bluuebunny/crossref_metadata_2025 huggingface dataset:
Vascular endothelial platelet endothelial adhesion molecule-1 (PECAM-1) expression is decreased by TNF-alpha and IFN-gamma. Evidence for cytokine-induced destabilization of messenger ribonucleic acid transcripts in bovine endothelial cells.
Authors: Stewart R J , Kashour T S , Marsden P A
Published: December 2022

Platelet endothelial cell-adhesion molecule-1 (PECAM-1, CD31) is constitutively expressed by vascular endothelium and concentrates at intercellular junctions. Regulation of PECAM-1 expression on endothelial cells may modulate leukocyte trafficking, angiogenesis, and vascular permeability. Given that cytokine activation induces profound alterations in endothelial phenotype, studies sought to determine whether cytokine treatment modulated PECAM-1 mRNA and protein content in macro- and microvascular endothelial cells. Northern blot analysis revealed expression of PECAM-1 mRNA transcripts in endothelial cells derived from bovine aorta, bovine glomeruli, and human umbilical vein under basal conditions. Treatment of endothelial cells with TNF-alpha and/or IFN-gamma led to dramatic decreases in steady-state levels of PECAM-1 mRNA transcripts. In contrast, reciprocal induction of ICAM-1 mRNA was evident. Actinomycin D chase experiments demonstrated that cytokines selectively destabilize PECAM-1 mRNA transcripts in bovine endothelial cells, decreasing the PECAM-1 mRNA transcript t1/2 from basal values of 15 +/- 2 h to 4 +/- 1 h in TNF-alpha- and IFN-gamma-treated cells (p < 0.005), an effect that appeared to be independent of new protein synthesis. Nuclear run-off analysis demonstrated no change in the rates of PECAM-1 gene transcription in response to cytokines treatment. Immunoblots and quantitative indirect immunofluorescence indicated decreased total cellular and cell-surface PECAM-1 protein expression following cytokine treatment. These findings provide evidence for cytokine-induced reciprocal regulation of transcripts of Ig-like adhesion molecules on vascular endothelium.
Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats.
Authors: Groen H , Klatter F A , Brons N H , Mesander G , Nieuwenhuis P , Kampinga J
Published: December 2022

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.
Mechanisms for the transendothelial migration of HIV-1-infected monocytes into brain.
Authors: Nottet H S , Persidsky Y , Sasseville V G , Nukuna A N , Bock P , Zhai Q H , Sharer L R , McComb R D , Swindells S , Soderland C , Gendelman H E
Published: December 2022

HIV-1 penetration of the brain is a pivotal event in the neuropathogenesis of AIDS-associated dementia. The establishment of productive viral replication or up-regulation of adhesion molecule expression on brain microvascular endothelial cells (BMVEC) could permit entry of HIV into the central nervous system. To investigate the contribution of both, we inoculated primary human BMVEC with high titer macrophage-tropic HIV-1 or cocultured them with virus-infected monocytes. In both instances, BMVEC failed to demonstrate productive viral replication. Cell to cell contact between monocytes and microvascular endothelium resulted in E-selectin expression on BMVEC. BMVEC. cocultured with LPS-activated HIV-infected monocytes expressed even higher levels of E-selectin and vascular cell adhesion molecule-1 (VCAM-1). Transwell assays supported a role of soluble factors, from virus-infected monocytes, for the induction of adhesion molecules on BMVEC. To verify the in vivo relevance of these findings, levels of adhesion molecules were compared with those of proinflammatory cytokines and HIV-1 gene products in brain tissue of AIDS patients with or without encephalitis and HIV-seronegative controls. E-Selectin, and to a lesser degree VCAM-1, paralleled the levels of HIV-1 gene products and proinflammatory cytokines in brain tissue of subjects with encephalitis. Most importantly, an association between macrophage infiltration and increased endothelial cell adhesion molecules was observed in encephalitic brains. Monocyte binding to encephalitic brain tissue was blocked with Abs to VCAM-1 and E-selectin. These data, taken together, suggest that HIV entry into brain is, in part, a consequence of the ability of virus-infected and immune-activated monocytes to induce adhesion molecules on brain endothelium.
Human T cell repertoire generation in the absence of MHC class II expression results in a circulating CD4+CD8- population with altered physicochemical properties of complementarity-determining region 3.
Authors: Henwood J , van Eggermond M C , van Boxel-Dezaire A H , Schipper R , den Hoedt M , Peijnenburg A , Sanal O , Ersoy F , Rijkers G T , Zegers B J , Vossen J M , van Tol M J , van den Elsen P J
Published: December 2022

In this study, we have investigated the impact of deficient MHC class II expression on the use of TCRBV6 and TCRBJ gene elements, and on the pattern of amino acid incorporation exhibited in the N1-D-N2 segments of the third complementarity-determining region (CDR3) of these TCRBV6 rearrangements. To this end, we have analyzed circulating T cells from three, nonrelated MHC class II-deficient (bare lymphocyte syndrome (BLS)) patients and three MHC class II-expressing family members. The patients and healthy controls exhibited similar, nonrandom usage profiles of TCRBV6 and TCRBJ gene elements in both the CD4+CD8- and the CD4-CD8+ subsets of peripheral blood T cells. No statistically significant differences between patients and controls were detected in the length of CDR3, or in the amount of non-germline modification at the sites of recombination. However, detailed analysis of the TCRBV6 rearrangements derived from the CD4+CD8- subsets from the BLS patients revealed patterns of amino acid incorporation into the N1-D-N2 region of CDR3 that resulted in altered charge and hydropathicity properties of the presumed Ag binding site. In this way, we have been able to demonstrate that human T cell repertoire development in the absence of MHC class II expression results in a circulating CD4+CD8- T cell population bearing TCRs with altered CDR3 profiles. Such altered profiles are likely to be a direct reflection of the lack of MHC class II-mediated selection processes in these BLS patients.
Isolation and characterization of cell lines with genetically distinct mutations downstream of protein kinase C that result in defective activation-dependent regulation of T cell integrin function.
Authors: Mobley J L , Ennis E , Shimizu Y
Published: December 2022

beta 1-integrins expressed on resting T cells support only minimal adhesion to integrin ligands. T cell activation through multiple stimuli, including phorbol ester treatment and Ab cross-linking of the CD3/TCR complex, results in a rapid and transient switch in integrin function from low to high avidity binding. The exact nature of the intracellular signals involved in this avidity switch remain poorly defined, but the ability of phorbol esters to induce such up-regulation implicates a role for protein kinase C (PKC). We have used a genetic approach to identify factors other than PKC that regulate activation-dependent beta 1-integrin function on T cells. We isolated mutants of the Jurkat T cell line that express beta 1- and beta 2-integrins but do not exhibit increased integrin activity in response to PMA stimulation or CD3 cross-linking. PKC activity appears to be normal in the mutants. One mutation is associated with an altered form of the mitogen-activated protein kinase ERK1 and an inability to produce IL-2. Another mutant with defective integrin function has IL-2 production intact. Complementation analysis verified that these two types of mutants are genetically distinct. Thus, two mutations downstream of PKC have been identified that alter the process of integrin regulation without affecting T cell viability or proliferative capacity. These mutants represent novel reagents for the identification of integrin regulatory factors and indicate possible sites of pharmacologic intervention that could prevent integrin-dependent migration and localization in the process of inflammation, while leaving other T cell functions intact.
The role of BALB/c donor CD8+ lymphocytes in graft-versus-host disease in (BALB/c x A/J)F1 (CAF1) mice.
Authors: Vidal S , Labrador M , Rodríguez-Sánchez J L , Gelpí C
Published: December 2022

To investigate the role of donor T lymphocyte subsets in the development of chronic graft-vs-host disease (GVHD) induced in (BALB/c x A/J)F1 (CAF1) mice by injecting BALB/c lymphoid cells, we analyzed the effect that CD8+ cell removal from donor inoculum has on the manifestation of the disease. Compared with age- and sex-matched CAF1 mice injected with whole lymphocyte inoculum, CAF1 mice injected with CD8(+)-depleted inoculum exhibited: 1) a higher incidence and exacerbation of nephritis by immunocomplexes; 2) higher (five- to sevenfold) spontaneous IL-4 production; 3) higher frequency titer and precocity of anti-dsDNA, anti-histone, and IgM and IgG rheumatoid factors; 4) a dramatic change in the frequency and titer of anti-U1 small nuclear ribonucleoprotein Abs; and 5) a markedly decreased engraftment (10- to 15-fold) on BALB/c donor lymphocytes. In contrast, rheumatoid arthritis-like disease, a later clinical manifestation of the GVHD in CAF1 + BALB/c model, is not present in the CD8(+)-depleted model (CAF1 + CD8-BALB/c). Considered together, these data suggest that CD8+ donor T lymphocytes play an important role in the degree of chimerism, modulation of the response to autoantigens, and clinical aspects developed in the GVHD model presented here.
Age-associated differences in TNF-alpha and nitric oxide production in endotoxic mice.
Authors: Chorinchath B B , Kong L Y , Mao L , McCallum R E
Published: December 2022

Gram-negative bacterial infection is a common cause of septic shock in the older population in the U.S. We employed an experimental model of sepsis to study the cause of increased lethality due to LPS in older animals. Three ages of male B6JC3J/Nia mice, young (2 mo old), mature (12 mo old), and senescent (24 mo old), were treated with bacterial LPS, and the older mice were found to be 10 times more sensitive to LPS lethality. Increased sensitivity to LPS in senescent mice correlated with significantly elevated plasma TNF-alpha and nitric oxide levels. Abs to TNF-alpha afforded aged animals passive protection against a supralethal dose of LPS, establishing a central role for TNF in the increased sensitivity to LPS seen in the aged animals. Other cytokines, such as IL-1 and IFN-gamma, appeared secondary to TNF and nitric oxide in the age-associated sensitivity to LPS. Plasma corticosterone levels were increased by LPS at a time when maximal levels of plasma TNF-alpha were observed in both age groups, although the kinetics of hormone production and the magnitude of TNF-alpha release varied among the age groups. Exogenously administered dexamethasone protected senescent animals given a high dose of LPS, by decreasing cytokine levels. The increased sensitivity to LPS observed in aged animals, therefore, seems to be due to excessive TNF and nitric oxide production, resulting from perturbed endogenous hormonal control of cytokine production.
Reactive oxygen intermediates as regulators of TNF-alpha production in rat lung inflammation induced by silica.
Authors: Gossart S , Cambon C , Orfila C , Séguélas M H , Lepert J C , Rami J , Carré P , Pipy B
Published: December 2022

Exposure to mineral dusts such as silica has been associated with progressive pulmonary inflammation and fibrosis. There is evidence that the release of reactive oxygen intermediates (ROI) and cytokines by alveolar macrophages (AM) is involved in lung injury associated with silica exposure. However, the chronology and relationship between these two mediators are poorly understood. In this study, an animal model of silicosis has been used, allowing simultaneous follow-up of lung histopathologic state, AM TNF-alpha production at the protein (biologic assay) and mRNA (reverse transcriptase-PCR) levels, and the release of ROI (luminol-dependent chemiluminescence), after bronchoalveolar lavages. In particular, it has been shown that intratracheal instillation of silica (50 mg/kg) in rats led to fibrosis characterized by cellular interstitial infiltrates with granulomas, and in AM, it led to 1) an early and continuous increase in 12-O-tetradecanoylphorbol-13-acetate- or zymosan-triggered ROI production (days 1, 3, 14, and 28 post-treatment), and 2) a rise of TNF-alpha mRNA expression and protein secretion on days 3 and 14. A free radical scavenger pretreatment (N-ter-butyl-alpha-phenylnitrone) reversed lung histopathologic changes and decreased AM ROI production and TNF-alpha expression at the level of mRNA. These findings suggest that ROI production is an important primary event determining the silica-induced inflammatory process. ROI may act in an autocrine or paracrine manner and regulate TNF-alpha production by a mechanism promoting gene expression. The critical role of this cytokine in the pathogenesis of silicosis was confirmed by anti-TNF-alpha Ab treatment.
The animal lectin galectin-3 interacts with bacterial lipopolysaccharides via two independent sites.
Authors: Mey A , Leffler H , Hmama Z , Normier G , Revillard J P
Published: December 2022

Galectin-3 is a beta-galactoside binding protein expressed by activated macrophages, epithelial cells, and certain other cell types. Galectin-3 has a C-terminal carbohydrate binding domain, an N-terminal part consisting of a proline- and glycine-rich repetitive domain, and a small N-terminal domain. Two independent LPS binding sites on galectin-3 were demonstrated by binding of biotinylated LPS to immobilized recombinant galectin-3. One appears to be the carbohydrate binding site in the C-terminal domain that confers binding of LPS from Klebsiella pneumoniae that has a beta-galactoside-containing polysaccharide chain. This binding is best demonstrated using galectin-3 immunocaptured by a mAb to the N-terminal part (M3/38) and is inhibited by lactose. In contrast, Salmonella minnesota R7 LPS (Rd mutant), which is devoid of beta-galactosides, appears to bind to a site within the N-terminal part of galectin-3. This interaction is best demonstrated using galectin-3 directly immobilized in wells, and it is inhibited by the Ab M3/38, but not by lactose. Binding inhibition by polymyxin B and the profile of inhibition by a panel of LPSs with different amounts of the inner and outer cores present indicate that this second binding site recognizes the lipid A/inner core region of LPSs.
Effect of overexpression of human Cu,Zn superoxide dismutase in transgenic mice on macrophage functions.
Authors: Mirochnitchenko O , Inouye M
Published: December 2022

Properties of macrophages from transgenic mice with the human Cu,Zn superoxide dismutase (SOD) gene under the control of the mouse hydroxyl-methyl coenzyme A reductase (HMGCR) promoter were studied. In these mice, a twofold overproduction of Cu,Zn SOD in intraperitoneal macrophages resulted in the significant reduction of their microbicidal and fungicidal activity. Intracellular production and release of H2O2 in macrophages from transgenic mice activated by PMA was found to be significantly increased, whereas extracellular release of O2- was inhibited. When treated with LPS or LPS plus IFN-gamma, macrophages from transgenic mice were found to produce less nitric oxide (NO) than normal mice, suggesting that the nitrocompound metabolism in macrophages overproducing Cu,Zn SOD was also affected. Analysis of NF-kappa B DNA-binding activity and antiphosphotyrosine immunoblotting experiments suggest that impairment of macrophage functions may be attributed to the inhibition of signal transduction pathways as well as to changes in oxygen radical metabolism. The present data support the notion that antioxidant enzymes play important roles in the function of macrophages.
Crucial role of 55-kilodalton TNF receptor in TNF-induced adhesion molecule expression and leukocyte organ infiltration.
Authors: Neumann B , Machleidt T , Lifka A , Pfeffer K , Vestweber D , Mak T W , Holzmann B , Krönke M
Published: December 2022

Stimulation of leukocyte adhesion to the endothelium by TNF is mediated by the up-regulation of adhesion molecules on the endothelial cell surface. C57BL/6 mice and syngenic 55-kDa TNF receptor-deficient mice (TNFRp55-/- mice) were challenged with TNF, and the kinetics of intracellular adhesion molecule-1, ICAM-1, mucosal addressin cell adhesion molecule-1, vascular adhesion molecule-1 (VCAM-1), and E-selectin expression were examined in various organs. TNF induced sustained VCAM-1 expression within 4 h in lung, liver, and kidney. In the lungs, but not in other organs, transient E-selectin expression was induced by TNF within 0.5 h and peaked at 4 h. The TNF-induced expression of VCAM-1 and E-selectin was found to be exclusively controlled by the 55-kDa TNF-receptor (TNFRp55) as demonstrated by analysis of TNFRp55-/- mice. Furthermore, TNF triggered mononuclear cell and neutrophil infiltration of lung, liver, and kidney in C57BL/6 mice but not TNFRp55-/- mice. Interestingly, MAdCAM-1 expression in the marginal sinus of the spleen was detected in wild-type mice but was absent in TNFRp55-/- mice. Together, the data suggest that in vivo the 55-kDa TNF receptor mediates the induction of VCAM-1 and E-selectin expression and is critically involved in the control of leukocyte organ infiltration.
Lupus autoantibodies to double-stranded DNA cross-react with ribosomal protein S1.
Authors: Tsuzaka K , Leu A K , Frank M B , Movafagh B F , Koscec M , Winkler T H , Kalden J R , Reichlin M
Published: December 2022

One cDNA clone (G7) was isolated from a lambda gt11 human liver cDNA library by the reaction with a serum containing anti-dsDNA Abs and was ligated into pGEX-1 lambda T vector. All the 10 SLE sera with anti-dsDNA, 2 samples of human monoclonal anti-dsDNA (33.C9 and 33.H11), and 2 affinity-purified anti-dsDNA Abs recognized the glutathione S-transferase fusion protein expressed by G7 (G7-FP). Ab binding to the recombinant protein expressed by G7 (G7-RP) and to G7-FP was inhibited completely by calf thymus dsDNA. The cDNA was 1314 nucleotides in length and contained an open reading frame encoding 352 amino acids. However, it seems to be a partial length cDNA because the affinity-purified Ab from G7-FP recognized only a 104-kDa protein on Western blot using MOLT4 cell extract. The nucleotide sequence of G7 was homologous (99% identity) to a cDNA encoding human ribosomal protein (r-protein) S1 homologue mRNA. The encoded protein contains repeating residues as a feature of r-proteins S1. Cytoplasmic and nucleolar staining of 33.H11 on indirect immunofluorescence (IF) using HEP 2 cells was inhibited by both G7-RP and calf thymus dsDNA. On ELISA, 33.H11 had a higher affinity for G7-RP than for DNA while 33.C9 had a higher affinity for DNA than for G7-RP and binds nuclei on IF. We conclude that G7 encodes a portion of human r-protein S1 and anti-dsDNA Abs cross-react with this protein.
Characterization and purification of a protein kinase C substrate in human B cells. Identification as lymphocyte-specific protein 1 (LSP1).
Authors: Carballo E , Colomer D , Vives-Corrons J L , Blackshear P J , Gil J
Published: December 2022

Incubation of B-chronic lymphocytic leukemia (B-CLL) cells with phorbol esters resulted in the phosphorylation of two major PKC substrates, MARCKS (myristoylated, alanine-rich C kinase substrate) and MRP (MARCKS-related protein), and of a third protein, with an apparent m.w. of 60,000 that was the most prominent protein kinase C substrate in these cells. p60 phosphorylation was time and PMA dose dependent, and was induced by cell-permeable diacylglycerol, but not by inactive phorbol esters. Two-dimensional electrophoretic analysis of the protein phosphorylation pattern from the B cell line CESS demonstrated the identity between the p60 protein expressed in this cell line and that expressed in B-CLL cells. p60 was purified from CESS cells and peptide microsequencing of this protein revealed that it was lymphocyte-specific protein 1 (LSP1), that is here characterized as the most prominent protein kinase C substrate in B cells.
IL-12 produced by antigen-presenting cells induces IL-2-independent proliferation of T helper cell clones.
Authors: Maruo S , Toyo-oka K , Oh-hora M , Tai X G , Iwata H , Takenaka H , Yamada S , Ono S , Hamaoka T , Kobayashi M , Wysocka M , Trinchieri G , Fujiwara H
Published: December 2022

We investigated the role of IL-12 in proliferation of various Th cell clones (class II-alloreactive (4-86 and 4-55) and keyhole limpet hemocyanin + self I-Ek-reactive (9-16)) following stimulation with Ag on APCs. These clones proliferated in response to stimulation with rIL-2, rIL-12, or Ag/APC. The proliferation induced by Ag/APC stimulation was not affected by anti-IL-2 Ab but was markedly inhibited by anti-IL-12 Abs. Consistent with this finding was the absence of detectable IL-2 activity in culture supernatants 12 to 48 h after Ag/APC stimulation, and the detection of significant levels of IL-12 in an Ab-capture bioassay. IL-12 was produced within 12 h after Ag/APC stimulation, reaching a peak after 18 to 24 h. The production of IL-12 in cultures of Th clones and APC contrasted with the production of IL-2 but not IL-12 upon allostimulation of primary T cells and the inhibition of their proliferation exclusively by anti-IL-2 Abs. Analysis of the expression of IL-12-binding sites on Th cells revealed low levels of IL-12 receptors in resting Th clones but high IL-12R levels 2 to 3 days after Ag/APC stimulation, declining gradually thereafter. The changes in IL-12R expression levels correlated closely with the IL-12 responsiveness of Th populations at various times after Ag/APC stimulation; Th populations obtained 3 and 10 days after Ag/APC stimulation exhibited very high and weak or marginal responsiveness to rIL-12, respectively, whereas the responses to rIL-2 were comparable in both Th populations. These results indicate that the Ag/APC-stimulated proliferation of terminally differentiated Th clones, in contrast to naive T cells, depends on the production of IL-12 by APC and on the simultaneous up-regulation of IL-12R on Th cells rather than on an IL-2 autocrine mechanism.
Residues in TAP2 peptide transporters controlling substrate specificity.
Authors: Momburg F , Armandola E A , Post M , Hammerling G J
Published: December 2022

The transporter associated with Ag processing (TAP) translocates peptides from the cytosol into the endoplasmic reticulum where they associate with MHC class I molecules. Two specificity patterns with regard to the C-terminal residue of transported peptides have been previously shown. While the u allele of rat TAP and the mouse TAP preferentially transport peptides with hydrophobic C-terminal residues, no such selection was reported for the a allele of rat TAP or for the human TAP. We were able to map two short stretches in rat TAP2, with two polymorphic residues each, that essentially control the differential peptide transport observed for the rat alleles by constructing several hybrids between rat TAP2a and TAP2u and co-expressing them with rat TAP1 in TAP-deficient T2 cells. The critical residues are located in putative cytoplasmic loops close to the membrane.
Engraftment of human tumor-infiltrating lymphocytes and the production of anti-tumor antibodies in SCID mice.
Authors: Williams S S , Chen F A , Kida H , Yokata S , Miya K , Kato M , Barcos M P , Wang H Q , Alosco T , Umemoto T , Croy B A , Repasky E A , Bankert R B
Published: December 2022

We report here the placement of nondisrupted 1-mm3 pieces of fresh human lung tumor biopsy tissue into the subcutis of severe combined immunodeficient (SCID) mice results in the engraftment of tumor-infiltrating leukocytes (TIL) in all but 5 of 148 mice inoculated with 39 different biopsy tissue specimens. In mice coengrafted with tumor and TIL the normal histologic architecture of the tumor and TIL interface was maintained for up to 22 wk. The TIL in the xenograft were shown to divide and were maintained exclusively at the site of tumor inoculation. It is established here that plasma cells in the TIL population produce Abs that react in western blots with tumor cell lysates. These Abs were shown to react with high and low m.w. proteins derived from both the membrane and cytosolic fractions of tumor cell lysates. The production of human Ig was found to be T cell dependent, and immunohistochemistry and in situ hybridization of DNA, using a human-specific cDNA probe, established the human identity of the tumor and TIL. High levels of human Ig in the sera of mice inoculated with tumor biopsy tissue are associated with the growth arrest of adenocarcinoma xenografts. Our results establish the co-engraftment of human tumors and TIL into SCID mice as new animal model with which to evaluate TIL function and novel therapeutic strategies that are designed to augment TIL anti-tumor activity.
The phosphatidylinositol 3-kinase inhibitor wortmannin blocks mast cell exocytosis but not IL-6 production.
Authors: Marquardt D L , Alongi J L , Walker L L
Published: December 2022

Phosphatidylinositol 3-kinase (PI3-kinase) activity has been shown to be important in cellular signaling via receptors associated with tyrosine kinases and receptors coupled to small or heterotrimeric G proteins. The importance of this activity in mast cell degranulation, leukotriene C4 generation, and IL-6 production was examined in mouse bone marrow-derived mast cells stimulated by high affinity IgE receptor cross-linking, direct influx of calcium, and/or adenosine receptor agonist exposure. Wortmannin, a fungal metabolite that at nanomolar concentrations inhibits PI3-kinase relatively specifically, blocked the release of granule-associated mast cell mediators independent of the secretory stimulus used. This inhibition was most prominent after a 2- to 5-min preincubation with wortmannin and was equally effective in cells additionally treated with exogenous N-ethylcarboxamidoadenosine to potentiate preformed mediator release. Mast cell production of leukotriene C4 20 min after activation or IL-6 16 h after activation was unaffected by up to 100 nM of wortmannin exposure. Mast cells preincubated with wortmannin failed to develop the classic electronmicroscopic evidence of granule swelling and fusion, increased membrane ruffling, or exocytosis upon Ag challenge. Activation of PI3-kinase appears to be critical for mast cell degranulation but is not required for arachidonic acid metabolism or cytokine production to occur. Furthermore, the inhibition of mast cell secretion by wortmannin is not stimulus specific but is evident for both IgE receptor cross-linking and direct calcium influx.
Role for macrophage inflammatory protein-2 in lipopolysaccharide-induced lung injury in rats.
Authors: Schmal H , Shanley T P , Jones M L , Friedl H P , Ward P A
Published: December 2022

Macrophage inflammatory protein-2 (MIP-2) is a C-X-C chemokine that possesses chemotactic activity for neutrophils. Rat MIP-2 was cloned and expressed as a 7.9-kDa peptide that exhibited dose-dependent neutrophil chemotactic activity at concentrations from 10 to 250 nM. Rabbit polyclonal Ab to the 7.9-kDa peptide showed reactivity by western blot analysis and suppressed its in vitro chemotactic activity. Cross-desensitization chemotaxis experiments suggested that the chemotactic responses elicited by MIP-2 and the related chemokine, cytokine-induced neutrophil chemoattractant, may be mediated through a common receptor. Also, chemotactic responses to human GRO-alpha were blocked by exposure of human neutrophils to either GRO-alpha or rat MIP-2, suggesting conservation of this receptor-mediated response. After LPS instillation into rat lung, mRNA for MIP-2 was up-regulated in a time-dependent manner, peaking at 6 h. MIP-2 protein was detected in bronchoalveolar lavage fluids of these animals and a significant amount of chemotactic activity present in these fluids was attributed to MIP-2. On the basis of intrapulmonary instillation of Ab to MIP-2, neutrophil accumulation in lungs after airway instillation of LPS was found to be MIP-2-dependent. These data indicate that MIP-2 plays a significant role in LPS-induced inflammatory response in rat lungs and is required for the full recruitment of neutrophils.
Protein S-thiolation and dethiolation during the respiratory burst in human monocytes. A reversible post-translational modification with potential for buffering the effects of oxidant stress.
Authors: Seres T , Ravichandran V , Moriguchi T , Rokutan K , Thomas J A , Johnston R B
Published: December 2022

Stimulation of the respiratory burst in mouse macrophages or human neutrophils results in the formation of disulfide bonds between low m.w. thiols and sulfhydryl groups on specific cytosolic proteins (S-thiolation). S-thiolation is reversible in certain chemical systems. The aim of the present study was to analyze the dynamic nature of this process in human monocytes under physiologic conditions. We report here that the extent of S-thiolation and the rate of respiratory burst stimulated by opsonized zymosan or phorbol diester increased for 10 to 20 min and then declined (dethiolation) in close association. Individual proteins underwent S-thiolation and dethiolation at different rates. H2O--appeared particularly effective in mediating S-thiolation, based on inhibition of S-thiolation by added catalase and accentuation by azide, which inhibits cellular catalase. S-thiolation did not occur in stimulated monocytes from patients with chronic granulomatous disease. The addition of H2O2 to monocytes or lymphocytes induced rapid S-thiolation (1 to 3 min); a subsequent dethiolation returned most of the proteins to baseline by 15 to 30 min. At 0 degrees C and after addition of 1,3-bis-(2-chloroethyl)-1-nitrosourea, there was effective S-thiolation on exposure to H2O2, but dethiolation was inhibited, suggesting a possible role for glutathione (GSH)/thioredoxin reductase systems in this process. GSH was determined to be the most abundant low m.w. thiol bound to S-thiolated proteins, but gamma-glutamylcysteine and cysteine were also bound. The time of maximal reduction in cytosolic GSH during the respiratory burst (10 min) coincided with the time at which protein-bound GSH was highest. S-thiolation-dethiolation represents a reversible post-translational modification that could protect cellular proteins from irreversible oxidative damage.
Up-regulation of VLA-5 expression during monocytic differentiation and its role in negative control of the survival of peripheral blood monocytes.
Authors: Terui Y , Furukawa Y , Sakai T , Kikuchi J , Sugahara H , Kanakura Y , Kitagawa S , Miura Y
Published: December 2022

Interaction between fibronectin (FN) and very late activation Ag-5 (VLA-5) integrin was recently reported to be involved in apoptosis of hematopoietic cells. In an effort to clarify the physiologic role of FN in the regulation of biologic behavior of terminally differentiated hematopoietic cells, we have examined the change of VLA-5 expression during myeloid cell differentiation and its effects on monocytes and granulocytes. VLA-5 alpha mRNA was up-regulated during monocytic differentiation, but not during granulocytic differentiation of HL-60 cells. Flow cytometric and immunocytochemical analysis revealed that surface expression of VLA-5 was selectively increased upon monocytic differentiation and that it was strongly positive on peripheral blood monocytes. Susceptibility to FN-induced apoptosis was greatly increased upon monocytic differentiation, and it was almost completely abrogated by anti-VLA-5 Ab or RGD peptide. Similarly, FN could significantly enhance apoptosis of normal monocytes but not of granulocytes. Finally, we have shown that anti-FN Ab could suppress spontaneous apoptosis of normal monocytes in culture and prolong their survival. These results suggest that FN might play an important role in negative regulation of the survival of monocytes through its interaction with VLA-5, which is selectively up-regulated during monocytic differentiation.
T lymphocyte indifference to extrathymic islet allografts.
Authors: Coulombe M , Gill R G
Published: December 2022

Although the deletion of self-reactive T cells maturing in the thymus is well documented, the nature of tolerance to Ags expressed exclusively on peripheral (extrathymic) tissues is less clear. Numerous studies have examined T cell reactivity to transgene-encoded Ags expressed in the periphery through tissue-specific promoters. These studies have yielded varied results concerning the degree and nature of tolerance induced to these Ags, ranging from T cell clonal deletion or inactivation to clonal "ignorance" of the peripheral transgene-encoded Ag. Furthermore, the relationship of these findings to the generation and maintenance of transplantation tolerance to extrathymic tissue allografts remains unclear. Whereas transgenic studies represent a developmental form of tolerance, grafted tissues are introduced into adult, nontolerant recipients. To relate these two model systems, we determined whether extrathymic tissue allografts could induce a developmental form of tolerance. As a model of extrathymic alloantigen expression, pancreatic islet allografts, depleted of donor APCs, were established in the periphery of severe combined immune-deficient (SCID) mice. We then examined the potential donor reactivity of adoptively transferred bone marrow-derived T cell precursors maturing in the presence of the established peripheral allograft. Such T cells were neither activated nor tolerized either in vitro or in vivo, suggesting that T cells are indifferent to Ags expressed by extrathymic islet allografts when presented in the absence of a second costimulatory signal.
Quantitative analysis of T cell activation: role of TCR/ligand density and TCR affinity.
Authors: Kim D T , Rothbard J B , Bloom D D , Fathman C G
Published: December 2022

(B6 X A)F1 mice were immunized with sperm whale myoglobin, and T cell clones and hybridomas were generated. Hybridoma 74a.e9 was specific for the sperm whale myoglobin 67-79 peptide and could be partially activated by a peptide analogue, equine myoglobin with a natural 74G substitution. Using this hybridoma in T cell activation assays, we studied the effects of varying the avidity of the TCR for its ligand, the concentration of MHC:peptide complex on the APC, and the density of TCR on the surface. Varying ligand concentration on the surface of the APC, the TCR avidity, or the density of TCR on the T cell were equally important parameters in driving T cell activation. The mouse myoglobin (74T) analogue, however, acted as an antagonist to the T cell response. Its effectiveness was also partially determined by its ability to bind to MHC. By independently altering each of these variables and following T cell activation, we describe the interrelationships among these three components (MHC:peptide:TCR) that control the activation of the T cell.
Plasma from patients with severe invasive group A streptococcal infections treated with normal polyspecific IgG inhibits streptococcal superantigen-induced T cell proliferation and cytokine production.
Authors: Norrby-Teglund A , Kaul R , Low D E , McGeer A , Newton D W , Andersson J , Andersson U , Kotb M
Published: December 2022

Previous studies have suggested a central role for superantigen-induced immune responses in the pathogenesis of streptococcal toxic shock syndrome. The production of streptococcal superantigens by clinical group A streptococcal (GAS) isolates was studied, and the ability of plasma collected from patients with severe invasive GAS infections to neutralize the proliferative- and cytokine-inducing activities of these superantigens was investigated. Overnight culture supernatants from all GAS isolates obtained from patients with invasive disease were found to contain superantigenic activity, as evident from their ability to drive potent T cell proliferation, induce high production of cytokines, and stimulate T cells in a V beta-specific manner. Twelve patients with severe invasive GAS infections, including 11 streptococcal toxic shock syndrome cases and one necrotizing fasciitis without shock, were treated with i.v. infusions of normal polyspecific Ig (IVIG). Plasma samples collected from each patient before and after IVIG administration were analyzed for their ability to neutralize the activity of streptococcal superantigens produced by the GAS isolate that caused their disease. In all IVIG-treated patients, the capacity to neutralize the superantigenic activity, produced by their respective GAS isolate or by purified streptococcal pyrogenic exotoxins, increased in plasma following IVIG administration. Of particular clinical relevance, post-IVIG plasma from each patient completely blocked cytokine production elicited by their respective GAS culture supernatants or by purified streptococcal pyrogenic exotoxins. This study shows that IVIG treatment confers in vivo inhibitory activity against a large array of streptococcal superantigens and suggests that IVIG may be useful in the treatment of severe invasive streptococcal infections.
IL-12 synergizes with IL-2 and other stimuli in inducing IL-10 production by human T cells.
Authors: Jeannin P , Delneste Y , Seveso M , Life P , Bonnefoy J Y
Published: December 2022

IL-12, a potent inducer of IFN-gamma production by T cells and NK cells, has been recently reported to exacerbate an established Th2 response in vivo. However, the effect of IL-12 on Th2-lymphokine production remains unclear. Since IL-10 is a lymphokine associated with Th2 responses which decreases both IL-12-induced IFN-gamma production and IL-12 production by macrophages, we have analyzed here, in an APC-free system, the ability of IL-12 to modulate the production of human IL-10 by established Th0, Th1, and Th2 T cell clones (TCC), T cell lines, and purified peripheral blood T cells. IL-12 synergized with anti-CD3 mAb, Con A, or IL-2 in inducing IL-10 production by Th0, Th1, and Th2 TCC and by T cell lines. This effect was dose dependent (from 0.1 to 50 U/ml) and associated with an increase of IL-10 mRNA transcription. As previously reported, IL-12 also enhanced IFN-gamma production by stimulated Th1 and Th0 TCC and, to a lesser extent, IL-4 production by stimulated Th0 and Th2 TCC. These observations were extended to peripheral blood T cells stimulated in the presence of exogenous IL-2. Moreover, using neutralizing anti-IL-2 Ab, we report that endogenous IL-2 produced by stimulated Th0 TCC could in part contribute to the effect of IL-12 on IL-10 and IL-4 production. In conclusion, IL-12 synergizes with IL-2 and other stimuli in inducing IL-4 and IL-10 production by T cells. This property may help to explain why IL-12 does not efficiently down-regulate an established Th2 response.
Allele-specific differences in the interaction of MHC class I molecules with transporters associated with antigen processing.
Authors: Neisig A , Wubbolts R , Zang X , Melief C , Neefjes J
Published: December 2022

MHC class I molecules bind peptides that are translocated from the cytosol into the endoplasmic reticulum by the peptide transporter associated with antigen processing (TAP). Class I heterodimers have been shown to associate with TAP and are released when loaded with peptide. Here, we show the existence of two pools of class I heterodimers, one associated with TAP and one that is free. Whereas the free pool is recognized by the class I-specific Ab W6/32, the TAP-associated pool is not. Analysis of several class I alleles shows binding to TAP with different efficiencies, even at the earliest time points of MHC class I assembly. Most HLA-A and -C alleles tested interacted efficiently with TAP, whereas a considerable number of HLA-B alleles associated very inefficiently or not at all with TAP. This was also observed in cells with nonfunctional TAP. Sequence comparison of the different class I alleles allowed the definition of amino acids in the peptide binding groove that might be involved in TAP association. Binding of peptides to two different pools of class I heterodimers may ensure efficient peptide association in an environment where peptides have a short life span.
Enhanced development of Th2-like primary CD4 effectors in response to sustained exposure to limited rIL-4 in vivo.
Authors: Yoshimoto K , Swain S L , Bradley L M
Published: December 2022

Previous studies have established that cytokines regulate development of Th cell subsets from naive CD4 cells, suggesting promising therapeutic potential for cytokines that has not been fully realized, in part due to high dose toxicity. Here we examined effects of sustained delivery of low doses of rIL-4 on development of cytokine-secreting primary CD4 cells in vivo. Diffusion chambers injected with X63.Ag-653 plasmacytoma transfectants that constitutively produce rIL-4 were implanted s.c. in mice 24 to 48 h before immunization with keyhole limpet hemocyanin (KLH). Five days later at the peak of the response, KLH-specific CD4 cells from controls, which received chambers containing untransfected plasmacytoma cells, secreted primarily IL-2 and IL-4, with low levels of IFN-gamma and no IL-5 following Ag restimulation. Administration of rIL-4 enhanced IL-2 and IL-4 production by two- to fivefold, with a corresponding decrease in IFN-gamma. As frequencies of KLH-specific precursors that secreted IL-4 were unaltered, exogenous cytokine apparently affected the magnitude of IL-4 secretion by primed CD4 cells. Although IL-4 was undetectable in sera of mice carrying chambers, titers of KLH-specific IgG1 and IgG3 Ab were increased by rIL-4, whereas IgM, IgG2a, and IgG2b levels were unaltered. The results indicate that sustained delivery of low doses of cytokines at sites distant from Ag exposure can selectively potentiate development of CD4 subsets.
Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability.
Authors: van der Burg S H , Visseren M J , Brandt R M , Kast W M , Melief C J
Published: December 2022

The impact of the MHC class I peptide binding stability on the immunogenicity of particular peptide Ags in class I-restricted cytotoxic T lymphocyte responses is not clearly established. Therefore, we have determined the dissociation rate of each peptide from MHC class I at 37 degrees C and compared this to that of a consensus CTL epitope. Newly defined immunogenic peptides formed relatively stable MHC-peptide complexes as shown by their low dissociation rates, whereas nonimmunogenic peptides displayed high dissociation rates. In addition virtually all previously described HLA-A*0201-restricted T cell epitopes showed low dissociation rates. Furthermore, we show that the immunogenicity of HIV-1-derived peptides can be predicted more accurately by their dissociation rate than by the MHC class I binding affinity. Selection of peptides based on affinity and their dissociation rate leads to a more precise identification of candidate CTL epitopes than selection based on affinity alone. These results help to understand why some peptides are recognized by CTL and, along with detailed knowledge of protein processing rules, therefore have important implications for the selection of peptides in peptide-based vaccines.
Antibodies to an autostimulatory growth factor (IL-2) or its receptor induce death of leukemogenic cells.
Authors: Orban P C , Schrader J W
Published: December 2022

We show for the first time that Ab-mediated antagonism of growth factor activity can induce death of all cells in clonal populations surviving and growing by an autostimulatory mechanism. Models of autostimulatory leukemia were generated by transfecting a mouse IL-2-dependent cell line (FD.C/2) with vectors directing production of IL-2 by these cells. One series of clones grew in a density-dependent manner in the absence of exogenous IL-2 and produced tumors in syngeneic mice. Although these clones released relatively small amounts of IL-2, their growth and survival was only partially inhibited by Abs to IL-2 or the IL-2R. Another autostimulatory clone was derived, using a different vector, which produced significantly less IL-2. Treatment of cells of this clone with Abs to IL-2 or its receptor resulted in death of all cells. These data demonstrate that Abs that antagonize growth factor action can induce the death of cells transformed by autostimulatory mechanisms. They suggest that while autostimulatory tumors are relatively resistant to Abs that block growth factor action, this is a quantitative phenomenon, and competitive antagonists of growth factor action of sufficiently high affinity may provide effective and specific adjuvants to the treatment of such tumors.
Resistance of naive mice to murine hepatitis virus strain 3 requires development of a Th1, but not a Th2, response, whereas pre-existing antibody partially protects against primary infection.
Authors: Pope M , Chung S W , Mosmann T , Leibowitz J L , Gorczynski R M , Levy G A
Published: December 2022

Murine hepatitis virus strain 3 (MHV-3) produces a strain-dependent spectrum of disease. The development of liver necrosis has been shown to be related to production of a unique macrophage procoagulant activity (PCA), encoded by the gene fgl-2, in susceptible mice. These studies were designed to examine the influence of Th1/Th2 cells on resistance/susceptibility and production of macrophage PCA in resistant (A/J) and susceptible (BALB/cJ) strains of mice following infection with MHV-3. Immunization of A/J mice with MHV-3 induced a Th1 cellular immune response, and one Th1 cell line (3E9.1) protected susceptible mice and inhibited PCA production by macrophages both in vitro and in vivo. In contrast, immunization of BALB/cJ mice with an attenuated variant of MHV-3 derived from passaging MHV-3 in YAC-1 cells resulted in a Th2 response. Transfer of spleen cells and T cell lines from immunized BALB/cJ mice failed to protect naive susceptible syngeneic mice from infection with MHV-3 and augmented macrophage PCA production to MHV-3 in vitro. However, serum from immunized BALB/cJ mice contained high titrated neutralizing Ab that protected naive BALB/cJ animals from lethal primary MHV-3 infection. These results demonstrate that susceptible BALB/cJ mice generate a Th2 response following MHV-3 infection and that these Th2 cells neither inhibit MHV-3-induced macrophage PCA production nor protect naive mice from MHV-3 infection. The results suggest that Ab protects against primary infection but cannot eradicate ongoing infection. Thus, these data define the differential role of Th1/Th2 lymphocytes in primary and secondary MHV-3 infection and emphasize the importance of PCA in the pathogenesis of MHV-3 infection.