bluuebunny/crossref_metadata_2025 huggingface dataset:
Signal transduction mediated by the reconstituted IL-2 receptor. Evidence for a cell type-specific function of IL-2 receptor beta-chain.
Authors: Minami Y , Oishi I , Liu Z J , Nakagawa S , Miyazaki T , Taniguchi T
Published: December 2022

The binding of IL-2 to its specific receptor (IL-2R) triggers various cellular events including the induction of nuclear proto-oncogenes (c-fos, c-jun and c-myc genes) and the proliferation of hemopoietic cells. In the present study, we have established NIH 3T3 fibroblasts in which the three IL-2R subunits, the alpha-chain (IL-2R alpha), the beta-chain (IL-2R beta), and the gamma-chain (IL-2R gamma), are constitutively expressed. The resulting cell lines express high affinity IL-2R on their cell surface at levels comparable with those of IL-2-responsive lymphoid cells. We observed that the high affinity IL-2R in NIH 3T3 fibroblasts can mediate the IL-2-stimulated tyrosine phosphorylation of p42/p44 (mitogen-activated protein kinase) and the induction of the c-fos, c-jun and c-myc genes. In NIH 3T3 fibroblasts the high affinity IL-2R bearing a deletion of a region rich in acidic amino acids (the "acidic" region) in the IL-2R beta-chain failed to induce the tyrosine phosphorylation of MAP kinase as well as the expression of the all three nuclear proto-oncogenes. On the other hand, our previous studies had demonstrated that the high affinity IL-2R bearing the same mutant IL-2R beta-chain retained the ability to induce c-myc gene in response to IL-2 in a murine IL-3-dependent pro-B cell line, BAF/B03. Hence, these results reveal the underlying complexity of signal transduction among different cell types. The inability of the reconstituted high affinity receptor to mediate the IL-2-induced proliferation of NIH 3T3 fibroblasts suggests that induction of the three nuclear proto-oncogenes and the tyrosine phosphorylation of mitogen-activated protein kinase in NIH 3T3 fibroblasts are not sufficient to induce cellular proliferation.
Structure, chromosomal localization, and methylation pattern of the human mb-1 gene.
Authors: Ha H , Barnoski B L , Sun L , Emanuel B S , Burrows P D
Published: December 2022

The Ag receptor on B lymphocytes is a multimeric complex that is composed of an Ag-specific component, surface Ig, which is noncovalently associated with at least two other proteins, Ig alpha and Ig beta. These are the glycoprotein products of the B lineage-restricted mb-1 and B29 genes and are crucial for the cell surface expression and function of the Ag receptor on B lymphocytes. To better understand the regulation of mb-1, we have cloned and sequenced a 5.7-kb genomic DNA fragment that contained the human gene. The overall structure of human mb-1 is very similar to that of the murine gene, including the number and approximate size of exons. The promoter region lacks a TATA element, but contains two copies of an early B cell factor-binding motif, which previously has been shown to be important for murine mb-1 expression. Other structural features include two nuclear factor-kappa B binding sites at the 5' end of the gene and a long stretch of AG rich-sequence between exons 3 and 4, downstream of an Alu repeat sequence that contains a potential stem-loop structure. The mb-1 gene was localized to chromosome 19q13.2-13.3 by a combination of two methods, PCR amplification of DNA from a somatic cell hybrid-mapping panel and fluorescence in situ hybridization. An examination of the methylation pattern revealed a striking correlation between demethylation in the 5' region of the gene and expression of mb-1. The demethylated HpaII/MspI sites are adjacent to the nuclear factor-kappa B-binding motifs, which suggests a role for this transcription factor in the regulation of human mb-1 gene expression.
Distinct effects of human herpesvirus 6 and human herpesvirus 7 on surface molecule expression and function of CD4+ T cells.
Authors: Furukawa M , Yasukawa M , Yakushijin Y , Fujita S
Published: December 2022

This study was undertaken to investigate the effects of newly isolated T lymphotropic viruses, human herpesvirus (HHV)-6 and HHV-7, on CD4+ T cells. We first examined changes in surface molecule expression on CD4+ T cells after infection with HHV-6 or HHV-7 by flow cytometry. Among surface molecules examined, CD3 expression appeared to decline markedly after infection with HHV-6 variant A (strain U1102) but the decreased level of CD3 expression after infection with HHV-6 variant B (strain Z29) was slight. Impairment of surface CD3 expression on HHV-6 variant A-infected cells was also demonstrated by measuring intracellular free Ca2+ concentration in response to anti-CD3 mAb. In contrast, HHV-7 infection induced a marked loss of surface CD4 expression, but the decline of CD3 expression was slight. Cytotoxic activity of virus-specific CD4+ CTL clones decreased after infection with both HHV-6 variant A or HHV-7 but the degree of reduction of cytotoxicity by HHV-6 variant B was not significant. Addition of lectin restored the cytotoxicity of HHV-7-infected CTL but not that of HHV-6 variant A-infected CTL. Northern blot analysis and immunoprecipitation showed that infection with HHV-6 and HHV-7 did not affect the transcription and protein synthesis of CD3 and CD4. These findings suggest that both HHV-6 and HHV-7 may directly cause T cell immunodeficiency but that the mechanisms of CD4+ T cell dysfunction mediated by HHV-6 and HHV-7 are different.
Administration of recombinant human IL-7 to mice alters the composition of B-lineage cells and T cell subsets, enhances T cell function, and induces regression of established metastases.
Authors: Komschlies K L , Gregorio T A , Gruys M E , Back T C , Faltynek C R , Wiltrout R H
Published: December 2022

These studies investigate the effects of exogenously administered recombinant human IL-7 (rhIL-7) on mouse leukocyte subsets in vivo in normal and tumor-bearing mice. The administration of rhIL-7 to normal mice caused a pronounced leukocytosis (three- to fivefold increase over background) in the spleen and lymph nodes, with B-lineage and T cells, NK cells, and macrophages all being increased. CD8+ T cells increased disproportionately, such that the CD4 to CD8 ratio decreased dramatically. The rhIL-7-induced effects were dose-dependent, increased with duration of treatment, and were reversible after cessation of rhIL-7 administration. T cell number increases after rhIL-7 treatment were primarily a result of an expansion of the peripheral T cell population. Importantly, splenocytes from rhIL-7-treated mice have enhanced proliferative responses to various T cell stimuli in vitro and were able to potentiate an allogeneic CTL response in vivo. The rhIL-7-induced changes in T cell number and the CD4 to CD8 ratio also were observed in mice bearing early Renca renal adenocarcinoma pulmonary metastases, and these changes coincided with up to a 75% reduction in pulmonary metastases. Overall, these results demonstrate that the administration of rhIL-7 to mice profoundly increases the number of B and T cells, and reduces the number of pulmonary metastases. The results also suggest that IL-7 may be useful for restoring lymphoid subsets in immunosuppressed hosts and in enhancing T cell-mediated immune responses. Such effects may be useful in the treatment of microbial diseases and cancer.
Monoclonal antibody to an activation neoepitope of alpha M beta 2 inhibits multiple alpha M beta 2 functions.
Authors: Elemer G S , Edgington T S
Published: December 2022

alpha M beta 2, a beta 2 integrin expressed on cells of myelomonocytic differentiation, functions as a receptor for factor X (X) and fibrinogen (Fg) and participates in leukocyte adhesion to vascular endothelium. Acquisition of high affinity ligand binding has been suggested to result from a conformational change of alpha M beta 2 in response to agonist-induced leukocyte stimulation. We now describe mAb 7A10, which preferentially binds to alpha M beta 2 on activated cells and reports the functional activation of this integrin. Agonist-stimulated monocytes and monocytic cells, but not resting cells, maximally expressed the 7A10 neoepitope, whereas expression of other selected alpha M beta 2 epitopes remained unchanged. The neoepitope was elicited equally by exposure of cells to either ADP or FMLP and did not require divalent cations for expression. Saturation of the 7A10 neoepitope by this Ab on stimulated THP-1 cells inhibited both X and Fg binding and abolished the alpha M beta 2-driven cellular coagulant response. Stimulated monocytic cells, which bound X and/or Fg, exhibited a sustained adhesion to unstimulated endothelial cell monolayers and Ab 7A10 inhibited this sustained adhesion. We conclude that activated conformers of alpha M beta 2 mediate X and Fg binding, that assembly of either X or Fg or both on alpha M beta 2 mediates leukocyte adhesion to unstimulated endothelium through sparse ICAM-1, and that mAb 7A10 can report and functionally inhibit this pathway of leukocyte adhesion.
Pathogenesis of hyperacute experimental autoimmune myasthenia gravis. Acetylcholine receptor/cholinergic site/receptor function/autoimmunity.
Authors: Mihovilovic M , Donnelly-Roberts D , Richman D P , Martinez-Carrion M
Published: December 2022

Three mAbs, mAbs 249E, 370, and 383C, directed against the alpha-bungarotoxin (alpha BgTx) binding site of the acetylcholine receptor (AChR) induce a hyperacute form of experimental autoimmune myasthenia gravis (EAMG), characterized by death within hours of mAb injection. To analyze the mechanisms of this effect, purified AChR-mAb complexes were investigated for their ability to bind the cholinergic agonist carbamoylcholine and to undergo agonist-induced activation of the cholinergic ionophore. The three mAbs inhibited carbamylcholine binding, and, conversely, their binding to AChR was inhibited by carbamylcholine. All three completely inhibited carbamylcholine-induced T1+ influxes to AChR-rich vesicles. These data indicate that the severe hyperacute EAMG induced by these mAbs results from blockage of AChR function and that the role of such potent Abs (even if present in small amounts) in the pathogenesis of human myasthenia gravis deserves further investigation.
Complement activation by recombinant HIV-1 glycoprotein gp120.
Authors: Süsal C , Kirschfink M , Kröpelin M , Daniel V , Opelz G
Published: December 2022

The mechanism of CD4+ cell depletion in HIV-infected patients is poorly understood. In this study we investigated whether rgp120 can activate the complement system in the absence of anti-gp120 Abs. We found that the complement proteins C4, C3d, C5b-9, and properdin bind to rgp 120-coated CD4+ T cells of healthy individuals when incubated in autologous serum. Activation of the complement system occurred primarily via the classical pathway and was abolished in sera deficient in C1q and C4 as well as in the presence of EDTA. No cell lysis was observed in a lymphocytotoxicity assay using human serum, possibly because of homologous restriction of complement lysis. In contrast, addition of rabbit sera induced lysis of the rgp 120-precoated cells. Cell lysis by rabbit serum was found to be because of naturally occurring IgM anti-gp 120 Abs. The rgp 120, which was immobilized on the surface of microtiter plates activated complement in the absence of lymphocytes. Complement activation by cell-bound HIV-1 envelope glycoprotein gp120 with subsequent opsonization may be relevant for the elimination of noninfected CD4+ T cells in HIV-infected patients.
TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide.
Authors: Bacik I , Cox J H , Anderson R , Yewdell J W , Bennink J R
Published: December 2022

Under most circumstances, cell surface MHC class I molecules display peptides derived from a cytosolic pool of proteins. The efficient presentation of such peptides requires the functioning of two MHC gene products [TAP1 and TAP2 (transporter-associated with Ag processing 1 and 2)] that form a complex that facilitates transmembrane movement of peptides from the cytosol to the endoplasmic reticulum, the site of peptide association with class I molecules. It has been previously shown that peptides can be presented in a TAP-independent manner in association with HLA A2.1 or H-2 Kd if they are expressed COOH-terminal to an endoplasmic reticulum insertion/signal sequence derived from the adenovirus E3/19K glycoprotein (Anderson et al., 1991. J. Exp. Med. 174: 489; Eisenlohr et al., 1992. Cell 71: 963). We show that: 1) the E3/19K signal sequence greatly enhances the presentation of each of four additional peptides tested in association with H-2 Kb or Kk, 2) the E3/19K signal sequence can be substituted by a signal sequence derived from beta-IFN, and 3) the E3/19K signal sequence does not function when located at the COOH terminus of antigenic peptides. These findings indicate that first, many peptides require TAP for efficient presentation to T cells, second, expression of peptides COOH-terminal to signal sequences is a generally applicable method of bypassing the TAP-dependence of peptide presentation and third, the leader sequence does not act to bypass TAP simply by increasing the hydrophobic nature of peptides.
Effect of anti-CD4 on CD4 subsets. I. Anti-CD4 preferentially deletes resting, naive CD4 cells and spares activated CD4 cells.
Authors: Chace J H , Cowdery J S , Field E H
Published: December 2022

Anti-CD4 has been extensively studied in murine models of autoimmunity and transplantation. The timing of anti-CD4 administration in these systems is critical because anti-CD4 effectively blocks primary T-dependent responses but does not diminish ongoing or memory responses in immunized animals. These differential effects suggest that anti-CD4 suppresses a subpopulation of CD4+ cells. We previously observed in vitro that simultaneous activation through TCR-T3 rescued CD4+ cells from anti-CD4 elimination. From this we hypothesized that activated CD4+ cells resisted the effects of anti-CD4. We now show that in vivo treatment with anti-CD4 preferentially eliminated resting, naive CD4+ cells rather than memory and effector CD4+ cells. The CD4+ cells that remained after anti-CD4 treatment exhibited evidence of recent activation, because a higher percentage expressed IL-2R, regardless of subset phenotype. Moreover, Mls-1-primed, anti-CD4-treated mice showed a higher percentage of V beta 6+ (Mls-1 reactive) CD4+ cells than either unprimed mice, anti-CD4-treated mice, or Mls-1-primed controls, implicating the importance of recent activation. These anti-CD4-resistant cells also retained their functional abilities. T cells from BALB/c mice treated with anti-CD4 after Mls-1 immunization maintained their MLR proliferation against DBA/2 stimulator cells. In addition, anti-CD4 did not reduce T-dependent antibody responses in mice previously primed against the Ag cholera toxin or SRBC. Thus, activated CD4+ cells resist the suppressive effects of anti-CD4. Our findings have critical implications for the ongoing clinical trials using anti-CD4.
Monocytes provide a novel costimulatory signal to T cells that is not mediated by the CD28/B7 interaction.
Authors: Johnson J G , Jenkins M K
Published: December 2022

Resting CD4+T cells must receive nonspecific costimulatory signals from APC to produce maximal amounts of IL-2 in response to TCR signaling. The T cell-specific surface molecule CD28 is one protein that transduces a costimulatory signal following interaction with its ligand B7. We report here the identification of another contact-mediated costimulatory signal provided by the human histiocytic line U937 and by purified monocytes. Although this monocyte-derived costimulus is not transduced by the CD28/B7 interaction, it synergizes with the CD28 signal to elicit maximal IL-2 production from freshly isolated T cells. IL-2 production by previously activated CD8+ T cells depends on the monocyte-derived costimulatory signal, although memory CD4+ T cells respond well to either the monocyte-derived costimulus or B7-derived costimulation. In contrast, IL-2 secretion by naive T cells appears to require the synergistic interaction between both costimulatory pathways. These results suggest that the array of costimulatory ligands expressed by various APC affects the magnitude of the T cell response and also which T cell subsets are stimulated.
Long term activation of natural killer cells results in modulation of beta 1-integrin expression and function.
Authors: Mainiero F , Gismondi A , Milella M , Morrone S , Palmieri G , Piccoli M , Frati L , Santoni A
Published: December 2022

Integrin expression and function is largely modulated by cell activation. Here we provide evidence that long term activation of human NK cells results in a marked modulation of beta 1-integrin expression and adhesive functions. By flow cytometry and immunochemical analysis we have detected induction of alpha 1 beta 1 and alpha 2 beta 1, increased expression of alpha 4 beta 1 and alpha 5 beta 1, and decline of alpha 6 beta 1 on CD3-CD56+ NK cells generated from 10-day coculture of nonadherent PBMC with irradiated RPMI 8866 EBV+ lymphoblastoid B cell line. Adhesion assays performed on extracellular matrix-coated plates showed that, unlike fresh NK cells, long term-activated NK cells bind to native collagen I via alpha 2 beta 1 and to heat-denatured collagen I in an RGD-dependent manner, although they lose the ability to bind to laminin. In regard to the adhesion to FN, no major quantitative changes are observed after long term NK cell activation. However, whereas alpha 4 beta 1 and alpha 5 beta 1 completely mediate the adhesion of fresh NK cells to fibronectin, binding of activated NK cells is only partially beta 1-dependent and seems to involve also non-beta 1-integrin(s) recognizing and RGD sequence. The modulation of beta 1-integrin expression and the acquisition of new adhesive properties on long term-activated NK cells may be relevant for their traffic and tissue localization during inflammation and immune response.
Expression and function of CD7 molecule on human natural killer cells.
Authors: Rabinowich H , Pricop L , Herberman R B , Whiteside T L
Published: December 2022

The CD7 molecule, one of the earliest T-lymphocyte Ag expressed during ontogeny, has recently been demonstrated to facilitate activation of T cells and to preferentially activate TCR-gamma/delta + subset of T cells. The CD7 Ag is also expressed on human NK cells, but its function has not been determined. In this study, expression and function of CD7 Ag on highly enriched NK cells (94 +/- 3% mean +/- SD, n = 12) obtained by negative selection from peripheral blood of normal donors were investigated. The CD7 Ag was found to be expressed at a significantly (p < 0.002) higher level on fresh NK cells than on IL-2-activated, NK cells. CD7 on human NK cells was found to be a signal-transducing molecule with a rapid increase in cytoplasmic free calcium observed on binding of anti-CD7 mAb to the surface of NK cells. Cross-linking of CD7 induced expression of surface activation molecules such as CD25, CD71, HLA-DR, CD69, and CD54. Activation by anti-CD7 mAb cross-linked to plastic or through goat anti-mouse Ig also induced a variety of NK cell functions: it stimulated secretion of IFN-gamma, led to proliferation of NK cells, as measured by [3H]thymidine incorporation, and significantly enhanced cytotoxicity of NK cells against K562 targets (p < 0.03). However, CD7 on NK cells did not seem to transduce a lytic signal, because it neither mediated redirected killing of Fc gamma R+ murine mastocytoma P815 cells nor triggered lysis of a hybridoma expressing the antibody in a membrane-bound form. CD7 molecules appeared to have a regulatory role in adhesion of NK cells to fibronectin, because cross-linking of CD7 on resting NK cells significantly augmented their adhesion to fibronectin-coated plastic surfaces. However, this induced adhesion was not associated with increased expression of beta 1-integrins on NK cells. Thus, CD7-mediated signals appear to augment function of adhesion molecules on NK cells, which may be involved in NK cell activation by providing both anchorage and costimulatory triggering.
Functional analysis of human Fc gamma RII (CD32) isoforms expressed in B lymphocytes.
Authors: Van Den Herik-Oudijk I E , Westerdaal N A , Henriquez N V , Capel P J , Van De Winkel J G
Published: December 2022

The low affinity IgG receptor Fc gamma RII (CD32) represents the most widely distributed class of human Fc gamma R. To analyze the biologic functions of different Fc gamma RII isoforms, we stably transfected Fc gamma RIIb1, IIb1*, IIb2, IIa, and a IIa tail- mutant to the mouse IIA1.6 B lymphoma cell line. Of these, Fc gamma RIIb1* represents a receptor variant that is identical to IIb1 except for a single amino acid difference in the cytoplasmic tail (amino acid position 11) where a tyrosine (IIb1) is replaced by an aspartic acid (IIb1*). Evaluation of capping ability showed the Fc gamma RIIb1 molecules to cap effectively, which was even more apparent with IIb1*. None of the Fc gamma RIIa, IIa tail-, or IIb2 isoforms capped significantly. Internalization of Fc gamma R-antibody complexes proved very efficient for both the Fc gamma RIIa and IIb2 isoforms, whereas the IIb1 molecules internalized moderately compared with IIb1*, which internalized less efficiently. Notably, human IgG aggregates were internalized effectively by Fc gamma RIIa and moderately by IIb2. Neither Fc gamma RIIb1 nor IIb1* proved capable of internalizing such IgG aggregates. Cross-linking of the different Fc gamma R molecules showed Fc gamma RIIa capable of triggering increases in [Ca2+]i. Fc gamma R expressed on B cells were able to down-regulate [Ca2+]i on co-cross-linking with slgG. Notably, all three Fc gamma RIIb receptors proved active in this respect, in contrast to Fc gamma RIIa. The cell distribution of these Fc gamma RII isoforms was analyzed in a panel of human B cell lines to complement the IIA1.6 B cell model. Fc gamma RIIa was found expressed both at message and protein levels in all tested human B cell lines. In the pre-B cell lines evaluated, no Fc gamma RIIb molecules were detectable, whereas both Fc gamma RIIb1 and IIb2 molecules were found present in more mature B cell lines. These data support both a complex expression pattern of Fc gamma RII isoforms in B cell lines and functional differences between these B cell molecules.
Expansion/deletion of mature T cells exposed to endogenous superantigens in vivo.
Authors: Webb S R , Hutchinson J , Hayden K , Sprent J
Published: December 2022

Previous studies have shown that in thymectomized hosts exposure of mature T cells to Mlsa (mtv-7) Ag in vivo leads to specific tolerance and the disappearance of Mlsa-reactive V beta 6+ T cells after an initial phase of T cell expansion. To investigate the factors controlling postthymic elimination of mature T cells, we examined T cell responses to Mlsa and other endogenous superantigens in a number of different strain combinations. The results show that the extent of T cell expansion/deletion is influenced by various factors, including the H-2 haplotype of the host and the particular V beta studied. Collectively, the results suggest that the extent of elimination of mature T cells is variable and may be a function of high avidity interactions with APC.
Endogenous peptides with distinct amino acid anchor residue motifs bind to HLA-A1 and HLA-B8.
Authors: DiBrino M , Parker K C , Shiloach J , Turner R V , Tsuchida T , Garfield M , Biddison W E , Coligan J E
Published: December 2022

Distinct amino acid (aa) residue motifs for peptides binding to HLA-A1 and HLA-B8 were identified by sequence analyses of reversed-phase HPLC fractions containing endogenous peptides derived from these HLA molecules. Fifteen different primary sequences were determined for HLA-A1-associated peptides, 12 of which were nine aa in length. Common features among these peptide sequences were Tyr at the COOH-terminus, a negatively charged aa (usually Glu) at position 3 (P3), and Pro at P4. Twenty-seven different primary sequence assignments were made for HLA-B8-associated peptides, most of which were eight aa in length. Lys, and in a few cases Arg, predominated at P3 and P5; Leu and Pro predominated at P2, and Leu was the preferred COOH-terminal residue. Unlike all other human class I molecules whose peptide-binding properties have been studied, both HLA-A1 and HLA-B8 endogenous peptide sequences have a dominant anchor residue at P3, and these aa are opposite in charge to the aa at position 156 of the peptide-binding site. Synthetic peptides corresponding to endogenous peptide sequences bound to their respective HLA molecules in vitro, indicating that they derive from peptides bound to HLA and not from copurifying contaminants. Eight of the HLA-A1 and HLA-B8 endogenous peptide sequences matched intracellularly expressed proteins found in protein sequence data bases. The HLA-A1 peptide-binding motif was then used to identify potential antigenic peptides from influenza A viral proteins that bound to HLA-A1 in vitro.
Structural analysis of the human VH locus using nonrepetitive intergenic probes and repetitive sequence probes. Evidence for recent reshuffling.
Authors: Matsumura R , Matsuda F , Nagaoka H , Fujikura J , Shin E K , Fukita Y , Haino M , Honjo T
Published: December 2022

The organization and evolution of the 0.8-Mb JH-proximal region in the human Ig VH locus were studied by mapping DNA fragments hybridized to non-repetitive intergenic probes and by determination of the content and distribution of repetitive sequences. Southern blot analysis of cloned DNA covering the 0.8-Mb region with intergenic probes allowed us to map two to seven cross-hybridizing fragments by each probe. Clusters of fragments detected by an identical set of probes appeared repeatedly within the 0.8-Mb JH-proximal region. Distantly located VH segments flanked by a cluster of DNA fragments hybridized by the same set of probes were highly homologous to each other, providing evidence for recent frequent duplication and translocation throughout the locus. DNA fragments detected by the same set of probes were orientated with the same 5' to 3' order within the cluster, suggesting little involvement of inversion upon recombination in the locus. The content of interspersed Alu and L1 sequences in the VH locus were not significantly greater than the average in the genome.
Cerebral expression of multiple cytokine genes in mice with lymphocytic choriomeningitis.
Authors: Campbell I L , Hobbs M V , Kemper P , Oldstone M B
Published: December 2022

The simultaneous expression of multiple cytokine genes was examined within the brain and peripheral organs of euthymic and congenic athymic mice during the course of lymphocytic choriomeningitis (LCM) induced by the intracranial inoculation of LCM virus. In euthymic mice, levels of TNF-alpha, IL-1 alpha, IL-6, and IL-1 beta mRNA in the brain increased early in infection and further increased with the progression of disease; the level of IFN-gamma mRNA was expressed at high levels late in infection and correlated with the onset of clinical disease. In the peripheral organs, TNF-alpha, IL-1 alpha, and IL-1 beta gene expression predominated early and then declined late in disease. In athymic mice (which do not develop LCM), expression of TNF-alpha, IL-1 alpha, IL-6, and IL-1 beta mRNA increased progressively in brain and peripheral organs from days 3 to 6 postinfection. The levels of these transcripts were significantly lower than those in infected euthymic animals. In euthymic mice, in situ hybridization revealed IFN-gamma-, TNF-alpha-, and IL-1 alpha-expressing cells throughout the inflammatory cell infiltrates associated with areas of LCMV nucleic acid. Therefore, in mice with LCM there is simultaneous expression of multiple cytokine genes, the pattern of which is distinct for brain vs peripheral organs and for euthymic vs athymic mice. The close spatiotemporal relationship between expression of cytokine genes and evolution of disease in brain implicate cytokines are not only key coordinators of the host response but also as possible mediators of neuropathology in LCM.
Taxol provides a second signal for murine macrophage tumoricidal activity.
Authors: Manthey C L , Perera P Y , Salkowski C A , Vogel S N
Published: December 2022

The anticancer drug, taxol, blocks cell division by stabilizing microtubules. However, taxol has distinct cell-cycle-independent effects. For example, taxol and bacterial LPS induce strikingly similar responses in murine macrophages. Here we report that taxol, like LPS, provides a "second" signal for murine macrophage activation to tumoricidal activity. Tumoricidal activity was determined by the release of 51Cr from prelabeled P815 mastocytoma target cells. Taxol or LPS alone weakly induced C3H/OuJ (Lpsn) murine macrophages to kill P815 mastocytoma cells, and tumoricidal activity was not induced by the classic "priming" signal, IFN-gamma. However, combinations of taxol or LPS with IFN-gamma synergized to activate macrophages to lyse tumor cells. Taxol activation of macrophages required an intact LPS signaling pathway, as taxol did not induce IFN-gamma-treated C3H/HeJ (Lpsd) macrophages to lyse target cells. In normal (Lpsn) murine macrophages, IFN-gamma, LPS, or taxol alone induced low or moderate levels of nitric oxide synthase gene expression and nitric oxide secretion. However, this gene and cytostatic metabolite were induced synergistically by combinations of taxol or LPS with IFN-gamma. Secretion of nitric oxide correlated with tumor cell killing, and taxol-activated macrophages failed to kill tumor targets in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase. The data illustrate the potential for taxol to activate macrophage mediated-antitumor mechanisms in addition to its better characterized role as an anti-mitotic agent.
Identification of autoantibody epitopes of glutamic acid decarboxylase in stiff-man syndrome patients.
Authors: Li L , Hagopian W A , Brashear H R , Daniels T , Lernmark A
Published: December 2022

Stiff-man syndrome is a neurologic disorder characterized by progressive rigidity of skeletal muscles. Deficiency of the neurotransmitter gamma-aminobutyric acid and autoantibodies to glutamic acid decarboxylase (GAD), the enzyme synthesizing gamma-aminobutyric acid, are closely associated with the disorder, although the relevant antigenic epitopes have not been identified. In the present study, sera from two patients with SMS was used in an immunoblotting assay with recombinant GAD67 (M(r) 67,000) and GAD65 (M(r) 65,000) isoforms to test whether SMS sera can recognize specific epitopes. We found that both SMS sera recognized the GAD65, but not the GAD67, isoform. Using 13 different synthetic GAD peptides to block the autoantibodies, two GAD65 epitopes were identified. One epitope recognized by both patients' sera, was blocked by the peptide representing amino acid residues 354-368. In one patient only, blocking was also observed by a peptide representing residues 390-402, which includes the binding site of the GAD cofactor, pyridoxal 5'-phosphate. A single amino acid substitution in GAD65 at position 401 (leucine to proline) and representing the analogous GAD67 sequence in this region significantly reduced the peptide's inhibitory effect. These findings suggest that SMS GAD autoantibodies share distinct GAD65 linear epitopes and that some SMS patients' autoantibodies may block the active site, explaining SMS GABA deficiency.
Expression of Mtv-7 sag gene in vivo using a retroviral vector results in selective inactivation of superantigen reactive T cells.
Authors: Kang J , Ido E , Pawling J , Beutner U , Huber B T , Hozumi N
Published: December 2022

T cells expressing specific TCR V beta chains are intrathymically eliminated in mice expressing the murine Mls (minor lymphocyte stimulating) superantigens. Recently, in vitro studies have shown that the endogenous mouse mammary tumor virus (MMTV)-7 sag gene encodes Mls-1 Ag. The demonstrated ability of MMTV superantigen proteins to react with TCRs has led to the postulate that other infectious retroviruses may use superantigen-like molecules to modify the host's immune system. In this report, successful retrovirus-mediated Mtv-7 sag gene transfer into pluripotent hematopoietic stem cells is described. In two different strains of Mls-1- host mice (CBA/Ca and BALB/c) reconstituted with Mtv-7 sag gene expressing bone marrow cells, low levels of ectopic Mtv-7 sag gene expression on syngeneic donor hematopoietic stem cell-derived population alone can induce partial clonal deletion of Mls-1 reactive V beta 6+ and V beta 8.1+ T cells, and complete clonal inactivation of V beta 8.1+ T cells.
Redox mechanism as alternative to ligand binding for receptor activation delivering disregulated cellular signals.
Authors: Nakashima I , Pu M Y , Nishizaki A , Rosila I , Ma L , Katano Y , Ohkusu K , Rahman S M , Isobe K , Hamaguchi M
Published: December 2022

Cross-linking with specific ligand is a general requirement for ordered activation of cell surface receptors. In this study we demonstrated a novel pathway for disregulated receptor activation through a redox mechanism. Treatment of murine thymocytes or spleen cells with thiol-reactive HgCl2, a known inducer of autoimmune proliferative lymphocyte disorders in rodents, was found to induce tyrosine phosphorylation of several cellular proteins, which was up to 100 times as extensive as that triggered by stimulation with antireceptor antibody or mitogen. Through the cross-linkage by thiol-reactive bivalent mercury, transmembrane CD4, CD3, and CD45 and glycosylphosphatidylinositol-anchored Thy-1 were aggregated together on thymocytes or T lymphocytes. Along with the aggregation of Thy-1 and CD4, nonreceptor protein tyrosine kinase p56lck was aggregated and activated. These events were linked to extensive protein tyrosine phosphorylation, which was visualized as a well localized spot beneath the membrane. Under appropriate conditions, this novel pathway of multiple receptor aggregation delivered a disregulated signal into T lymphocytes, which cross-talked to the antireceptor antibody-induced signal, for prolonged cell proliferation and IL-2 production. These results suggest a novel mechanism of disregulation of the ligand-dependent receptor function.
The novel mAb QR6.6 detects a surface molecule on immature thymocytes and inhibits their proliferation on thymic epithelial cells.
Authors: Tamura H , Kuzuhara H , Takeuchi T , Yamamoto H
Published: December 2022

We previously reported that the nude mouse-derived splenic T cell clone N-9F exhibits a proliferative response when cultured on thymic stromal cells. This N-9F proliferation is mediated by direct cell-to-cell interactions between T and thymic stromal cells. A thymic epithelial cell clone, SL10.3, also supports N-9F growth. To identify the molecule involved in T cell development in the thymus, we established mAb specific to the N-9F clone. One of these mAb, QR6.6, was found to inhibit the N-9F proliferative response on SL10.3. QR6.6-positive cells were detected in thymus but not in other lymphoid organs such as bone marrow, lymph nodes, or spleen. QR6.6-positive cells accounted for 3 to 5% of the cells in adult thymuses whereas higher percentages were found in neonatal (10-20%) and fetal thymuses (70% at E17 and 10-20% at E15). The positive cells were primarily CD4-8- thymocytes in fetuses and CD4-8- to CD4+8+ thymocytes in adults. The QR6.6 mAb precipitates a 100 kDa molecule from the N-9F clone. The addition of the mAb to fetal thymus organ culture reduces the recovery of cells at culture day 4. It was also found that the mAb inhibits fetal thymocyte proliferation on the SL10.3 thymic epithelial cell line. These results suggest that the 100 kDa molecule detected by the QR6.6 mAb may play a crucial role in the early stage of thymocyte development.
Stimulator cell type influences the response of T cells to staphylococcal enterotoxins.
Authors: Yagi J , Uchiyama T , Janeway C A
Published: December 2022

Responses to the superantigen Mls are characterized by proliferation of a significant percentage of T cells expressing receptors encoded by one or a few V beta gene segments. Apparently similar responses are elicited by the staphylococcal enterotoxins (SEs) and other bacterial superantigens. We have observed that T cells can be stimulated by the bacterial superantigen SEs presented by either spleen cells or fibroblasts transfected with the appropriate MHC class II genes. However, the results in this study showed that T cells required more than 100-fold higher concentrations of SEA in the presence of L cell transfectants than spleen APC, although T cell responses to SEB and several other toxins presented by the two types of APC were equivalent. Thus, L cell transfectants have a selective defect in presenting SEA. These data suggest that fibroblasts lack a component required by SEA to stimulate certain T cells, and lead us to propose an alternative model for bacterial superantigen mitogenesis in which the superantigen binds to and modifies the behavior of an endogenous co-ligand.
Mechanism of differential regulation of IL-2 in murine Th1 and Th2 T cell subsets. 1. Induction of IL-2 transcription in Th2 cells by up-regulation of transcription factors with the protein synthesis initiation factor 4E.
Authors: Barve S S , Cohen D A , De Benedetti A , Rhoads R E , Kaplan A M
Published: December 2022

Regulation of IL-2 gene expression in response to receptor-mediated stimuli is known to be mediated primarily by the IL-2 transcriptional enhancer and multiple transcription factors. However, the mechanism that controls the differential expression of the IL-2 gene in both human and murine CD4+ Th cell subsets (Th1-IL-2+ and Th2-IL-2-) is not clearly understood. Differential IL-2 gene expression was assessed in murine Th1 and Th2 subsets by analyzing the expression of a Escherichia coli lacZ reporter gene under control of the human IL-2 enhancer (IL2ZH) transfected in both T cell subsets. Stimulation of transfected T cells with the mitogen Con A, anti-CD3 Ab, or PMA plus ionomycin activated the IL2ZH construct in Th1 but not Th2 cells. However, IL2ZH was activated in stimulated Th2 cells that were co-transfected with a vector that overexpressed the eukaryotic initiation factor 4E (eIF-4E). It has been shown that eIF-4E is rate limiting for protein synthesis and its overexpression leads to increased rates of protein synthesis. Hence, eIF-4E overexpression could have overcome a deficiency in transcriptionally active levels of IL-2 regulatory factors in Th2 cells leading to IL-2 enhancer activation. This possibility was supported by demonstrating that transcriptionally active levels of the critical IL-2 transcription factor, nuclear factor of activated T cells (NF-AT), occurred only in Th2 cells overexpressing eIF-4E but not in normal Th2 cells, thus indicating that the inability of Th2 cells to express IL-2 was associated with inadequate levels of at least one transcription factor, NF-AT. Moreover, these results were confirmed by the observation that eIF-4E overexpression augmented NF-AT binding activity in Th2 cells. These data suggest that concentrations of inducible transcription factors are a major component of the regulatory mechanisms dictating IL-2 expression and may be under translational control in Th1/Th2 T cell subsets.
The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex.
Authors: Ziegler S F , Levin S D , Johnson L , Copeland N G , Gilbert D J , Jenkins N A , Baker E , Sutherland G R , Feldhaus A L , Ramsdell F
Published: December 2022

CD69 is a rapidly induced T cell activation Ag that is also expressed in an inducible fashion on cells of most, if not all, hematopoietic lineages. Molecular cloning has shown that CD69 is a type II membrane glycoprotein that is a member of the C-type lectin family. In this report we have shown that induction of CD69 mRNA in activated murine thymocytes and T cells is very rapid, peaking between 30 and 60 min poststimulation, and transient, dropping to nearly resting levels by 8 h. An analysis of the mouse CD69 gene structure showed the gene to consist of 5 exons and have a phorbol ester-inducible promoter element within the first 700 bp upstream of the start of transcription. Chromosomal mapping placed the mouse CD69 gene on the long arm of chromosome 6 near the NK gene complex that contains the related NKR-P1 and Ly-49 gene families. The human CD69 gene mapped to chromosome 12p13 near the related NKG2 gene cluster and in a region associated with rearrangements in approximately 10% of cases of childhood acute lymphocytic leukemia.
Changes in cell adhesion molecule expression on T cells associated with systemic virus infection.
Authors: Andersson E C , Christensen J P , Marker O , Thomsen A R
Published: December 2022

Virus-induced changes in adhesion molecule expression on T cells were investigated to understand how antiviral effector cells migrate into infectious foci. FACS analysis revealed that after systemic infection with lymphocytic choriomeningitis virus a number of cell adhesion molecules, including VLA-4, LFA-1, and ICAM-1, are up-regulated on CD8+ cells, whereas the lymph node homing receptor MEL-14 is down-regulated during the infection; only marginal changes were observed for CD4+ cells. Basically similar but less marked results were obtained in mice infected with Pichinde virus. Further analyses showed that T cells with a changed adhesion molecule profile tended to present other cell surface markers indicating a state of cellular activation, e.g., IL-2R, and included all virus-specific CTL effectors. Regarding the physiologic significance of these changes in adhesion molecule expression, it was found that up-regulation of VLA-4 expression on splenic T cells correlated with influx of inflammatory cells into the cerebrospinal fluid of intracerebrally infected animals, and that the number of CD8+VLA-4hi cells increased from lymph nodes and spleen to blood and cerebrospinal fluid. These results support the hypothesis that up-regulation of VLA-4 is important for effector T cell homing to sites of inflammation.
Intracerebral cytokine mRNA expression during fatal and nonfatal alphavirus encephalitis suggests a predominant type 2 T cell response.
Authors: Wesselingh S L , Levine B , Fox R J , Choi S , Griffin D E
Published: December 2022

Sindbis virus (SV) causes an acute encephalomyelitis in mice. A T cell-dependent inflammatory response is first detected 3 days after infection and includes T cells, B cells, and macrophages. The cytokines produced locally by intrinsic cells of the brain in response to infection and by infiltrating mononuclear cells and their contributions to outcome of infection have not been identified. Semiquantitative reverse transcriptase-PCR was used to evaluate the expression of mRNAs for IL-1 beta, IL-2, IL-4, IL-6, IL-10, TNF-alpha, leukemia inhibitory factor (LIF), and TGF-beta in the brain during fatal and nonfatal SV encephalitis of immunocompetent BALB/cJ and immunodeficient scid/CB17 mice. IL-1 beta and IL-6 mRNAs were detected in uninfected mice before infection and were up-regulated within 24 h. TGF-beta mRNA was also constitutively expressed in uninfected mice. LIF mRNA was occasionally detected in uninfected mice but increased in amounts only in BALB/cJ not scid mice after infection. TNF-alpha, IL-4, and IL-10 mRNAs were not found in uninfected mice but were induced within 24 h and continued to rise through 7 days after infection with substantially higher levels in BALB/cJ than scid mice. These data suggest that intrinsic brain cells produce IL-1, IL-4, IL-6, IL-10, LIF, and TGF-beta mRNAs in response to viral infection. IFN-gamma and IL-2 mRNAs were detected only in BALB/cJ mice and not until 3 days after infection with the initiation of inflammation. IL-4 and IL-10 mRNAs were more persistent and more easily detectable than IL-2 and IFN-gamma mRNAs. These data suggest a predominant type 2 cytokine response in the brain during SV encephalitis. BALB/cJ mice infected with a neurovirulent strain of SV (NSV), had 100% mortality, whereas NSV-infected scid mice developed persistent nonfatal infection. Inflammation was more intense in NSV-infected mice, however, no substantial differences in cytokine mRNA levels were detected when compared with mice with nonfatal SV infection suggesting that the cytokines measured do not in and of themselves lead to fatal central nervous system disease.
Macrophage-colony-stimulating factor expression by anti-CD45 stimulated human monocytes is transcriptionally up-regulated by IL-1 beta and inhibited by IL-4 and IL-10.
Authors: Gruber M F , Williams C C , Gerrard T L
Published: December 2022

Macrophage (M)-CSF is a survival and differentiation factor for mononuclear phagocytes. Stimulation of human monocytes with immobilized mAb directed to CD45 induces M-CSF message and small amounts of protein, which is strongly augmented by costimulation with IL-1 beta. This study was undertaken to study the mechanisms leading to the IL-1 beta-induced up-regulation of M-CSF production and to determine how the antiinflammatory cytokines, IL-4 and IL-10, affect M-CSF production in this system. We demonstrate that IL-1 beta enhanced M-CSF mRNA levels, in part, by increasing M-CSF gene transcription but had no effect on M-CSF message half-life. The enhancement of M-CSF message levels in the presence of IL-1 beta was blocked by cycloheximide, suggesting that de novo protein synthesis was required. Moreover, soluble IL-1 receptors inhibited the effect of IL-1 beta on M-CSF production thus confirming that these effects were IL-1 receptor mediated. Both IL-4 and IL-10 strongly inhibited M-CSF secretion by anti-CD45/IL-1 beta-induced monocytes that was accompanied by decreased M-CSF message levels. IL-4 and IL-10 repressed M-CSF gene transcription but did not affect M-CSF message half-life. These findings demonstrate that IL-1 beta, at least in part, transcriptionally up-regulates M-CSF production in anti-CD45-stimulated human monocytes, a process that can be negatively regulated by both IL-4 and IL-10. These results suggest that IL-1 beta, IL-4, and IL-10 control the survival and differentiation of human monocytes through a regulation of autocrine M-CSF production.
Differential roles for triglyceride and phospholipid pools of arachidonic acid in human lung macrophages.
Authors: Triggiani M , Oriente A , Marone G
Published: December 2022

Arachidonic acid (AA) incorporation into and release from cellular glycerolipids are thought to be crucial events for the regulation of eicosanoid biosynthesis in inflammatory cells. The goal of our study was to determine the distribution of AA in the lipid pools of human lung macrophages (HLM) isolated from human lung parenchyma and to define the changes in AA pools occurring during cell activation. Mass spectrometry analysis indicated that the major pools of AA in HLM were located in phosphatidylethanolamine (PE), phosphatidylcholine (PC), and triglycerides (TG), and, to a lesser extent, in phosphatidylinositol/phosphatidylserine (PI/PS) and in a phospholipid similar to bis-(monoacylglyceryl)-phosphate (BMP). Exogenous AA was initially incorporated into TG and subsequently distributed within phospholipids. After 24 h of labeling, the distribution of exogenous AA in glycerolipid classes closely mimicked that of endogenous AA. Under these equilibrium conditions, HLM released 18.7 and 20.2% of cellular AA when stimulated with TPA or A23187, respectively. Free AA was the major product released by TPA- or A23187-stimulated HLM. However, A23187 but not TPA also induced the formation of leukotriene B4 and 5-HETE. AA was released from PC > PI/PS > or = PE > BMP. In contrast to phospholipids, the amount of AA in TG increased 15 to 90 min after cell activation and returned to prestimulation levels after 180 min. These data indicate that AA is stored in several glycerolipid pools of HLM. Among these pools, BMP is unique of macrophages and TG are particularly enriched in AA in HLM. During the early stage of cell activation, phospholipids act as a source of AA, whereas TG function as a reacylation pool for AA released from phospholipids. These data indicate that TG and phospholipid pools have a differential role in the control of free AA levels generated during HLM activation.