bluuebunny/crossref_metadata_2025 huggingface dataset:
Antimyeloperoxidase-associated proliferative glomerulonephritis: an animal model.
Authors: Brouwer E , Huitema M G , Klok P A , de Weerd H , Tervaert J W , Weening J J , Kallenberg C G
Published: June 2004

To develop an animal model for antimyeloperoxidase (MPO)-associated necrotizing crescentic glomerulonephritis (NCGN), we immunized Brown Norway rats with MPO and localized a neutrophil lysosomal enzyme extract, primarily consisting of MPO and elastinolytic enzymes, plus H2O2, the substrate of MPO, to the glomerular basement membrane (GBM). Upon immunization rats developed antibodies and positive skin tests to MPO. After unilateral perfusion of the left kidney with the lysosomal enzyme extract and H2O2, MPO and immunoglobulin (Ig)G localized transiently along the GMB. At the time of maximal inflammation, at 4 and 10 d after perfusion, MPO, IgG, and C3 could not be detected anymore. MPO-immunized rats perfused with the lysosomal enzyme extract and H2O2, in contrast to control-immunized and/or control-perfused rats, developed a proliferative GN characterized by intra- and extracapillary cell proliferation, ruptured Bowman's capsule, periglomerular granulomatous inflammation, and formation of giant cells. Monocytes, polymorphonuclear leukocytes (PMN), and to a far lesser extent T cells were found in the glomeruli. Interstitial infiltrates consisted of monocytes, PMN, and T cells. Granulomatous vasculitis of small vessels was found at 10 d after perfusion. The proliferative NCGN in this rat model closely resembles human anti-MPO-associated pauci-immune NCGN, and enables the study of the pathophysiology of anti-MPO-associated NCGN.
Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA.
Authors: Ikeyama S , Koyama M , Yamaoko M , Sasada R , Miyake M
Published: June 2004

Previously we showed that motility-related protein (MRP-1) is an antigen recognized by monoclonal antibody (mAb) M31-15 inhibiting cell motility and that the sequence of MRP-1 coincides with that of CD9. In the present study, plasmid was constructed in which human MRP-1/CD9 cDNA is expressed under the control of the Abelson murine leukemia virus promoter sequence. The expression plasmid for MRP-1/CD9 was introduced into Chinese hamster ovary cells, human lung adenocarcinoma cell line MAC10 (MRP-1 positive), and human myeloma cell line ARH77 (MRP-1 negative). All of the MRP-1/CD9 (over)expressing clones obtained from these transfected cells showed suppressed cell motility (penetration and phagokinetic track assays) depending on the degree of expression of MRP-1/CD9. Overexpression of MRP-1/CD9 by MAC10 cells resulted in the suppression of cell motility (maximally 73%) associated with considerable inhibition of the cell growth (maximally 48%). However, the inhibition of the growth of MAC10 cells by mAb M31-15 was < 17% at an antibody concentration of 1-5 micrograms/ml, which inhibits cell motility by > 90%. These results suggest that MRP-1/CD9 directly regulates cell motility and may also affect cell growth. Effects on metastasis by the expression of MRP-1 CD9 were investigated with mouse melanoma BL6 cells-BALB/c nu/nu mouse system. Metastatic potential of all transformants expressing MRP-1/CD9 was lower than that of parent BL6 cells.
Glucocorticoid-mediated control of the activation and clonal deletion of peripheral T cells in vivo.
Authors: Gonzalo J A , González-García A , Martínez C , Kroemer G
Published: June 2004

Poly- and oligoclonal T cell stimuli like anti-CD3 epsilon monoclonal antibody or Staphylococcus aureus enterotoxin B (SEB), injected at doses that per se are not lethal, provoke acute death within less than 24 h, provided that endogenous glucocorticoids (GC) are depleted by adrenalectomy or by injection of saturating amounts of the GC receptor antagonist RU-38486 (mifepristone). Pharmacological doses of the GC agonist dexamethasone (DEX) alter the in vivo response of splenic V beta 8+ T cells to SEB, thus impeding the expansion of such cells and causing their rapid (3 d) clonal deletion. In contrast, coadministration of RU-38486 counteracts a SEB-induced early (12 h) reduction of V beta 8+CD4+ and V beta 8+CD8+ spleen cells. In vivo T cell stimulation by injection of bacterial superantigen induces a rapid (peak at 90-120 min) increase in corticosterone serum levels, suggesting that endogenous GC might control early T cell activation. Accordingly, kinetic studies revealed that RU-38486 has to be administered within 2 h after superantigen administration to exert its lethal effect. Similarly, exogenous GC must be injected during this critical phase (2 h) to rescue animals from acute death induced by coinjection of SEB and D-galactosamine (GalN). Adrenalectomy, injection of RU-38486 and priming with GalN per se provoke the programmed death of peripheral CD4+ and CD8+ T cells. Thus, three manipulations that sensitize mice for the lethal effect of T cell stimulation also exert a proapoptotic effect on peripheral T cells. In synthesis, endogenous and exogenous GC regulate T cell responses and determine the propensity of peripheral T cells to undergo apoptosis.
Characterization of a novel Hodgkin cell line, HD-MyZ, with myelomonocytic features mimicking Hodgkin's disease in severe combined immunodeficient mice.
Authors: Bargou R C , Mapara M Y , Zugck C , Daniel P T , Pawlita M , Döhner H , Dörken B
Published: June 2004

A novel Hodgkin cell line, designated HD-MyZ, was established from the pleural effusion of a 29-yr-old patient with Hodgkin's disease (HD) of nodular sclerosing type. The majority of cells grow adherently and display typical morphological characteristics of Reed-Sternberg (RS) and Hodgkin (H) cells, i.e., large multi- and mononucleated cells with prominent nucleoli. Immunofluorescence analysis revealed a myelomonocytoid immunophenotype (expression of CD13 and CD68, and lack of lymphoid markers). HD-MyZ cells strongly expressed restin, a recently described intermediate filament-associated protein, the expression of which is restricted to H cells, RS cells, and in vitro cultivated peripheral blood monocytes. In addition mRNA expression of c-fms (colony-stimulating factor 1 receptor) could be induced in HD-MyZ cells by phorbol myristate acetate (PMA) stimulation. Southern blot analysis did not detect rearrangement of T cell receptor beta and immunoglobulin H loci, thus demonstrating the lack of lymphoid commitment. HD-MyZ cells were also devoid of Epstein-Barr virus genomes. HD-MyZ cells constitutively express mRNAs for interleukin 1 alpha (IL-1 alpha), IL-1 beta, IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 receptor (type I), and IL-6 receptor. Stimulation of cells with PMA increased mRNA expression as well as the secretion of IL-1 beta, IL-6, and IL-8, and induced the de novo expression of IL-8 receptors. Xenotransplantation into severe combined immunodeficient (SCID) mice by intravenous or subcutaneous inoculation led to development of disseminated tumors with infiltrative and destructive growth. In addition lymphadenopathy, pleural effusion, and infiltration of spleen were observed. Morphological and immunological analysis of tumor cells revealed the same features as HD-MyZ cells. This cell line might be an important tool for understanding the pathogenesis and biology of HD. In addition the SCID mice model might prove helpful in developing new therapeutic strategies.
Peripheral T cell activation and deletion induced by transfer of lymphocyte subsets expressing endogenous or exogenous mouse mammary tumor virus.
Authors: Waanders G A , Shakhov A N , Held W , Karapetian O , Acha-Orbea H , MacDonald H R
Published: June 2004

Murine T cell reactivity with products of the minor lymphocyte stimulatory (Mls) locus correlates with the expression of particular variable (V) domains of the T cell receptor (TCR) beta chain. It was recently demonstrated that Mls antigens are encoded by an open reading frame (ORF) in the 3' long terminal repeat of either endogenous or exogenous mouse mammary tumor virus (MMTV). Immature thymocytes expressing reactive TCR-V beta domains are clonally deleted upon exposure to endogenous Mtv's. Mature T cells proliferate vigorously in response to Mls-1a (Mtv-7) in vivo, but induction of specific anergy and deletion after exposure to Mtv-7-expressing cells in the periphery has also been described. We show here that B cells and CD8+ (but not CD4+) T cells from Mtv-7+ mice efficiently induce peripheral deletion of reactive T cells upon transfer to Mtv-7- recipients, whereas only B cells stimulate specific T cell proliferation in vivo. In contrast to endogenous Mtv-7, transfer of B, CD4+, or CD8+ lymphocyte subsets from mice maternally infected with MMTV(SW), an infectious homologue of Mtv-7, results in specific T cell deletion in the absence of a detectable proliferative response. Finally, we show by secondary transfers of infected cells that exogenous MMTV(SW) is transmitted multidirectionally between lymphocyte subsets and ultimately to the mammary gland. Collectively our data demonstrate heterogeneity in the expression and/or presentation of endogenous and exogenous MMTV ORF by lymphocyte subsets and emphasize the low threshold required for induction of peripheral T cell deletion by these gene products.
The role of macrophage inflammatory protein 1 alpha in Schistosoma mansoni egg-induced granulomatous inflammation.
Authors: Lukacs N W , Kunkel S L , Strieter R M , Warmington K , Chensue S W
Published: June 2004

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a 6-8-kD, lipopolysaccharide-inducible monocyte and neutrophil chemotactic protein that may be important in acute and chronic inflammation. The present study determined the sequential production, source, and in vivo contribution of murine MIP-1 alpha in synchronized Schistosoma mansoni egg pulmonary granuloma formation. Granulomas were examined under conditions of primary, secondary vigorous, and secondary immunomodulated immunity. Secreted MIP-1 alpha was measured in 24-h supernatants from intact granulomas (700/ml) cultured with or without soluble egg antigen (SEA). Primary granulomas isolated from naive mice over a 16-d period showed low spontaneous MIP-1 alpha production (< 1 ng/ml). However, when primary granulomas were challenged with SEA, significant MIP-1 alpha production was observed beginning at day 4 and peaking at day 16. Intact vigorous (isolated from 8-wk-infected mice) and modulated (isolated from 20-wk-infected mice) secondary pulmonary granulomas demonstrated comparable spontaneous MIP-1 alpha production. Addition of SEA to vigorous stage granulomas augmented expression of MIP-1 alpha at all time points, whereas stimulated modulated stage granulomas did not increase production. The latter observation is likely related to endogenous immunoregulatory mechanisms reported for modulated stage animals. Immunohistochemical localization of MIP-1 alpha in granuloma sections and cytocentrifuge preparations from vigorous lesions localized MIP-1 alpha production to macrophages within granulomas. Treatment of mice with rabbit anti-mouse MIP-1 alpha antibodies significantly decreased 8-d primary granuloma formation (> 40%) when compared with control mice. Anti-MIP-1 alpha sera also decreased vigorous (> 20%), but not modulated granuloma formation. These findings demonstrate that MIP-1 alpha contributes to cellular recruitment during schistosome egg granuloma formation.
Alteration of the glycolipid binding specificity of the pig edema toxin from globotetraosyl to globotriaosyl ceramide alters in vivo tissue targetting and results in a verotoxin 1-like disease in pigs.
Authors: Boyd B , Tyrrell G , Maloney M , Gyles C , Brunton J , Lingwood C
Published: June 2004

All members of the verotoxin (VT) family specifically recognize globo-series glycolipids on the surface of susceptible cells. Those toxins that are associated with human disease, VT1, VT2, and VT2c, bind to globotriaosyl ceramide (Gb3) while VT2e, which is associated with edema disease of swine, binds preferentially to globotetraosyl ceramide (Gb4). We were recently able to identify, using site-directed mutagenesis, amino acids in the binding subunit of these toxins that are important in defining their glycosphingolipid (GSL) binding specificity (Tyrrell, G. J., K. Ramotar, B. Boyd, B. W. Toye, C. A. Lingwood, and J. L. Brunton. 1992. Proc. Natl. Acad. Sci. USA. 89:524). The concomitant mutation of Gln64 and Lys66 in the VT2e binding subunit to the corresponding residues (Glu and Gln, respectively) found in VT2 effectively converted the GSL binding specificity of the mutant toxin from Gb4 to Gb3 in vitro. We now report that the altered carbohydrate recognition of the mutant toxin (termed GT3) has biological significance, resulting in a unique disease after intravascular injection into pigs as compared with classical VT2e-induced edema disease. The tissue localization of radiolabeled GT3 after intravascular injection was elevated in neural tissues compared with VT2e accumulation, while localization of GT3 to the gastrointestinal tract was relatively reduced. Accordingly, the pathological lesions after challenge with GT3 involved gross edema of the cerebrum, cerebellum, and brain stem, while purified VT2e caused hemorrhage and edema of the cerebellum, and submucosa of the stomach and large intestine. In addition, both radiolabeled toxins bound extensively to tissues not directly involved in the pathology of disease. VT2e, unlike GT3 or VT1, bound extensively to red cells, which have high levels of Gb4. The overall tissue distribution of VT2e was thus found to be influenced by regional blood flow to each organ and not solely by the Gb4 levels of these tissues. Conversely, the distribution of GT3 (and VT1), which cleared more rapidly from the circulation, correlated with respective tissue Gb3 levels rather than blood flow. These studies indicate the primary role of carbohydrate binding specificity in determining systemic pathology, suggest that the red cells act as a toxin carrier in edema disease, and indicate that red cell binding does not protect against the pathology of systemic verotoxemia.
Tolerance of T cell receptor gamma/delta cells in the intestine.
Authors: Barrett T A , Tatsumi Y , Bluestone J A
Published: June 2004

The present study examined the mechanism(s) of tolerance induction for intestinal intraepithelial lymphocytes (iIELs) using an alloantigen (Ag)-specific gamma/delta T cell receptor (TCR gamma/delta) transgenic (Tg) model. In Tg Ag-bearing H-2b/d mice (Tgb/d), Tg iIELs were Thy-1-, CD44+, CD45R (B220)+, and CD5+, whereas in syngeneic Tgd/d mice, iIELs were Thy-1+, CD44-, and CD45R- with a subset of CD5+ cells. Previously, we had shown that tolerance for Tgb/d iIELs involved functional anergy and deletion (Barrett, T. A., M. L. Delvy, D. M. Kennedy, L. Lefrancois, L. A. Matis, A. L. Dent, S. M. Hedrick, and J. A. Bluestone. 1992. J. Exp. Med. 175:65). In this study we demonstrate that Tgb/d iIELs expressing dull levels of Thy-1 proliferated in the presence of exogenous rIL-2. A direct precursor-product relationship between the Thy-1+-responsive iIELs and the tolerant Thy-1dul/- iIELs was demonstrated by adoptive transfer into severe combined immunodeficient (SCID) mice. Tg Thy-1+ iIELs reconstituting Ag+ but not Ag- SCID mice downregulated Thy-1 after Ag exposure in vivo. Analysis of bone marrow (BM) chimeras demonstrated the persistence of Tg IELs in all Ag+ chimeras although a modest degree of clonal deletion was apparent. The greatest percentage of Tg IELs were detected when Ag was restricted to radioresistant cells (e.g., epithelial cells) compared with BM-derived antigen-presenting cells (APC). This was especially apparent in thymectomized chimeric mice. Consistent with the notion that Ag-bearing epithelial cells may be poor APC, isolated intestinal epithelial cells from Ag-bearing mice failed to stimulate Tg iIELs compared with splenic APC. These studies suggest that the major population of TCR gamma/delta iIELs were probably extrathymically derived and encountered self-Ag on intestinal epithelial cells. The induction of tolerance likely involved an activation event resulting in downregulation of Thy-1. These mechanisms of tolerance for TCR gamma/delta iIELs led to the persistence of a reservoir of self-reactive T cells with the potential for mediating autoimmune disease.
In vivo persistence of expanded clones specific for bacterial antigens within the human T cell receptor alpha/beta CD4-8- subset.
Authors: Dellabona P , Casorati G , Friedli B , Angman L , Sallusto F , Tunnacliffe A , Roosneek E , Lanzavecchia A
Published: June 2004

We analyzed the T cell receptor (TCR) rearrangements of 100 TCR-alpha/beta CD4-CD8- (double negative [DN]) T cell clones from normal individuals. We found that in four out of six donors this subset contains expanded clones that often account for 0.5% and, in one individual, even 7% of all peripheral blood lymphocytes. By combining limiting dilution analysis and N region oligotyping of polymerase chain reaction amplified TCR cDNA, we could measure the clonal size and show that two of these expanded clones remain stable in size for up to 4 yr in peripheral blood. The expanded clones analyzed ex vivo are not cycling and CD45 RAhi ROlo, but express high levels of alpha 4/beta 1 integrins, suggesting that they may have reverted to resting cells after activation. One of these expanded DN clones proliferates in vitro in response to Escherichia coli presented by monocytes cultured in GM-CSF plus IL-4 and kills CD1a+ Molt-4 cells. In contrast to what was found in the alpha/beta DN subset, alpha/beta CD4+ T cell clones specific for a tetanus toxin epitope showed a very small clonal size (< 1 in 10(7)) and could not be reisolated after 2 yr. Taken together, these results indicate that large clonal size and persistence are distinctive features of alpha/beta DN cells specific for bacterial antigens. These cells may use antigen-presenting cells, restriction molecules, and selection routes different from those used by antigen-specific CD4+ T cells.
CD28-B7 interactions allow the induction of CD8+ cytotoxic T lymphocytes in the absence of exogenous help.
Authors: Harding F A , Allison J P
Published: June 2004

The activation requirements for the generation of CD8+ cytotoxic T cells (CTL) are poorly understood. Here we demonstrate that in the absence of exogenous help, a CD28-B7 interaction is necessary and sufficient for generation of class I major histocompatibility complex-specific CTL. Costimulation is required only during the inductive phase of the response, and not during the effector phase. Transfection of the CD28 counter receptor, B7, into nonstimulatory P815 cells confers the ability to elicit P815-specific CTL, and this response can be inhibited by anti-CD28 Fab or by the chimeric B7-binding protein CTLA4Ig. Anti-CD28 monoclonal antibody (mAb) can provide a costimulatory signal to CD8+ T cells when the costimulatory capacity of splenic stimulators is destroyed by chemical fixation. CD28-mediated signaling provokes the release of interleukin 2 (IL-2) from the CD8+ CTL precursors, as anti-CD28 mAb could be substituted for by the addition of IL-2, and an anti-IL-2 mAb can block the generation of anti-CD28-induced CTL. CD4+ cells are not involved in the costimulatory response in the systems examined. We conclude that CD8+ T cell activation requires two signals: an antigen-specific signal mediated by the T cell receptor, and an additional antigen nonspecific signal provided via a CD28-B7 interaction.
Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells.
Authors: Taub D D , Lloyd A R , Conlon K , Wang J M , Ortaldo J R , Harada A , Matsushima K , Kelvin D J , Oppenheim J J
Published: June 2004

The human cytokine interferon-inducible protein 10 (IP-10) is a small glycoprotein secreted by activated T cells, monocytes, endothelial cells, and keratinocytes, and is structurally related to a family of chemotactic cytokines called chemokines. Although this protein is present in sites of delayed-type hypersensitivity reactions and lepromatous leprosy lesions, the biological activity of IP-10 remains unknown. We report here that recombinant human IP-10 stimulated significant in vitro chemotaxis of human peripheral blood monocytes but not neutrophils. Recombinant human IP-10 also stimulated chemotaxis of stimulated, but not unstimulated, human peripheral blood T lymphocytes. Phenotypic analysis of the stimulated T cell population responsive to IP-10 demonstrated that stimulated CD4+ and CD29+ T cells migrated in response to IP-10. This resembles the biological activity of the previously described T cell chemoattractant RANTES. Using an endothelial cell adhesion assay, we demonstrated that stimulated T cells pretreated with optimal doses of IP-10 exhibited a greatly enhanced ability to bind to an interleukin 1-treated endothelial cell monolayer. These results demonstrate that the IP-10 gene encodes for an inflammatory mediator that specifically stimulates the directional migration of T cells and monocytes as well as potentiates T cell adhesion to endothelium.
Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain.
Authors: Porcelli S , Yockey C E , Brenner M B , Balk S P
Published: June 2004

CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules.
Peripheral selection of V delta 1+ cells with restricted T cell receptor delta gene junctional repertoire in the peripheral blood of healthy donors.
Authors: Beldjord K , Beldjord C , Macintyre E , Even P , Sigaux F
Published: June 2004

To characterize the T cell receptor (TCR) repertoire expressed by the V delta 1+ gamma/delta T cell population, we have studied the V delta 1-J delta 1 junctional sequences from peripheral blood samples of healthy donors. We show that, surprisingly, this repertoire is restricted in most healthy adults, with a donor-specific and relatively stable pattern, whereas this repertoire remains unrestricted in infants, and is similar to that of thymocytes. These data contrast with the general assumption that the junctional repertoire of V delta 1+ gamma/delta T cells is extensive, and strongly suggest that peripheral recruitment of V delta 1+ cells bearing particular TCR occurs in humans during the postnatal stage.
Effect of downregulation of germline transcripts on immunoglobulin A isotype differentiation.
Authors: Wakatsuki Y , Strober W
Published: June 2004

In this study we determined the role of immunoglobulin (Ig) germline transcripts in the isotype switch differentiation of the cloned lymphoma B cell line CH12.LX. In initial studies, we showed that addition of transforming growth factor beta (TGF-beta) and interleukin 4 (IL-4), either alone or in combination, augment switching from membrane (m)IgM+ to mIgA+ cells, and that increased switching is preceded and paralleled by an increase in the steady-state level of alpha germline transcripts (alpha GLT). Interestingly, TGF-beta and IL-4 affect switching in different ways, as shown by the fact that IL-4 increases and TGF-beta decreases the number of dual-positive (mIgM+/mIgA+) cells; in addition, TGF-beta and IL-4 have different effects on the time course of induction of alpha GLT. In subsequent studies, we established that we could downregulate alpha GLT levels in CH12.LX B cells by transfecting an expression vector that can be induced to produce transcripts antisense to the I alpha exon. Using this approach we downregulated alpha GLT in CH12.LX B cells undergoing switching in the presence of TGF-beta and IL-4 and showed that such downregulation led to decreased switching, as evidenced by decreased appearance of dual-positive B cells as well as decreased IgA synthesis relative to IgM synthesis. This result was corroborated by the fact that incubation of CH12.LX cells with phosphorothio-oligo antisense DNA to I alpha sequence also led to a decrease in the number of dual-positive cells and in the IgA/IgM secretion ratio. In summary, IgA isotype differentiation in CH12.LX B cell, particularly the steps necessary for the elaboration of mIgM+/mIgA+ switch intermediate cells, is inhibited by downregulation of alpha GLT; it is therefore apparent that alpha GLT plays a key role in the initial stage of isotype switch differentiation.
Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4+ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C.
Authors: Minutello M A , Pileri P , Unutmaz D , Censini S , Kuo G , Houghton M , Brunetto M R , Bonino F , Abrignani S
Published: June 2004

The adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4+ T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCR) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCR identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCR transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein NS4 is highly immunogenic for intrahepatic CD4+ T cells primed by HCV in vivo, and that there can be compartmentalization of some NS4-specific CD4+ T cells to the liver of patients with chronic hepatitis C.
Cytokine mRNA expression during an in vitro response of human B lymphocytes: kinetics of B cell tumor necrosis factor alpha, interleukin (IL)6, IL-10, and transforming growth factor beta 1 mRNAs.
Authors: Matthes T , Werner-Favre C , Tang H , Zhang X , Kindler V , Zubler R H
Published: June 2004

Expression of mRNA for eight cytokines was analyzed in an in vitro response-proliferation and Ig-secretion--of normal human B lymphocytes. This was made possible by the use of murine thymoma cells as helper cells in conjunction with human T cell supernatant, and the design of human DNA sequence-specific primers for RT-polymerase chain reaction. mRNAs for interleukin (IL)2 and IL-4, but also for IL-1 alpha and IL-1 beta remained undetectable during the whole culture period in highly purified B cells prepared by a three-step purification protocol. However, tumor necrosis factor alpha and IL-6 mRNAs peaked during days 1-3 after culture start and became undetectable after 5-6 d, shortly before bulk B cell proliferation started to decline. In contrast, transforming growth factor beta 1 mRNA, after a progressive increase during the first few days, and IL-10 mRNA, after a peak on days 1-3, remained detectable in immunoglobulin (Ig)-secreting cultures throughout the observation period of 22 d. Clonal analysis on 8-d cultures that had been seeded with single B cells by autocloning with the cell sorter, revealed that 85% of 77 B cell clones studied, expressed TGF-beta 1 mRNA, and only 19% IL-10 mRNA. These findings show a differentiation stage-related cytokine program during a B cell response, whereby (a) B cells can become activated without IL-1 alpha or IL-1 beta expression; (b) mRNA for positive (IL-10) and negative (TGF-beta 1) autoregulatory factors coexists in cell populations during the later phase of the response, although not necessarily in all B cell clones; and (c) normal Ig-secreting cells cease IL-6 expression in contrast to their malignant counterparts, myeloma cells.
Molecular cloning, expression, and chromosomal localization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the C-type animal lectin superfamily of signal-transmitting receptors.
Authors: López-Cabrera M , Santis A G , Fernández-Ruiz E , Blacher R , Esch F , Sánchez-Mateos P , Sánchez-Madrid F
Published: June 2004

The activation of T lymphocytes, both in vivo and in vitro, induces the expression of CD69. This molecule, which appears to be the earliest inducible cell surface glycoprotein acquired during lymphoid activation, is involved in lymphocyte proliferation and functions as a signal transmitting receptor in lymphocytes, natural killer (NK) cells, and platelets. To determine the structural basis for CD69 function, the cDNA coding for CD69 was isolated by a polymerase chain reaction-based strategy using oligonucleotides deduced from peptide sequences of the purified protein. The isolated cDNA exhibited a single open reading frame of 597 bp coding for CD69, and predicted a 199-amino acid protein of type II membrane topology, with extracellular (COOH-terminal), transmembrane, and intracellular domains. The CD69 clone hybridized to a 1.7-kb mRNA species, which was rapidly induced and degraded after lymphocyte stimulation, consistent with the presence of rapid degradation signals at the 3' untranslated region. Transient expression of the polypeptide encoded by CD69 cDNA in COS-7 cells demonstrated that it presented properties comparable to native CD69 protein. The CD69 gene was regionally mapped to chromosome 12 p13-p12 by both somatic cell hybrid DNA analysis and fluorescence in situ hybridization coupled with GTG banding (G bands by trypsin using Giemsa). Protein sequence homology search revealed that CD69 is a new member of the Ca(2+)-dependent (C-type) lectin superfamily of type II transmembrane receptors, which includes the human NKG2, the rat NKR-P1, and the mouse NKR-P1 families of NK cell-specific genes. CD69 also has a structural homology with other type II lectin cell surface receptors, such as the T cell antigen Ly49, the low avidity immunoglobulin E receptor (CD23), and the hepatic asialoglycoprotein receptors. The CD69 protein also shares functional characteristics with most members of this superfamily, which act as transmembrane signaling receptors in early phases of cellular activation.
Acquired tolerance to experimental autoimmune encephalomyelitis by intrathymic injection of myelin basic protein or its major encephalitogenic peptide.
Authors: Khoury S J , Sayegh M H , Hancock W W , Gallon L , Carpenter C B , Weiner H L
Published: June 2004

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system that can be induced in a number of species by immunization with myelin basic protein (MBP) in adjuvant, and serves as an experimental model for the study of multiple sclerosis. The role of the thymus in acquired tolerance in autoimmune models has not been thoroughly investigated. In this study, we examined the effects of intrathymic injection of MBP or its major encephalitogenic peptide on the course of EAE in Lewis rats. A single intrathymic injection of MBP 48 h pre- but not postimmunization protects animals from actively induced EAE. An intact MBP-primed thymus was required up to 10 d postimmunization, as thymectomy on days 1, 2, and 7 postimmunization abrogated the protective effect, whereas thymectomy on day 10 did not. The proliferative response of primed lymphocytes was significantly reduced in animals that were intrathymically injected with MBP. Protection against clinical EAE was induced by thymic injection of the major encephalitogenic region (residues 71-90) but not a nonencephalitogenic (21-40) MBP epitope. Immunohistologic examination of the brain from rats intrathymically injected with encephalitogenic peptide showed markedly reduced cellular infiltrate and virtual absence of activation and inflammatory cytokines as compared with rats intrathymically injected with the nonencephalitogenic peptide. These results indicate that the thymus may play an active role in acquired systemic immunologic tolerance in T cell-mediated experimental autoimmune diseases. This effect may be mediated by a process of clonal inactivation of autoreactive T cell clones circulating through the thymus.
Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta.
Authors: Vodovotz Y , Bogdan C , Paik J , Xie Q W , Nathan C
Published: June 2004

Activated mouse peritoneal macrophages produce nitric oxide (NO) via a nitric oxide synthase that is inducible by interferon gamma (IFN-gamma): iNOS. We have studied the mechanisms by which transforming growth factor beta 1 (TGF-beta) suppresses IFN-gamma-stimulated NO production. TGF-beta treatment reduced iNOS specific activity and iNOS protein in both cytosolic and particulate fractions as assessed by Western blot with monospecific anti-iNOS immunoglobulin G. TGF-beta reduced iNOS mRNA without affecting the transcription of iNOS by decreasing iNOS mRNA stability. Even after iNOS was already expressed, TGF-beta reduced the amount of iNOS protein. This was due to reduction of iNOS mRNA translation and increased degradation of iNOS protein. The potency of TGF-beta as a deactivator of NO production (50% inhibitory concentration, 5.6 +/- 2 pM) may reflect its ability to suppress iNOS expression by three distinct mechanisms: decreased stability and translation of iNOS mRNA, and increased degradation of iNOS protein. This is the first evidence that iNOS is subject to other than transcriptional regulation.
Small amounts of superantigen, when presented on dendritic cells, are sufficient to initiate T cell responses.
Authors: Bhardwaj N , Young J W , Nisanian A J , Baggers J , Steinman R M
Published: June 2004

Dendritic cells are potent antigen-presenting cells for several primary immune responses and therefore provide an opportunity for evaluating the amounts of cell-associated antigens that are required for inducing T cell-mediated immunity. Because dendritic cells express very high levels of major histocompatibility complex (MHC) class II products, it has been assumed that high levels of ligands bound to MHC products ("signal one") are needed to stimulate quiescent T cells. Here we describe quantitative aspects underlying the stimulation of human blood T cells by a bacterial superantigen, staphylococcal enterotoxin A (SEA). The advantages of superantigens for quantitative studies of signal one are that these ligands: (a) engage MHC class II and the T cell receptor but do not require processing; (b) are efficiently presented to large numbers of quiescent T cells; and (c) can be pulsed onto dendritic cells before their application to T cells. Thus one can relate amounts of dendritic cell-associated SEA to subsequent lymphocyte stimulation. Using radioiodinated SEA, we noted that dendritic cells can bind 30-200 times more superantigen than B cells and monocytes. Nevertheless, this high SEA binding does not underlie the strong potency of dendritic cells to present antigen to T cells. Dendritic cells can sensitize quiescent T cells, isolated using monoclonals to appropriate CD45R epitopes, after a pulse of SEA that occupies a maximum of 0.1% of surface MHC class II molecules. This corresponds to an average of 2,000 molecules per dendritic cell. At these low doses of bound SEA, monoclonal antibodies to CD3, CD4, and CD28 almost completely block T cell proliferation. In addition to suggesting new roles for MHC class II on dendritic cells, especially the capture and retention of ligands at low external concentrations, the data reveal that primary T cells can generate a response to exceptionally low levels of signal one as long as these are delivered on dendritic cells.
Lipopolysaccharide antagonists block taxol-induced signaling in murine macrophages.
Authors: Manthey C L , Qureshi N , Stütz P L , Vogel S N
Published: June 2004

Taxol is the prototype of a new class of microtubule stabilizing agents with promising anticancer activity. Several studies show that taxol mimics the actions of lipopolysaccharide (LPS) on murine macrophages. To investigate the mechanism of taxol-induced macrophage stimulation, we evaluated the ability of Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) and SDZ 880.431 to block taxol-induced effects. RsDPLA and SDZ 880.431 are lipid A analogues that lack LPS-like activity, but inhibit the actions of LPS, presumably by blocking critical cellular binding sites. We report that RsDPLA and SDZ 880.431 potently inhibited taxol-induced TNF secretion, gene activation, and protein-tyrosine phosphorylation. The role of microtubules in taxol signaling was investigated. Taxol-induced microtubule bundling in primary and transformed RAW 264.7 macrophages was not blocked by RsDPLA or SDZ 880.431. Taxotere, a semisynthetic taxoid, was more potent than taxol as an inducer of microtubule bundling, but did not induce tumor necrosis factor alpha secretion and gene activation. These data dissociate the microtubule effects of taxol from macrophage stimulation and suggest that taxol stimulates macrophages through an LPS receptor-dependent mechanism. The results underscore the potential of taxol as a tool for studying LPS receptor activation and provide insights into possible therapeutic actions of this new class of drugs.
Direct evidence for the role of COOH terminus of mouse mammary tumor virus superantigen in determining T cell receptor V beta specificity.
Authors: Yazdanbakhsh K , Park C G , Winslow G M , Choi Y
Published: June 2004

It has recently been shown that open reading frames in the 3' long terminal repeats of mouse mammary tumor viruses encode superantigens. These viral superantigens (vSAGs) stimulate most T cells expressing appropriate V beta s almost regardless of the rest of the variable components of the T cell receptors (TCR) expressed by those cells. vSAGs produce a type II integral membrane protein with a nonessential short cytoplasmic domain and a large glycosylated extracellular COOH-terminal domain, which is predicted to interact with major histocompatibility complex class II molecules and the TCR. The transmembrane region of vSAG also has an internal positively charged lysine residue of unknown significance. A set of chimeric and mutant vSAG genes has been used in transfection experiments to show that only the extreme COOH-terminal portion of vSAGs determine their TCR V beta specificities, and to show that the lysine residue in the transmembrane domain is not essential for the function of vSAG.
B cell activation via CD40 is required for specific antibody production by antigen-stimulated human B cells.
Authors: Nonoyama S , Hollenbaugh D , Aruffo A , Ledbetter J A , Ochs H D
Published: June 2004

Costimulatory signals provided by T cells are required for B cells to produce specific antibody (Ab) to T-dependent antigen (Ag) bacteriophage phi x 174. In this study, we demonstrate that if cultured in the presence of anti-CD40, interleukin 10 (IL-10), and Ag, purified B cells can produce antiphage Ab in quantities comparable to those synthesized by B cells cocultured with Ag and T cells. Isotypes produced by B cells in this culture system correspond to those observed in sera of B cell donors. Culture of immunoglobulin (Ig)D- and IgD+ B cells reveals that Ag-induced production of antiphage Ab is restricted to IgD- subset of B cells. In the absence of Ag, anti-CD40/IL-10-stimulated B cells produce only minute amounts of antiphage Ab, indicating that Ag stimulation is indispensable and provides a signal that is synergistic with anti-CD40 and IL-10. Addition of a soluble form of the CD40 ligand (sgp39) to the culture system has a similar effect on specific Ab synthesis as anti-CD40; addition of the soluble construct, CD40 Ig, known to inhibit gp39/CD40 interaction, suppresses in vitro antiphage Ab production by Ag exposed peripheral blood mononuclear cells. Finally, in vivo requirement of gp39/CD40 interaction for specific Ab production was demonstrated by the finding that activated T cells from patients with x-linked hyper IgM syndrome express functionally defective gp39 and respond with depressed Ab titers and fail to switch from IgM to IgG after multiple phage immunizations. These observations illustrate that in vitro and possibly in vivo Ag-specific Ab synthesis requires the presence of Ag and IL-10, and activation signals via CD40.
Human keratinocytes release the endogenous beta-galactoside-binding soluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms.
Authors: Wollenberg A , de la Salle H , Hanau D , Liu F T , Bieber T
Published: June 2004

A better understanding of the pathophysiological role of Langerhans cells (LC) in atopic diseases is dictated by the characterization of the structures involved in immunoglobulin (IgE)-binding on their cell surface. We previously reported that human LC express the high affinity receptor for IgE (Fc epsilon RI), as well as the low affinity receptor for IgE (Fc epsilon RII/CD23). In the present study, we document the presence of a third IgE-binding structure on human LC, the IgE-binding protein (epsilon BP), an endogenous soluble beta-galactoside binding lectin. Immunohistochemical studies performed on normal human skin revealed an anti-epsilon BP reactivity in the cytoplasm of keratinocytes and in that of acinous cells of eccrine sweat glands. epsilon BP was also found on the cell surface of LC, as shown by anti-epsilon BP/anti-CD1a double labeling and flow cytometric analysis. Anti-epsilon BP binding to the surface of LC was completely abolished by preincubation with lactose and restored by addition of recombinant human epsilon BP, indicating that epsilon BP binds to LC surface by virtue of its lectin property. Immunoblot analysis of anti-epsilon BP-reactive material in keratinocytes and purified LC disclosed a protein with an apparent molecular weight of 33,000 consistent with epsilon BP. Interestingly, mRNA transcripts for epsilon BP were detected only in keratinocytes but not in purified LC isolated from normal skin. epsilon BP was found to be released in culture supernatants of keratinocytes. Incubation of LC with these supernatants resulted in epsilon BP-binding to LC surface via protein-carbohydrate interaction. Most importantly, we could show that binding of human myeloma IgE to LC was inhibited by epsilon BP. In contrast, neuraminidase-treated human myeloma IgE binds to LC only in the presence of epsilon BP. In situ binding studies revealed that keratinocytes, although containing epsilon BP intracytoplasmatically, failed to exhibit any IgE-binding properties. Collectively, our results suggest that human keratinocytes produce the beta-galactoside-binding lectin epsilon BP, which subsequently binds to the surface of LC where it is functional in modulating their binding capacity for IgE glycoforms.
A first or dominant immunization. I. Suppression of simultaneous cytolytic T cell responses to unrelated alloantigens.
Authors: Rowley D A , Stach R M
Published: June 2004

A first or dominant immunization with one antigen markedly inhibited specific cytolytic T lymphocyte (CTL) responses to a second unrelated alloantigen without suppressing antibody responses to other antigens. Suppression was induced rapidly, became systemic, and could be transferred passively with only serum. Suppression did not result from elimination of cells capable of responding to the second antigen. The mechanisms responsible for this "priority of the first response" may be the same that help protect the fetus during pregnancy, promote renal allograft survival after multiple blood transfusions, and prevent effective CTL-mediated immunity to variants of tumor cells or infectious agents that arise during tumor progression or chronic infections.
Functional GATA-3 binding sites within murine CD8 alpha upstream regulatory sequences.
Authors: Landry D B , Engel J D , Sen R
Published: June 2004

Genes encoding the accessory molecules CD8 and CD4 are activated early in thymocyte development, generating CD4+8+ double positive intermediates, which give rise to two functionally distinct mature T cell subsets that express either CD4 or CD8. The mechanisms that govern the activation or suppression of the CD8 gene are likely to be central to the T cell development program. To identify the key regulatory factors, we have initiated an analysis of the transcriptional regulation of the murine CD8 alpha gene. We have identified three CD8+ cell-specific DNAase I hypersensitive sites (HSS) located upstream of the murine CD8 alpha gene. In vitro mobility shift analysis of the -4.0-kb HSS region has revealed multiple binding sites for the T cell-restricted transcription factor GATA-3. In vitro translated murine GATA-3 binds specifically to both CD8 GATA sites, and coexpression of this factor in transient transfection assays transactivates a reporter construct containing these sequences. These results provide the first evidence for the role of a T cell-restricted factor in the regulation of either CD8 or CD4 genes.