
HIV-1-Tat modulates the function of monocytes and alters their interactions with microvessel endothelial cells. A mechanism of HIV pathogenesis.
Authors: Lafrenie R M , Wahl L M , Epstein J S , Hewlett I K , Yamada K M , Dhawan S
Published: December 2022

Monocytes are major targets of HIV infection in patients with AIDS. In vitro infection of monocytes with HIV is associated with increased expression of beta 2 integrins, which increases both monocyte aggregation and monocyte/endothelial adhesion as well as monocyte metalloproteinase (MMP-9) expression. Treatment of primary monocytes with soluble HIV-Tat protein mimicked many of the properties of HIV infection of monocytes. Tat treatment up-regulated the expression of the beta 2 integrins, which was associated with the formation of large aggregates of monocytes and increased adhesion to endothelial monolayers. Treatment of monocytes with Tat increased their adhesion to both untreated and TNF-alpha-treated endothelial monolayers, and adhesion was inhibited by inclusion of anti-beta 2 and anti-ICAM-1 Abs. The increased adhesion of activated monocytes was accompanied by substantial disruption of the endothelial monolayers, with retraction or detachment of individual endothelial cells. Tat treatment of monocytes up-regulated the synthesis and release of the protease MMP-9, providing a potential mechanism to explain endothelial cell/basement membrane detachment. Thus, extracellular Tat is capable of activating monocytes even in the absence of HIV infection. Our studies demonstrate that many of the effects of HIV infection on monocyte homotypic and heterotypic adhesion, protease secretion, and disruption of the endothelium can be mimicked by treatment with HIV-Tat protein alone. These results suggest a mechanism where monocytes could be inappropriately activated by HIV-Tat, secreted by HIV-infected cells, causing them to extravasate into underlying tissues and ultimately contribute to tissue damage as seen during the progression of AIDS.
Serum high molecular weight dipeptidyl peptidase IV (CD26) is similar to a novel antigen DPPT-L released from activated T cells.
Authors: Duke-Cohan J S , Morimoto C , Rocker J A , Schlossman S F
Published: December 2022

This study demonstrates that the 175-kDa form of dipeptidyl peptidase IV (DPPIV) found in normal human serum is identical with a similarly-sized Ag, DPPT-L, found to be rapidly expressed on the surface of activated T cells. As activation progresses, the expression of DPPT-L reaches a peak on day 3, after which expression falls, whereas expression of the 105-kDa CD26/DPPIV detected by the mAb 1F7 increases, as does the ability to bind adenosine deaminase. The loss of DPPT-L from the surface of activated T cells correlates exactly with the appearance of DPPT-L and DPPIV activity in serum-free tissue culture medium. The release of DPPIV was generally greater from CD4+ cells than from CD8+ T cells, and within the CD4+ subset, the CD45RO+ subset was the major source, which correlated with surface expression before culture. We show that the DPPIV released from activated T cells is antigenically, biochemically, and enzymatically similar to DPPIV circulating in the serum and is distinct from the DPPIV activity of 105-kDa CD26. The T cell-released DPPIV is able to function as a costimulating molecule for the response to the recall Ag, tetanus toxoid, at levels similar to those at which recombinant soluble CD26 and serum DPPIV exhibit costimulatory function, suggesting that the released DPPIV may serve an important immunoregulatory function in vivo, both locally and within the systemic circulation.
Biochemical characterization of antigen-specific glycosylation-inhibiting factor from antigen-specific suppressor T cells. I. Identification of a 55-kilodalton glycosylation-inhibiting factor peptide with TCR alpha-chain determinant.
Authors: Nakano T , Ishii Y , Ishizaka K
Published: December 2022

Stimulation of OVA-specific suppressor T cell (Ts) hybridoma and bee venom phospholipase A2 (PLA2)-specific Ts hybridoma with Ag-pulsed APC or by cross-linking of CD3 resulted in the formation of Ag-specific glycosylation-inhibiting factor (GIF). Affinity-purified Ag-specific GIF preparations, obtained by using Ag-coupled Sepharose or anti-TCR alpha-chain-coupled Affi-Gel, contained a 55-kDa peptide, which bound both polyclonal anti-GIF Abs and anti-TCR-alpha mAb in immunoblotting. The same hybridomas constitutively secrete 13-kDa bioactive GIF peptide that has no affinity for homologous Ag, but neither the Ag-specific GIF activity nor 55-kDa GIF peptide was detectable in culture supernatants of unstimulated cells. Northern blot analysis of mRNA from the anti-CD3-stimulated hybridoma with 32P-labeled GIF cDNA revealed only 0.6 kb mRNA, which encodes the 13-kDa nonspecific GIF. No mRNA of the 55-kDa GIF was detectable. A representative OVA-specific Th hybridoma, DO 11.10 cells contain the 0.6 kb GIF mRNA and constitutively secrete inactive GIF peptide. However, the Th hybridoma failed to secrete the 55-kDa peptide or any peptide with the TCR-alpha determinant upon stimulation with anti-CD3. It appears that the formation of the 55-kDa peptide with the TCR-alpha determinant is unique for a subset of T cells including Ts cells that form bioactive GIF.
Cells treated with TAP-2 antisense oligonucleotides are potent antigen-presenting cells in vitro and in vivo.
Authors: Nair S K , Snyder D , Gilboa E
Published: December 2022

Treatment of RMA and EL4 cells or freshly isolated splenocytes with antisense (AS) oligonucleotides directed against the TAP-2 gene recreates the phenotype seen in cells that are genetically deficient in TAP function. Cells incubated with AS oligonucleotides exhibit reduced MHC class I expression on the cell surface, which can be increased by incubating the oligonucleotide-treated cells at 28 degrees C or by adding MHC haplotype-matched peptides to the culture medium. RMA cells or splenocytes treated with AS oligonucleotides and incubated with peptide were highly effective in generating primary CTL responses in vitro. The bulk of the AS oligonucleotide-responsive and CTL-inducing cells resided in the adherent fraction of splenocytes. Moreover, TAP-2 AS oligonucleotide-treated adherent splenocytes pulsed with OVA peptide elicited potent OVA-specific CTL responses in vivo and provided effective protection from challenge with tumor cells expressing the corresponding Ag. AS oligonucleotide technology provides a simple approach to develop broadly applicable methods for generating potent APC to study TAP function in normal cells and to identify other gene products involved in MHC class I presentation.