bluuebunny/crossref_metadata_2025 huggingface dataset:
A filarial cysteine protease inhibitor down‐regulates T cell proliferation and enhances interleukin‐10 production
Authors: Hartmann Susanne , Kyewski Bruno , Sonnenburg Bettina , Lucius Richard
Published: March 2007

Filarial nematodes are a cause of chronic debilitating diseases in the tropics. A hallmark of filariasis is the marked down‐regulation and polarization of host immune responses, yet molecular constituents of parasites causing this state have remained undefined. We describe a 17‐kDa antigen (Av17) of the rodent filarial parasite Acanthocheilonema viteae, which shows amino acid homologies to cystatin C, a major cysteine protease inhibitor belonging to family 2 of the cystatin superfamily. Av17 is released by filariae in vitro. Exported molecules of A. viteae worms are shown to markedly suppress mitogen‐induced T cell proliferation of mice and jirds. Av17 accounts for 45.5% of this suppressive activity in the murine system. Recombinant Av17 (rAv17), expressed in Escherichia coli, exhibits biological activity as a cysteine protease inhibitor and was used to examine the immunomodulatory effects. rAv17 induces down‐regulation of murine T cell responses to mitogens, to T cell receptor cross‐linking by anti‐CD3 antibodies and to specific antigens, and at the same time up‐regulation of interleukin‐10. Hence, this filarial cystatin is a likely effector molecule of immunomodulation and a potential target for antifilarial intervention.
Evidence for a CD4‐associated calcium influx independent of the phosphoinositide transduction pathway in human T cells
Authors: Lafont Virginie , Fischer Thierry , Zumbihl Robert , Faure Sandrine , Hivroz Claire , Rouot Bruno , Favero Jean
Published: March 2007

We recently showed, using human Jurkat T cell variants lacking the T cell receptor (TCR)/CD3 complex, that the lectin jacalin is able to trigger intracellular calcium increase provided that CD4 is expressed on the cell surface. Involvement of the CD4 molecule in jacalin‐induced biological effects was furthermore demonstrated in differentiated U937 myelomonocytic cells expressing or not expressing CD4, and is confirmed here in human CD4‐transfected mouse thymoma cells. In the present paper, we analyze the CD4‐associated calcium response triggered by jacalin independently of the TCR/CD3 complex. We show that the observed calcium rise results from a direct long‐lasting calcium influx from the outside without release of calcium from intracellular stores. We demonstrate that it is independent of the phosphoinositide phospholipase C transduction pathway. Moreover, we show that this peculiar calcium response can be blocked by protein tyrosine kinase inhibitors (herbimycin and genistein) giving evidence of the involvement of a protein tyrosine kinase, the best candidate of which is the CD4‐associated p56lck. Altogether, our results suggest that, independently of the TCR/CD3 complex, CD4 may be involved in the triggering of a calcium signal dependent on a protein tyrosine kinase and independent of the phosphoinositide transduction pathway.
Proteasome regulation of Fas ligand cytotoxicity
Authors: Matsui Ken , Omura Satoshi , Cui Haili , Schauer Stephanie L. , Sonenshein Gail E. , Ju Shyr‐Te
Published: March 2007

The role of NF‐xB in regulating FasL‐mediated cytotoxicity was investigated by using lactacystin. Lactacystin is a microbial metabolite known to inhibit only the protease activity of the proteasome, which is required for NF‐xB translocation. When activated by immobilized anti‐CD3 monoclonal antibody, hybridoma T cells (5D5) degraded IxBβ, translocated NF‐xB into the nucleus, transcribed immediate‐early genes and the Fas ligand (FasL) gene, and expressed FasL‐mediated cytotoxicity. Lactacystin strongly blocked IxBβ degradation and the translocation of NF‐xB (p50/RelA heterodimer), but had little effect on the expression of the transcription factors, Oct‐1 and AP‐1. Moreover, lactacystin did not inhibit the nuclear translocation of NF‐ATp whereas cyclosporin A inhibited the translocation of both NF‐xB and NF‐ATp. The expression of c‐myc and nur77, two immediate‐early genes implicated in FasL gene activation, was blocked by lactacystin. Subsequently, the expression of FasL gene and FasL‐mediated cytotoxicity was inhibited. LLnL, a well‐known peptide aldehyde which inhibits the protease activities of the proteasome and cysteine proteases, also inhibited NF‐xB translocation and FasL‐mediated cytotoxicity. However, these events were not inhibited by the highly specific cysteine protease inhibitor E64. These observations provide further evidence that FasL cytotoxicity is regulated by the proteasome. Furthermore, lactacystin must be added early in order to efficiently inhibit the induction of FasL cytotoxicity, indicating that the early events are critical for FasL gene activation. Our study integrates the proteasome‐dependent IxB degradation and NF‐xB translocation into a T cell activation cascade which results in FasL gene activation and the expression of FasL‐mediated cytotoxicity.
Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18
Authors: Lecoanet‐Henchoz Sybille , Plater‐Zyberk Christine , Graber Pierre , Gretener Denise , Aubry Jean‐Pierre , Conrad Daniel H. , Bonnefoy Jean‐Yves
Published: March 2007

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the α chain of the β2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ‐CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ‐CD23 and CD11b/CD 18‐expressing cells were significantly inhibited by anti‐CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up‐regulation of interleukin‐6 (IL‐6) production following ZZ‐CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL‐6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activites of murine myeloid cells.
Human mast cells express functional TrkA and are a source of nerve growth factor
Authors: Nilsson Gunnar , Forsberg‐Nilsson Karin , Xiang Zou , Hallböök Finn , Nilsson Kenneth , Metcalfe Dean D.
Published: March 2007

Mast cells are the principal effector cells in IgE‐dependent hypersensitivity reactions. Despite reports that rodent mast cells proliferate in the presence of nerve growth factor (NGF), human mast cells reportedly do not respond to this factor. To determine if human mast cells express the NGF receptors, TrkA tyrosine receptor and the low affinity NGF receptor (LNGFR), we first analyzed the mRNA expression by RT‐PCR of TrkA and LNGFR in a human mast cell line (HMC‐1) and in human mast cells cultured in the presence of stem cell factor. Both HMC‐1 and cultured human mast cells were found to express TrkA but not LNGFR. TrkA protein was demonstrated by Western blot analysis of HMC‐1 lysates. Using flow cytometric analysis and mast cell tryptase as a mast cell marker, both HMC‐1 cells and cultured human mast cells were shown to co‐express tryptase and TrkA. Treatment of mast cells with NGF resulted in phosphorylation of TrkA on tyrosine residues as detected by immunoblotting with an antiphosphotyrosine antibody. Furthermore, NGF induced the immediate early gene c‐fos in HMC‐1 cells. HMC‐1 cells and cultured human mast cells were also found to express NGF mRNA, and conditioned medium from HMC‐1 cells stimulated neurite outgrowth from chicken embryonic sensory ganglia in culture. This effect was blocked by anti‐NGF. Thus, mast cells express functional TrkA and synthesize NGF, suggesting a mechanism by which NGF may act as an autocrine factor for human mast cells, and by which mast cells and nerves may interact.
Antagonism of cytotoxic T lymphocyte‐mediated lysis by natural HIV‐1 altered peptide ligands requires simultaneous presentation of agonist and antagonist peptides
Authors: Sewell Andrew K. , Harcourt Gillian C. , Goulder Philip J. R. , Price David A. , Phillips Rodney E.
Published: March 2007

Mutations in human immunodeficiency virus (HIV) cluster in cytotoxic T lymphocyte (CTL) epitopes (Phillips, R. E. et al., Nature 1991. 354: 453) and are subject to immune‐mediated positive selection (Price, D. A. et al., Proc. Natl. Acad. Sci. USA 1997. 94: 1890). We studied the effects of naturally occurring mutations in the HIV‐1 p17 Gag HLA A2 restricted epitope SLYNTVATL on recognition by anti‐HIV CTL. Most of these naturally occurring mutants escaped killing by one CTL line and the majority acted as CTL antagonists. We also investigated whether CTL exposed to a strict antagonist peptide restricted by HLA A2 were unresponsive when exposed to targets presenting the wild‐type sequence. The results show that antagonism of anti‐HIV CTL killing requires the simultaneous presence of agonist and antagonist peptide. We found no evidence that CTL exposed to an antagonist received a functionally negative signal since these CTL retained an unimpaired capacity to lyse targets bearing wild‐type peptide.
CpG‐containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: A new class of vaccine adjuvants
Authors: Lipford Grayson B. , Bauer Marc , Blank Christian , Reiter Rudi , Wagner Hermann , Heeg Klaus
Published: March 2007

Foreign DNA has been shown to impinge on immune cell function by an as yet unidentified mechanism. We and others have demonstrated that single‐stranded (ss) DNA containing the motif CpG flanked by two 5′ purines and two 3′ pyrimidines are mitogenic for B cells and activate macrophages to release tumor necrosis factor‐α, interferon‐γ, interleukin (IL)‐6 or IL‐12. Because of these proinflammatory responses we investigated if ssDNA would serve as a potential vaccine adjuvant. Here we show that CpG‐containing oligonucleotides represent a powerful adjuvant for both humoral and cellular immune responses. When ssDNA was incorporated into inocula, specific antibody titers of the IgG2 isotype were enhanced by greater than 100‐fold. Primary cytotoxic T lymphocyte responses generated to either unprocessed protein antigen or major histocompatibility complex class I‐restricted peptide were exceedingly strong. Evidence is also provided that oligomers directly influenced T cell receptor‐triggered T cell proliferation. Thus ssDNA oligomers may serve as inexpensive and safe vaccine adjuvants and, in addition, differential effects due to sequence may allow for directed responses.
An Fcγ receptor I (CD64)‐negative subpopulation of human peripheral blood monocytes is resistant to killing by antigen‐activated CD4‐positive cytotoxic T cells
Authors: Grage‐Griebenow Evelin , Baran Jaroslaw , Loppnow Harald , Los Marek , Ernst Martin , Flad Hans‐Dieter , Pryjma Juliusz
Published: March 2007

It has been demonstrated that in monocyte/T cell co‐cultures activated with recall antigens, cytotoxic T cells were generated which are able to reduce the number of antigen‐presenting monocytes. In previous studies we could show that a minor subset of monocytes, the Fcγ receptor I‐negative (CD64−) monocytes, exhibits significantly higher antigen‐presenting capacity than the main population of monocytes (< 90%) which are Fcγ receptor I‐positive (CD64+). Therefore, we addressed the question whether they are also differentially susceptible to T cell‐mediated killing. In the present study we demonstrate that the CD64− monocyte subset is more resistant to killing by antigen‐activated T cells than CD64+ monocytes, as indicated by a higher viability and recovery of CD64− monocytes. This mechanism involves CD95 (Fas) antigen, since monocyte death in co‐cultures with antigen‐activated T cells could be partially reduced by blocking anti‐Fas monoclonal antibodies (mAb). In agreement with this finding, although CD95 antigen was expressed on CD64+ and CD64− monocytes at comparable levels, killing of CD64− monocytes by activating anti‐Fas mAb was lower than of CD64+ monocytes.
Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses
Authors: Oliver Alyce M. , Martin Flavius , Gartland G. Larry , Carter Robert H. , Kearney John F.
Published: March 2007

Mouse splenic B cells can be separated based on their distinctive expression of cell surface antigens. Marginal zone (MZ) B cells are CD38high CD21high CD23low/−, while follicular (FO) B cells are CD21int CD23int and newly formed (NF) B cells are CD21dim/− CD23−. Exploiting these phenotypic distinctions, we isolated the three B cell subsets and assessed their other phenotypic differences and functional capabilities in vitro. FO B cells proliferate more than the other B cell subsets in response to either IgM or CD38 cross‐linking. MZ B cells proliferate better than FO B cells when stimulated with lipopolysaccharide (LPS), sub‐optimal levels of LPS and CD38 cross‐linking or CD40 ligation. When NF, FO and MZ B cells were stimulated with either LPS or LPS and interleukin‐4, MZ B cells secreted more IgM and IgG3 than the other two subsets. Similarly, calcium fluxes measured within MZ B cells are larger in amplitude and more sustained after B cell receptor cross‐linking than those found in the other two subsets. Collectively, these results indicate that CD38high CD21high CD23low/− MZ B cells are specially suited to play a unique role in response to antigens delivered to the MZ area.
Genetic modification of a carcinoma with the IL‐4 gene increases the influx of dendritic cells relative to other cytokines
Authors: Stoppacciaro Antonella , Paglia Paola , Lombardi Luciano , Parmiani Giorgio , Baroni Carlo , Colombo Mario P.
Published: March 2007

Tumor cells genetically modified with certain cytokine genes gain immunogenic properties that allow the development of systemic anti‐tumor immunity. Whether different cytokines may influence infiltration of transduced tumors by dendritic cells (DC) has not been investigated. Therefore, we analyzed the C26 murine colon carcinoma genetically modified to release interleukin (IL)‐2, IL‐4, IL‐12, granulocyte colony‐stimulating‐factor (CSF) or granulocyte‐macrophage (GM)‐CSF for immunostaining with the monoclonal antibody NDLC145 recognizing the DEC205 determinant which, on tumor sections, is virtually restricted to DC. Infiltrating leukocytes were also characterized for expression of co‐stimulatory molecules like CD54, CD86 and major histocompatibility complex class II. The intratumoral DC content was dependent on the type of transduced cytokines with C26/IL‐4 being the most abundant in DEC205+ cells. The effect of IL‐4 in recruiting DC did not depend on the type of tumor since it was confirmed in the TSA mammary carcinoma. In comparison with C26/GM‐CSF, C26/IL‐4 had more B7.2+ cells but less Ia+ cells. Furthermore, the hypertrophic skin overlaying tumors producing GM‐CSF showed numerous Langerhans cells stained by NDLC145 and the draining lymph nodes showed abundance and paucity of DC in C26/GM‐CSF and C26/IL‐4, respectively. When injected into the ear pinna, C26/GM‐CSF stimulated, whereas C26/IL‐4 inhibited DC‐mediated priming of delayed‐type hypersensitivity reaction by 2,4‐dinitro‐1‐fluorobenzene. These findings prove that transduced cytokines differently influence DC recruitment at the tumor site and DC function in nearby tissues. Along with the other leukocytes and their secondary produced cytokines, DC create an environment in which T cells can be differently modulated. Such a phenomenon may have implications on genetic modification of tumor cells to be used as cancer vaccine.
Priming of cytotoxic T lymphocytes by five heat‐aggregated antigens in vivo: Conditions, efficiency, and relation to antibody responses
Authors: Speidel Katharina , Osen Wolfram , Faath Stefan , Hilgert Ivan , Obst Reinhard , Braspenning Joris , Momburg Frank , Hämmerling Günter J. , Rammensee Hans‐Georg
Published: March 2007

Mice were immunized i.p. with soluble or heat‐denatured protein antigens [ovalbumin, β‐galactosidase, or recombinant E7 protein of human papilloma virus type 16 (HBV)]. Heat‐denatured (100°C) preparations of these proteins were able to induce cytotoxic T lymphocytes (CTL) that recognize cells expressing the respective genes, whereas native protein was either inefficient or required up to 30‐fold higher doses. If the heat‐treated proteins were separated into aggregated and soluble fractions by ultracentrifugation, only the aggregated fractions were able to induce specific CTL; this is probably because of the easier access to one of the major histocompatibility complex class I loading pathways for exogenous antigen. Addition of the adjuvant aluminium hydroxide (alum) to aggregated proteins abolished their ability to induce CTL; thus, a condition leading to a strong antibody response appeared to inhibit CTL induction. Interestingly, immunization with heat‐denatured ovalbumin plus alum increased the IgM/IgG1 ratio compared to immunization with native ovalbumin and alum. Immunization of B6 mice transgenic for an HLA‐A2/H‐2Kb hybrid gene with heat‐denatured, recombinant HPV 16‐E7 protein induced Db‐restricted CTL specific for the peptide 49–57 of E7, indicating that this epitope is immunodominant over any A2‐restricted E7 epitope in these mice. A whole influenza virus preparation heated to 100°C or even autoclaved was still able to induce virus‐specific CTL and BALB/c spleen cells heated to 100°C could still cross‐prime minor H‐specific CTL in B6 mice, although with lower efficiency than fresh spleen cells. Thus, aggregated proteins can be considered as components for future vaccines.
Priming of helper T cell‐dependent antibody responses by hemagglutinin‐transgenic B cells
Authors: Kalberer Christian P. , Reininger Luc , Melchers Fritz , Rolink Antonius G.
Published: March 2007

Mice expressing the hemagglutinin (HA) gene of influenza virus PR8 (H1 subtype) under the control of x light chain promoter and enhancer have been generated. They express HA in and on B cells, and are tolerant to HA. In vitro, only lipopolysaccharide (LPS) blasts but not resting B cells of transgenic mice can stimulate HA‐specific helper T cells of HA‐specific α/β T cell receptor (TCR)‐transgenic mice. Transfer of HA‐transgenic LPS blasts into syngeneic, non‐transgenic recipients primes HA‐specific antibody responses. Resting, small HA‐transgenic B cells, which were purified by fluorescence‐activated cell sorting, prime lower antibody responses. Host B cells produce the HA‐specific antibody response. The donor HA‐transgenic B cells need to express major histocompatibility complex (MHC) class II molecules and need to be alive to induce the antibody response in the host. Most notably, the host antibody response never produces detectable levels of IgM, but only of switched IgG isotypes. Neither resting nor activated HA‐transgenic B cells induce tolerance in antibody responses. These results suggest that HA‐transgenic B cells, presenting both the intact antigen on the cell surface and peptides of the antigen on MHC class II, are effective inducers of helper T cell responses, and as judged by the Ig‐isotype response pattern, which is mainly IgG1, of Th2 type.
Mannose receptor‐mediated uptake of antigens strongly enhances HLA class II‐restricted antigen presentation by cultured dendritic cells
Authors: Tan M. C. Agnes A. , Mommaas A. Mieke , Drijfhout Jan Wouter , Jordens Reina , Onderwater Jos J. M. , Verwoerd Desiree , Mulder Aat A. , van der Heiden Annette N. , Scheidegger Doris , Oomen Lauran C. J. M. , Ottenhoff Tom H. M. , Tulp Abraham , Neefjes Jacques J. , Koning Frits
Published: March 2007

Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor‐mediated endocytosis. Here we show that endocytosis of mannose receptor‐antigen complexes takes place via small coated vesicles, while non‐mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non‐mannosylated antigens co‐localized with typical markers for major histocompatibility complex class II‐enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200–10 000‐fold enhanced potency to stimulate HLA class II‐restricted peptide‐specific T cell clones compared to non‐mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.
Monoclonal gammopathies in aging μ, x‐transgenic mice: Involvement of the B‐1 cell lineage
Authors: van Arkel Cornelis , Hopstaken Cornelis M. , Zurcher Chris , Bos Nicolaas A. , Kroese Frans G. M. , Savelkoul Huub F. J. , Benner Robbert , Radl Jiri
Published: March 2007

Monoclonal gammopathies (MG) are monoclonal proliferative disorders of B cells at the differentiation stage of Ig production. They can be detected in the serum, either as transient or as persistent homogenous immunoglobulin (H‐Ig) components. The exact phenotype, localization, and cell lineage origin of the precursor cells of MG are unknown, but may be crucial for both the correct diagnosis and for timely efficient treatment of the malignant forms. We used for the first time transgenic (Tg) mice (Sp6; μ/x) to study the origin of MG. In the μ, x Tg mice a small proportion of B cells can still produce endogenous IgM. These cells are of B‐1 cell origin. The MG in Tg mice showed a later onset and a lower frequency than those in littermate control mice, mainly due to a four times lower frequency of benign monoclonal gammopathy. The 10% of B‐1 cells that were able to produce endogenous Ig led to the development of MG in a frequency that was half the number of MG found in normal littermates. None of the MG in Tg mice produced an Ig of the Tg origin and therefore it can be concluded that they originated from B‐1 cells.
Down‐regulation of LFA‐1‐mediated T cell adhesion induced by the HIV envelope glycoprotein gp160 requires phosphatidylinositol‐3‐kinase activity
Authors: Mazerolles Fabienne , Barbat Christiane , Fischer Alain
Published: March 2007

Human immunodeficiency virus binds to CD4+ T lymphocyte by the interaction, in part, between its gp120 envelope glycoprotein and the CD4 molecule. We and others have reported that the lipid kinase phosphatidylinositol‐3‐kinase (PI3‐kinase) is associated with the CD4‐p56lck complex and can be activated by various CD4 ligands. In a previous report we showed that the gp160 envelope down‐regulates lymphocyte function‐associated antigen‐1 (LFA‐1)‐dependent adhesion between CD4+ T cells and B cells. This down‐regulation was shown to be p56lck‐dependent. Here we investigate the role of PI3‐kinase in the inhibition of adhesion induced by gp160 binding to CD4. We found that gp160 activates the PI3‐kinase of HUT78 CD4+ T cell lines in a way dependent on CD4‐p56lck association, since no activation was detected when the interaction between CD4 and p56lck was disrupted. It was also shown, using different inhibitors of the PI3‐kinase (wortmannin, Ly294002 and antisense oligonucleotides), that this lipid kinase was necessary for the down‐regulation of LFA‐1‐mediated adhesion induced by gp160. These results strongly suggest that PI3‐kinase activation induced by gp160 leads to down‐regulation of LFA‐1‐mediated T cell adhesion to B cells. Inhibition by gp160 of cytoskeleton rearrangement‐dependent, anti‐CD3‐mediated T cell adhesion to B cells was blocked by neutralization of PI3‐kinase activity, while inhibition of cytoskeleton rearrangement‐independent, Mg2+‐induced T cell adhesion was not. These results emphasize the role of PI3‐kinase in the regulation of cytoskeleton structure. It is proposed that gp160 activates both p56lck and PI3‐kinase which lead to a cytoskeleton organization unfavorable for LFA‐1 function.
Interleukin‐9 is involved in host protective immunity to intestinal nematode infection
Authors: Faulkner Helen , Humphreys Neil , Renauld Jean‐Christophe , van Snick Jacques , Grencis Richard
Published: March 2007

Murine studies have demonstrated that, as with other nematodes, infection with the intestinal nematode Trichinella spiralis is associated with a pronounced intestinal mastocytosis, eosinophilia and an elevation in serum levels of total IgE. Both interleukin (IL)‐4 and IL‐5 are clearly important in the generation of IgE responses and eosinophilia, respectively, but the control of mucosal mastocytosis in vivo is not as well defined. Mucosal mast cells appear to be particularly important with regard to T. spiralis infections as there is good evidence to suggest their involvement in expulsion of the parasite from the host. In this study we examined the effect of the overproduction of the Th2 cytokine IL‐9 on infection with this nematode. We demonstrate that naive IL‐9‐transgenic mice have an intense intestinal mastocytosis and high serum levels of mouse mast cell protease‐1. Moreover, upon infection high titers of parasite‐specific IgG1 were observed with a heightened mast cell response, which was associated with the rapid expulsion of T. spiralis from the gut. Furthermore, as depression of this mast cell response, using anti‐c‐kit antibodies, resulted in the inability of these mice to expel the parasite, this study clearly demonstrates an activity of IL‐9 on mucosal mastocytosis and the host protective immune response in vivo.
Induction of T cell adhesion to extracellular matrix or endothelial cell ligands by soluble or matrix‐bound interleukin‐7
Authors: Ariel Amiram , Hershkoviz Rami , Cahalon Liora , Williams Douglas E. , Akiyama Steven K. , Yamada Kenneth M. , Chen Chun , Alon Ronen , Lapidot Tsvee , Lider Ofer
Published: March 2007

The putative effects of interleukin (IL)‐7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human IL‐7 was found to bind ECM or fibronectin (FN) with IC50 values of 10–100 nM. Nanogram amounts of both soluble and, especially, FN‐ or ECM‐bound IL‐7, which differentially affected the morphologies of FN‐adherent T cells, induced the adhesion of resting CD4+ and CD8+ T cells in dose‐dependent and β1 integrin‐dependent manners. Under static and flow conditions, soluble IL‐7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule‐1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL‐7 of FN‐specific β1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN‐bound IL‐7 or by an integrin‐specific affinity‐modulating monoclonal antibody, but not by soluble IL‐7 or phorbol 12‐myristate 13‐acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio‐active state.
Chemoattractant receptor cross talk as a regulatory mechanism in leukocyte adhesion and migration
Authors: Campbell James J. , Foxman Ellen F. , Butcher Eugene C.
Published: March 2007

Leukocytes express multiple chemoattractant receptors that can trigger adhesion and direct their migration. Regulation of such proadhesive and migratory responses must often occur in a complex cytokine milieu in vivo, in which multiple receptors may be engaged simultaneously or sequentially, Here we have examined the interplay between interleukin‐8 (IL‐8) receptor and formyl peptide receptor (fPR) stimulation and its consequences for leukocyte adhesion and chemotactic responses. IL‐8 has no significant effect on fMLP‐stimulated adhesion and migration of human neutrophils, indicating that leukocytes have the potential to respond to sequential proadhesive and chemoattractant stimuli during homing and targeted migration. In contrast, fMLP at ⩾ 10 nM totally abrogated proadhesive and chemoattractant responses to IL‐8, a trans effect to which the fPR itself is relatively resistant. N‐formyl peptides are released by invasive bacteria and lysed cells, and the dominance of the fPR may ensure that signals from these terminal phagocyte targets can override host‐derived recruitment signaling through IL‐8 and other chemokine receptors. Asymmetric inhibition of adhesion‐triggering responses is also observed in lymphoid cells transfected with IL‐8 receptor A and fPR, but in this cellular context chemotactic responses are bidirectionally abrogated, suggesting the potential for downstream desensitization of motility programs as well. Cross talk between chemoattractant receptors and their signaling pathways may help target leukocyte migration in the context of complex chemoattractant arrays in vivo.
Distinct roles for lymphotoxin‐α and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue
Authors: Körner Heinrich , Cook Matthew , Riminton D. Sean , Lemckert Frances A. , Hoek Robert M. , Ledermann Birgit , Köntgen Frank , de St Groth Barbara Fazekas , Sedgwick Jonathon D.
Published: March 2007

Specialized roles for the pro‐inflammatory cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) were characterized in TNF/LTα−/− and TNF−/− mice established by direct gene targeting of C57BL/6 ES cells. The requirement for LT early in lymphoid tissue organogenesis is shown to be distinct from the more subtle and varied role of TNF in promoting correct microarchitectural organization of leukocytes in LN and spleen. Development of normal Peyer's patch (PP) structure, in contrast, is substantially dependent on TNF. Only mice lacking LT exhibit retarded B cell maturation in vivo and serum immunoglobulin deficiencies. A temporal hierarchy in lymphoid tissue development can now be defined, with LT being an essential participant in general lymphoid tissue organogenesis, developmentally preceeding TNF that has a more varied and subtle role in promotion of correct spatial organization of leukocytes in LN and spleen. PP development in TNF−/− mice is unusual, indicating that TNF is a more critical participant for this structure than it is for other lymphoid tissues.
Co‐localization of Fyn with CD3 complex, CD45 or CD28 depends on different mechanisms
Authors: zur Hausen Jan Dirk , Burn Paul , Amrein Kurt E.
Published: March 2007

The Src family protein tyrosine kinase Fyn (p59fyn) plays an important role in thymocyte development and T cell receptor (TCR) signal transduction. Fyn has been shown to associate with the TCR‐CD3 complex, the protein tyrosine phosphatase CD45 and several co‐receptors such as CD28 which are crucial for initiating T cell activation and proliferation. The molecular basis of how Fyn is associated with these transmembrane proteins is largely unknown. To investigate the Fyn association with the TCR‐CD3 complex, CD45 and CD28 at the molecular level, various Fyn/β‐galactosidase fusion proteins were constructed and expressed in Jurkat cells. Co‐localization experiments applying antibody‐induced co‐capping and double immunofluorescence staining techniques were used to study the association of these fusion proteins with the TCR‐CD3 complex, CD45 and CD28. Our results revealed that co‐localization of Fyn with the TCR‐CD3 complex requires the unique N terminus whereas co‐localization with CD45 depends on the unique N terminus, the Src homology (SH)3‐ and a functional SH2 domain. CD28 co‐localizes with Fyn molecules that contain the N terminus and a functional SH2 domain. These results suggest that Fyn association with the TCR‐CD3 complex, CD45 and CD28 is mediated by different molecular mechanisms.
Ultrastructural analysis of mouse thymocyte subpopulations
Authors: Yagi Hideki , Nakamura Masanori , Ishii Tadashi , Kasahara Shigeru , Itoh Tsunetoshi
Published: March 2007

To understand the lineage relationship and to define morphological characteristics of each thymocyte subset, we have performed ultrastructural analysis of highly purified thymocyte subpopulations. By flow cytometry, five subpopulations were sorted based on the expression of CD4 and CD8 and on cell size (forward scatter): large and small CD4+8+, CD4−8−, CD4+8−, and CD4−8+ thymocytes. Small CD4+8+ thymocytes were the smallest among lymphoid cells, and had a round and smooth cell outline with condensed nuclei, the cytoplasm was scanty and the cell organelles were not developed, suggesting the majority of this subset might be inactive by morphological criteria. CD4+8− thymocytes appeared to be similar to peripheral CD4+ T cells. The CD4−8− thymocyte subset contained morphologically immature cells in terms of cell size, presence of cell surface villi, and euchromatic appearance of the nucleus. CD4−8+ thymocytes, heterogeneous in cell size, nuclear chromatin contents and amount of cytoplasm, could be divided into two distinct types. Type 1 CD4−8+ thymocytes were intermediate in size, and therefore similar to peripheral mature CD8+ T cells. Type 2 CD4−8+ thymocytes were large and irregular in shape (large CD4−8+) with irregular‐shaped and euchromatic nuclei. Large CD4−8+ cells were, thus, considered to be at the transitional stage from CD4−8− to CD4+8+. At least two groups of large CD4+8+ cells were ultrastructurally classified by the nuclear chromatin content. Large CD4+8+ cells with heterochromatic nuclei were round with a smooth cell membrane, whereas large CD4+8+ cells with euchromatic nuclei were spherical with projections. Cytological features of heterochromatic large CD4+8+ cells are similar to those of small CD4+8+ thymocytes except for cell size. Euchromatic large CD4+8+ cells could be regarded as active blasts potentially leading to mature cells. Taken together, this is the first report that describes the ultrastructural characteristics of each thymocyte subset highly purified by flow cytometry.
Host cell factor CD59 restricts complement lysis of Plasmodium falciparum‐infected erythrocytes
Authors: Wiesner Jochen , Jomaa Hassan , Wilhelm Martin , Tony Hans‐Peter , Kremsner Peter G. , Horrocks Paul , Lanzer Michael
Published: March 2007

Here we demonstrate that components of the entire complement cascade are fixed on the surface of erythrocytes infected with the human malarial parasite Plasmodium falciparum. Despite the activation of lytic complement factors, no complement‐mediated lysis of parasitized erythrocytes was observed. Complement‐mediated lysis of P. falciparum‐infected erythrocytes occurred only in the absence of functional intrinsic CD59. These data suggest that the restriction of the complement attack of P. falciparum‐infected erythrocytes is principally mediated by intrinsic host cell factors, in particular CD59.
Rho prevents apoptosis through Bcl‐2 expression: Implications for interleukin‐2 receptor signal transduction
Authors: Gómez Javier , Martínez‐A. Carlos , Giry Murielle , García Alphonse , Rebollo Angelita
Published: March 2007

Here we describe a Rho‐mediated apoptosis suppression pathway driven by Bcl‐2 expression in the interleukin (IL)‐4‐ or IL‐2‐dependent murine T cell line TS1αβ. IL‐2, but not IL‐4, induces Bcl‐2 expression through RhoA activation which is inhibited by the specific Rho family inhibitor, Clostridium difficile Toxin B, as well as by a dominant negative RhoA mutant. Using transient transfections of RhoA mutants tagged with the vesicular stomatitis virus glycoprotein, we show that a constitutively active RhoA mutant induces Bcl‐2 expression and prevents apoptosis upon IL‐4 withdrawal. Finally, we have identified the signaling pathway involved together with RhoA in Bcl‐2 induction and show compelling evidence for the implication of phosphatidylinositol 3 kinase and protein kinase C.
Induction of heat shock protein 72 synthesis by endogenous tumor necrosis factor via enhancement of the heat shock element‐binding activity of heat shock factor 1
Authors: Watanabe Naoki , Tsuji Naoki , Akiyama Shinichiro , Sasaki Hiroyoshi , Okamoto Tetsuro , Kobayashi Daisuke , Sato Tsutomu , Hagino Tsukasa , Yamauchi Naofumi , Niitsu Yoshiro , Nakai Akira , Nagata Kazuhiro
Published: March 2007

Endogenous tumor necrosis factor (enTNF) acts as a resistance factor against cytotoxicity caused by heat by inducing manganous superoxide dismutase (MnSOD), thereby scavenging reactive oxygen free radicals. On the other hand, it is also well known that heat shock proteins (HSP) which are induced by heat stress behave as cytoprotective factor against this stress. However, the relationship of these two resistance factors is not elucidated yet. In the present study, we therefore proposed the possibility that enTNF enhances HSP72 expression. Heat‐sensitive L‐M (mouse tumorigenic fibroblast) cells, which normally do not express enTNF, were transfected with a nonsecretory‐type human TNF‐α expression vector to produce enTNF. Stable transfectants showed resistance to heat treatment and an increase of HSP72 expression. Conversely, when HeLa (human uterine cervical cancer) cells, which normally produce an appreciable amount of enTNF, were transfected with an antisense TNF‐α mRNA expression vector to inhibit enTNF synthesis, their heat sensitivity was enhanced and HSP72 expression was reduced by half. Although enTNF caused no difference in the level of heat shock factor (HSF) 1 in these cells, enTNF expression correlated well with the binding activity of HSF‐1 to a 32P‐labeled synthetic oligonucleotide containing the human heat shock element (HSE). These results indicate that enTNF participates not only in intrinsic resistance against heat via induction of MnSOD but also via enhancement of the HSE‐binding activity of HSF 1 followed by augmentation of HSP72 expression.
Analysis of susceptibility of NOD mice to spontaneous and experimentally induced thyroiditis
Authors: Damotte Diane , Colomb Evelyne , Cailleau Catherine , Brousse Nicole , Charreire Jeannine , Carnaud Claude
Published: March 2007

Beside diabetes, non‐obese diabetic (NOD) mice develop sporadic lymphoid infiltration of the thyroid gland, mimicking Hashimoto's thyroiditis. We have examined the prevalence of those manifestations in NOD mice, the influence of the major histocompatibility complex (MHC) and the association with autoantibodies. The incidence at 1 year is of 14.3% in wild‐type NOD mice versus 19.6% in congenic NOD.H2k mice. The moderate, but statistically significant difference, based on the analysis of 161 NOD and 169 NOD.H2k mice, suggests that MHC genes partially control spontaneous NOD thyroiditis. Autoantibodies against thyroglobulin (Tg) are mouse specific and their presence correlates closely with thyroiditis. The strong correlation between cellular and humoral anomalies therefore resembles Hashimoto's thyroiditis. NOD and NOD.H2k mice actively immunized against Tg develop severe chronic lesions with epithelium necrosis and interstitial tissue fibrosis. Most interestingly, those lesions do not regress spontaneously as in CBA/J mice. Paradoxically, the response to Tg of lymph node cells from NOD mice is weaker both in proliferation and cytokine production. The defect is most evident for interferon‐γ‐producing T cells and is reflected in the marked deficit in IgG2a antibodies. Thus a moderate anti‐Tg response seems to favor chronicity of thyroiditis. In conclusion, NOD and NOD.H2k mice offer a unique opportunity of analyzing the factors leading to immune chronicity in a genetic context which promotes autoimmune endocrinopathies.
In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis
Authors: Küsters Sabine , Tiegs Gisa , Alexopoulou Lena , Pasparakis Manolis , Douni Eleni , Künstle Gerald , Bluethmann Horst , Wendel Albrecht , Pfizenmaier Klaus , Kollias George , Grell Matthias
Published: March 2007

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)‐induced, CD4+ T cell‐dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A‐induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild‐type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.
Interleukin‐15 preferentially promotes the growth of intestinal intraepithelial lymphocytes bearing γδ T cell receptor in mice
Authors: Inagaki‐Ohara Kyoko , Nishimura Hitoshi , Mitani Akio , Yoshikai Yasunobu
Published: March 2007

Several cytokines including stem cell factor (SCF) and interleukin (IL)‐7 are known to be required for development of γδ T cell receptor (TCR) intestinal intraepithelial lymphocytes (i‐IEL) in mice. We show here the effects of IL‐15 on the proliferation and maintenance of murine γδ i‐IEL in vitro. γδ i‐IEL constitutively expressed a high level of IL‐15 receptor α mRNA and proliferated in response to IL‐15 more vigorously than αβ i‐IEL. Vγ/δ repertoire analysis revealed that IL‐15, like IL‐2, induced polyclonal expansion of γδ i‐IEL, whereas γδ i‐IEL responding to IL‐7 showed a Vγ/δ repertoire skewed towards Vγ1/Vδ4, Vδ5. IL‐15 efficiently prevented γδ i‐IEL from apoptosis induced by growth factor deprivation. This rescue was accompanied by up‐regulation of Bcl‐2 expression. These results suggest that IL‐15 plays important roles in proliferation and maintenance of γδ i‐IEL.
The CC chemokine antagonist Met‐RANTES inhibits eosinophil effector functions through the chemokine receptors CCR1 and CCR3
Authors: Elsner Jörn , Petering Holger , Höchstetter Renate , Kimmig Daniela , Wells Timothy N. C. , Kapp Alexander , Proudfoot Amanda E. J.
Published: March 2007

Eosinophils are predominant effector cells not only in allergic diseases but also in connective tissue diseases. The recruitment of eosinophils to the site of inflammation and release of reactive oxygen species leading to tissue damage and propagation of the inflammatory response are mediated by chemokines. Thus, agents that would be able to inhibit or antagonize chemokine‐induced eosinophil activation are interesting as therapeutical agents. We describe the effect of a chemokine receptor antagonist, Met‐RANTES, on human eosinophil effector functions in response to RANTES, monocyte chemoattractant protein (MCP)‐3 and eotaxin. Met‐RANTES was able to inhibit dose‐dependently [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP‐3 and eotaxin. Whereas maximal and half‐maximal inhibitory effect of Met‐RANTES following stimulation with RANTES and MCP‐3 were observed at 2 μg/ml and 1 μg/ml, respectively, maximal and half‐maximal inhibitory effects of Met‐RANTES in response to eotaxin were detected at 10 μg/ml and 3 μg/ml. Moreover, eotaxin‐induced [Ca2+]i transients were only half reduced at a Met‐RANTES concentration at which RANTES and MCP‐3 were completely blocked. Besides its effect on [Ca2+]i transients, Met‐RANTES dose‐dependently inhibited actin polymerization in eosinophils following chemokine stimulation. Whereas Met‐RANTES totally inhibited RANTES‐ and MCP‐3‐induced actin polymerization at 5 μg/ml, the eotaxin‐induced response was only reduced by 50%. However, Met‐RANTES inhibited dose‐dependently the release of reactive oxygen species in response to RANTES, MCP‐3 and eotaxin. Again, eotaxin‐induced release of reactive oxygen species, however, was only half reduced at a Met‐RANTES concentration (10 μg/ml) at which RANTES and MCP‐3 were completely blocked. The results of this study show that (1) Met‐RANTES is an effective and powerful antagonist of effector functions of human eosinophils following stimulation with RANTES, MCP‐3 and eotaxin; (2) Met‐RANTES seems to be able to antagonize the response of eosinophils through chemokine receptor 1 (CCR1) preferentially to CCR3; (3) Met‐RANTES antagonizes eosinophil but not neutrophil effector functions and might be therefore of interest for a new therapeutical approach to prevent the invasion and destructive power of eosinophils in diseases that are accompanied by eosinophil infiltration such as allergic asthma and connective tissue diseases.
Relapsing and remitting experimental allergic encephalomyelitis: A focused response to the encephalitogenic peptide rather than epitope spread
Authors: Takács Katalin , Chandler Phillip , Altmann Daniel M.
Published: March 2007

The progression of experimental allergic encephalomyelitis (EAE) in certain mouse strains has been reported to involve a broadening of the response to myelin antigens, apparently resulting from priming to endogenous determinants of the myelin sheath. The phenomenon has been termed determinant spread. Interest in this effect has centered on the mechanism it offers to explain the progressive, relapsing and remitting course of EAE and indeed of multiple sclerosis. We have conducted a systematic, longitudinal study in SJL mice to look for determinant spread during relapsing and remitting EAE, correlating epitope recognition and cytokine production with disease severity. Disease was induced using three of the four encephalitogenic proteolipid protein or myelin basic protein epitopes, and responses to each of four epitopes recognized by SJL T cells were tracked through acute disease, remission and relapse. The responses of lymph node cells, splenocytes and central nervous system (CNS)‐infiltrating T cells were analyzed. While marginal, transient responses to secondary epitopes were detectable in splenocytes, CNS‐infiltrating cells showed a dominant response to the original disease‐inducing epitope without evidence of a shift to other determinants during relapse. Disease relapse was correlated with an increase in CNS‐infiltrating cells and a high proliferative and interferon (IFN)‐γ response to the disease‐inducing peptide. During remission, there was a decrease in numbers of cells infiltrating the CNS. These cells were down‐regulated, showing low if any response to the myelin peptides tested as measured by proliferation, production of IFN‐γ or production of IL‐4. Our findings argue strongly against a causal role for determinant spread in disease relapse as observed in these models of EAE.