bluuebunny/crossref_metadata_2025 huggingface dataset:
Csk overexpression reduces several monokines and nitric oxide productions but enhances prostaglandin E2 production in response to lipopolysaccharide in the macrophage cell line J774A.1
Authors: Iwabuchi Kazuya , Hatakeyama Shigetsugu , Takahashi Akio , Ato Manabu , Okada Masato , Kajino Yuri , Kajino Ki‐Ichi , Ogasawara Kazumasa , Takami Kimitaka , Nakagawa Hachiro , Onoé Kazunori
Published: March 2007

The catalytic activity of src‐family protein tyrosine kinases (src‐PTK) is suppressed when a C‐terminal tyrosine is phosphorylated by an intracellular PTK, C‐terminal Src kinase (Csk). In the present report, to study the regulatory functions of the Csk in cells of monocyte/macrophage lineage, we transfected a eukaryotic expression vector containing rat csk cDNA in a macrophage cell line, J774A.1, and examined alterations of the response to lipopolysaccharide (LPS) in the transfectants which overexpressed Csk. Csk overexpression resulted primarily in a down‐regulation of Fgr activity, an src‐PTK expressed in J774A.1, and hyperphosphorylation of several cellular proteins of 35, 57, 66, 97 and 120–130 kDa. Furthermore, in these Csk transfectants, production of interleukin (IL)‐1α, IL‐6, tumor necrosis factor‐α, and nitric oxide (NO) following LPS stimulation were reduced compared with those in parental J774A.1 or J774A.1 transfected with the vector alone. The extent of reduction paralleled the amounts of Csk proteins expressed in the Csk‐transfected J774A.1. The reduced NO production in these cells was associated with low levels of mRNA of inducible NO synthetase. On the other hand, an enhancement of prostaglandin E2 production was observed in the Csk‐transfected J774A.1 cells upon stimulation with LPS, which appeared to result from the high level of prostaglandin‐H synthetase in the transfectants. The present findings indicate that overexpression of Csk has differential effects on the regulation of production of chemical mediators and monokines, probably via modulation of signal transduction downstream of LPS‐mediated signals.
Immunomodulatory effects of a plasmid expressing B7‐2 on human immunodeficiency virus‐1‐specific cell‐mediated immunity induced by a plasmid encoding the viral antigen
Authors: Tsuji Takashi , Hamajima Kenji , Ishii Norihisa , Aoki Ichiro , Fukushima Jun , Xin Ke‐qin , Kawamoto Susumu , Sasaki Shin , Matsunaga Kei‐Ichiro , Ishigatsubo Yoshiaki , Tani Kenji , Okubo Takao , Okuda Kenji
Published: March 2007

B7 co‐stimulation is essential for activating resting T cells following antigen recognition by the T cell receptor. To determine whether B7 has adjuvant activities on human immunodeficiency virus type‐1 (HIV‐1)‐specific immunity induced by inoculation of a plasmid encoding HIV‐1 env and rev (DNA vaccine), B7‐1 and B7‐2 expression plasmids were co‐inoculated with the DNA vaccine. The delayed‐type hypersensitivity response and cytotoxic T lymphocyte (CTL) activity were significantly enhanced when B7‐2 expression plasmid was co‐inoculated with the DNA vaccine, but were unaffected when the B7‐1 expression plasmid was used with the vaccine instead. The immunological response enhanced by B7‐2 decreased below the level of mice immunized with the DNA vaccine in combination with CTLA4Ig, an inhibitor of the B7/CD28 co‐stimulatory signal, suggesting that this signal is critical for the enhanced response induced by co‐inoculation of the DNA vaccine and B7‐2 expression plasmid. This enhancement appeared to occur via an interferon‐γ (IFN‐γ)‐dependent mechanism, as combined administration of the B7‐2 plasmid and neutralizing anti‐IFN‐γ antibody abrogated the virus‐specific cell‐mediated immunity. These results suggest that this gene‐based co‐inoculation strategy using HIV‐1 viral antigen and B7‐2 co‐stimulatory molecule could be a powerful means of combating HIV‐1 infection.
Activation of the gp130 signaling pathway by monoclonal antibodies directed against the gp130 molecule
Authors: Autissier Patrick , Liautard Janny , Brochier Jean , Gaillard Jean Philippe
Published: March 2007

Six cytokines of the interleukin (IL)‐6 family involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. We made a panel of anti‐gp130 monoclonal antibodies (mAb) to study the structure and function of the gp130 molecule. These mAb recognized different epitopes of the gp130 that we called A to J. Most of the mAb were found to be inhibitors and we studied whether some of them could also induce gp130 activation. When used alone, none of them was able to initiate the proliferation of IL‐6‐dependent cell lines. However, some particular associations of the mAb were able to induce a proliferative response. mAb B1 could activate the lines in association with F1 or with I2 but not with I1, which in ELISA was similar to I2. In contrast mAb B2, which in ELISA appeared to be very similar to B1, was able to activate the cells in association with I1 but not with F1 or I2. Two other mAb belonging to specificities A and C were found to be activators either in association with I1 only, or with I1 or B2, respectively. These associations of mAb appeared to be nearly as potent activators as IL‐6 itself. Although we still have no precise idea of the mechanisms involved, they are interesting tools to study the molecular interactions leading to gp130 activation and, from a practical point of view, valuable growth factors of hematopoietic stem cells.
Selectivity of the major histocompatibility complex class II presentation pathway of cortical thymic epithelial cell lines
Authors: Oukka Mohamed , Andre Patrice , Tunnel Pascale , Besnard Nelly , Angevin Veronique , Karlsson Lars , Trans Phuong L. , Charron Dominique , Bihain Bernard , Kosmatopoulos Kostadinos , Lotteau Vincent
Published: March 2007

Major histocompatibility complex (MHC) restriction of the immune response is established during positive selection of T cells in the thymus. This occurs mainly through interactions of T cell receptor of developing thymocytes with MHC/peptide ligands on cortical thymic epithelial cells (TEC). An ongoing controversy concerns the origin and the role of peptides involved in the positive selection of thymocytes. Evidence provided here shows that processing of MHC class II complexes in cortical TEC differs from that of medullary TEC. Removal of the invariant chain associated with MHC class II complexes was rapid and complete in medullary TEC which present peptides from both exogenous and cytosolic origin. In cortical TEC, a large fraction of class II dimers remained associated with a 10–12‐kDa fragment of invariant chain (Ii). Incomplete removal of Ii correlated with the inability of cortical TEC to present peptides from exogenous origin. However, presentation of peptides from cytosolic proteins by cortical TEC remained possible. Thus, most peptides from exogenous proteins may be excluded from participating in positive selection of CD4+ T cells by a mechanism limiting Ii breakdown.
The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics
Authors: Grommé Monique , van der Valk Richard , Sliedregt Karen , Vernie Leen , Liskamp Rob , Hämmerling Günter , Koopmann Jens‐Oliver , Momburg Frank , Neefjes Jacques
Published: March 2007

The major histocompatibility complex (MHC)‐encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ∼ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.
A novel 4‐kb interleukin‐13 receptor α mRNA expressed in human B, T, and endothelial cells encoding an alternate type‐II interleukin‐4/ interleukin‐13 receptor
Authors: Gauchat Jean‐François , Schlagenhauf Edith , Feng Ning‐Ping , Moser René , Yamage Mat , Jeannin Pascale , Alouani Sami , Elson Greg , Notarangelo Luigi D. , Wells Timothy , Eugster Hans‐Pietro , Bonnefoy Jean‐Yves
Published: March 2007

A 4 kb human interleukin‐13 receptor (IL‐13R) chain cDNA was cloned from a B cell cDNA library using expressed sequence tags homologous to mouse IL‐13R as probes. The deduced protein sequence shows a significant level of sequence identity with the IL‐5R and the human IL‐13R identified recently by expression cloning. The cytoplasmic region is very highly conserved between human and mouse homologs and contains a consensus binding motif for a signal transducer and activator of transcription. The cDNA encodes a protein binding IL‐13 when expressed alone which participates in a receptor complex for both IL‐4 and IL‐13 when expressed in conjunction with the IL‐4Rα chain. Transcripts for this IL‐13R chain could be detected in most tissues and organs studied and in T, B, endothelial cells, basophilic, immature mast cell, and monocytic cell lines. The pattern of expression is different from the other recently cloned IL‐13R molecule, and correlates with sites where IL‐4 and IL‐13 signaling is known to occur. This novel receptor is therefore likely to be implicated in reactions involved in IgE responses, T helper 2 differentiation, adhesion of leukocytes to endothelium, and therefore in pathological phenomena such as allergy, atopy, and asthma.
Presentation of abundant endogenous class II DR‐restricted antigens by DM‐negative B cell lines
Authors: Kovats Susan , Whiteley Phyllis E. , Concannon Patrick , Rudensky Alexander Y. , Blum Janice S.
Published: March 2007

Peptides derived from endogenous and exogenous antigens compete for binding and presentation via class II molecules. Studies with mutant B cell lines defective in exogenous antigen presentation suggest that HLA‐DM molecules facilitate the interaction of foreign peptides and class II molecules. In contrast, presentation of self antigens is not strictly dependent upon HLA‐DM, as demonstrated by the ability of these mutant cells to activate T cells specific for endogenous antigens. Two distinct classes of DM‐negative cells, T2 cells generated by in vitro mutagenesis and lines derived from bare lymphocyte syndrome (BLS) patients, were able to present epitopes derived from self proteins. Transfection of DM genes into the mutant cells enhanced the presentation of some, but not all, endogenous antigens, suggesting that formation of select endogenous peptide/class II complexes is not dependent upon DM. The efficiency of endogenous antigen presentation in the absence of DM was also dependent on the mutant antigen‐presenting cell studied, as the TxB hybrid T2 presented greater amounts of self peptides compared to cells from BLS patients. Thus, additional genes, aside from DM, may regulate the pathway for endogenous antigen presentation.
T cells recognize the VH complementarity‐determining region 3 of the idiotypic protein of B cell non‐Hodgkin's lymphoma
Authors: Wen Yue‐Jin , Lim Seah H.
Published: March 2007

The idiotypic protein expressed by B lymphoma cells is a clone‐specific tumor antigen which may be suitable for immune targeting by T cells. In this study, we cloned the immunoglobulin heavy chain variable gene (VH) of the idiotypic protein from a patient with B cell lymphoma and used a synthetic peptide of 22 amino acids corresponding to the VH complementarity‐determining region (CDR)‐3 of the idiotypic protein to investigate whether autologous T cells could recognize this unique peptide. We demonstrated that autologous T cells possessing both CD4+ and CD8+ phenotypes could be propagated. The T cells were able to proliferate, secrete cytokines, and lyse autologous cells presensitized with the specific peptide in a major histocompatibility complex‐dependent manner. Moreover, these CDR3 peptide‐primed T cells were also able to kill autologous lymphoma cells. Our results therefore offer new perspectives for specific therapeutic vaccination for the treatment of B cell lymphoma.
Post‐translational up‐regulation of the cell surface‐associated α component of the human type I interferon receptor during differentiation of peripheral blood monocytes: role in the biological response to type I interferon
Authors: Fantuzzi Laura , Eid Pierre , Malorni Walter , Rainaldi Gabriella , Gauzzi Maria C. , Pellegrini Sandra , Belardelli Filippo , Gessani Sandra
Published: March 2007

Human peripheral blood monocytes cultured in vitro exhibit a greater sensitivity to the antiviral effect of type I interferon (IFN) compared to freshly isolated monocytes. We evaluated the effect of macrophage differentiation on the expression of type I IFN receptors (IFN‐R). Binding studies with iodinated IFN‐α2 and Scatchard plot analysis revealed that a single class of high‐affinity receptors was present in freshly isolated monocytes. Monocyte differentiation to macrophages resulted in a three‐ to fourfold increase in the number of cell surface receptors with no change in their affinity. Polymerase chain reaction analysis of RNA revealed that comparable levels of mRNA for the IFN‐R α (IFNAR1) and IFNAR2 components were expressed in freshly isolated monocytes and 7‐day cultured macrophages. Likewise, the levels of IFNAR1 protein remained constant over time in culture. Immunofluorescence studies revealed that IFNAR1 was localized in intracellular compartments of freshly isolated monocytes, whereas it was predominantly detected on the cell surface in 7‐day cultured macrophages. The increased expression of IFN‐R on the plasma membrane of cultured macrophages may, at least in part, account for the increased antiviral efect of type I IFN in these cells. These modifications represent one of the events occurring during monocyte differentiation that may play a role in the regulation of macrophage functions.
Constitutive and inducible protein/DNA interactions of the interferon‐γ promoter in in vivo CD45RA and CD45RO T helper subsets
Authors: Barbulescu Karina , Zum Büschenfelde Karl‐Hermann Meyer , Neurath Markus F.
Published: March 2007

Interferon‐γ (IFN‐γ) is a key cytokine of T lymphocytes with major regulatory functions in the immune system. To determine and compare protein/DNA interactions at the native IFN‐γ locus in T cells, we analyzed the human IFN‐γ promoter by ligation‐mediated polymerase chain reaction (LM‐PCR) techniques. Accordingly, Jurkat T cells and primary CD45RA and CD45R0 CD4+ T cell subsets isolated from peripheral blood using immunomagnetic beads were cultured and analyzed by LM‐PCR. Constitutive and inducible protein/DNA interactions of the IFN‐γ promoter in vivo were detected in all T cells tested. Interestingly, an inducible footprint between − 183 and − 196 was consistently observed in Jurkat T cells and CD45RA and CD45R0 T helper subsets upon stimulation with phorbol 12‐myristate 13‐acetate + phytohemagglutinin (PMA + PHA) that was highly sensitive to treatment with corticosteroids. This novel target site, denoted the C‐site, was shown by several criteria, including cell distribution studies, stimulation experiments, supershift assays, and cross‐competition electrophoretic mobility shift assays to bind the transcription factor AP‐1. Mutation of the C‐site that prevented AP‐1 binding to this site was sufficient strikingly to reduce inducible promoter activity in primary CD45R0 T cells. In summary, the data demonstrate that IFN‐γ gene transcription in primary T cells is regulated in vivo at the level of constitutive and inducible protein/DNA interactions. We propose a model where basal transcription is maintained by binding of various transcription factors to the IFN‐γ promoter, whereas PMA + PHA‐inducible IFN‐γ transcription in CD45R0 T cells is associated with binding of AP‐1 to the C‐site.
Influences on the lifespan of B cell subpopulations defined by different phenotypes
Authors: Fulcher David A. , Basten Antony
Published: March 2007

The turnover of mature and immature B cells defined by a range of cell surface markers was investigated by feeding normal or bcl ‐2‐transgenic (bcl‐2‐Tg) mice 5'‐bromo‐2‐deoxyuridine (BrdUrd) for up to 6 weeks. In peripheral lymphoid tissue, B cells accumulated BrdUrd with a 50% labeling time of 4.3 weeks and a pattern of uptake indicative of the presence of both long‐lived and short‐lived cells. These two kinetic populations could be resolved into immature B220lo/heat‐stable antigen (HSA)hi cells which labeled rapidly, and B220hiHSAlo cells which were uniformly long‐lived with a half‐life of about 6 weeks. During loading and pulse‐chase experiments, BrdUrd uptake by cells within the mature B220hiHSAlo population clearly followed an exponential kinetic pattern, suggesting that their loss was governed by stochastic processes. Using other surface markers, the long‐lived population could also be defined by high expression of IgD, representing cells in the follicular mantle zone of the spleen, and by the phenotype IgMhiIgDloHSAlo which most likely represented marginal zone memory B cells. CD23 expression on B cells did not differentiate well between long and short‐lived cells. Only about half of newly labeled B cells appearing in the spleen progressed to the long‐lived compartment, a proportion which was not altered significantly in bcl ‐2‐Tg mice. The most likely explanation was that a combination of both positive and negative selection was operating at this site which was mediated by pathways not regulated by bcl‐2. On the other hand, overexpression of bcl‐2 did result in a two‐to threefold increase in the rate of appearance of newly labeled B cells in the spleen, consistent with a possible role for this protein during early selection events within the bone marrow. Selection processes appeared to be very active in young mice during the shaping of the B cell repertoire, since B cells from 6‐week‐old non‐Ig mice displayed a rapid rate of turnover irrespective of their surface phenotype, and a significant population of long‐lived cells did not become evident until the mice had reached about 12 weeks of age.
Regulation of transforming growth factor‐β production by interleukin‐12
Authors: Marth Thomas , Strober Warren , Seder Robert A. , Kelsall Brian L.
Published: March 2007

The induction of peripheral tolerance following oral antigen administration in several autoimmune disease and conventional animal models correlates with the production of transforming growth factor‐β (TGF‐β) and T helper type 2 (Th2) cytokines. The factors regulating TGF‐β production and its relation to the Th2 response, however, have not been defined. We demonstrate that the systemic administration of antibodies to interleukin (IL)‐12 to ovalbumin (OVA)‐T cell receptor (TCR) transgenic mice fed high doses of OVA, followed by systemic OVA challenge, substantially enhances TGF‐β, but not IL‐4 production by peripheral T cells. Furthermore, we demonstrate in an in vitro T cell differentiation model that naive (CD4+/Mel‐14hi) OVA‐TCR‐T cells stimulated with OVA‐pulsed dendritic cells (DC) produce four‐to fivefold higher amounts of TGF‐β when cultured with anti‐IL‐12 or anti‐interferon‐γ (IFN‐γ). In this in vitro system, IL‐4 was not required for TGF‐β production by T cells; however, it appeared to enhance levels of TGF‐β by promoting the growth of TGF‐β‐producing cells. Our findings demonstrate that IL‐12 and IFN‐γ are important negative regulators of TGF‐β production both in vivo and in vitro, and that their modulation may be of benefit for the treatment of autoimmune disorders.
Sox‐4 facilitates thymocyte differentiation
Authors: Schilham Marco W. , Moerer Petra , Cumano Ana , Clevers Hans C.
Published: March 2007

1he mouse Sry‐like transcription factor Sox‐4 is expressed in thymus, bone marrow, and gonads of adult mice. Sox‐4‐deficient mice die at embryonic day E14 due to cardiac malformation. In transfer experiments to irradiated recipients, B cell development was shown to be severely impaired in Sox‐4‐deficient progenitor cells. However, no drastic effects on T lymphocyte development were noted, despite the high level expression of the Sox‐4 gene in the thymus of normal mice. Here, we report a detailed analysis of T cell development from Sox‐4‐deficient progenitors. Explanted fetal thymic organ cultures (FTOC) of Sox‐4‐deficient thymi yielded 10–50‐fold fewer CD4 CD8 double‐positive and single‐positive cells than FTOC of littermates. This effect was T cell‐autonomous, since similar observations were made when FTOC were performed by culturing of Sox‐4‐deficient progenitors in wild‐type thymus lobes. When Sox‐4‐deficient fetal liver cells were injected together with normal cells intrathymically, they did not compete efficiently for reconstitution. It is concluded that Sox‐4 facilitates thymocyte development.
Maturation of Peyer's patch dendritic cells in vitro upon stimulation via cytokines or CD40 triggering
Authors: Ruedl Christiane , Hubele Sabine
Published: March 2007

In mouse Peyer's patches (PP), dendritic cells (DC) are localized in T cell areas as NLDC145+ CD11c+ cells, and in the dome and corona region of the follicle as NLDC145− CD11c+ cells, respectively, suggesting the presence of two different DC populations with distinct roles in antigen uptake, processing, and presentation. However, it is not clear how this relates to DC maturation. In this report, we demonstrate that freshly‐isolated CD11c+ DC have the properties of immature DC since they endocytose soluble antigens, phagocytose particulate material such as latex beads, synthetize major histocompatibility complex (MHC) class II and invariant chain, but, at the same time, display low stimulatory activity for resting T cells, as shown in mixed‐lymphocyte reaction and oxidative mitogenesis assays. When cultured for 24 h in the presence of the cytokines granulocyte‐macrophage colony‐stimulating factor and tumor necrosis factor or anti‐CD40, the cells undergo dramatic phenotypic and functional changes characteristic of DC maturation. After 24 h stimulation in vitro, CD11c+ cells lose the ability to take up proteins such as ovalbumin, and in parallel with this decline, the biosynthesis of MHC class II and invariant chain is dramatically down‐regulated or eliminated. On the other hand cells treated in vitro exhibit on the cell surface higher levels of MHC class II, of co‐stimulatory molecules (CD80, CD86), of adhesion molecules (CD44, intercellular adhesion molecule‐1), and acquire expression of the interdigitating DC surface marker NLDC145. Concomitantly, the ability to stimulate naive T cells drastically increased after in vitro treatment with both stimuli. Taken together, our results indicate that the majority of DC in the PP are immature in terms of their antigen‐uptake capacity. These sentinel antigen presenting cells are strategically positioned at the dome region of PP, where antigens are transcytosed via the M cells from the gut lumen. A second population of mature interdigitating NLDC145+ CD11c+ DC stimulates naive unprimed T cells in interfollicular areas by up‐regulation of surface ligands and accessory signals.
HLA‐B27 modulates intracellular survival of Salmonella enteritidis in human monocytic cells
Authors: Laitio PÄIvi , Virtala Mika , Salmi Marko , Pelliniemi Lauri J. , Yu David T. Y. , Granfors Kaisa
Published: March 2007

Human major histocompatibility complex class I allele HLA‐B27 is associated with a group of diseases called spondyloarthropathies. In reactive arthritis (ReA), the disease is triggered by certain infections, e.g. gastroenteritis caused by Salmonella. The host/microbe interaction is abnormal in susceptible individuals leading to inefficient elimination of arthritis‐triggering bacteria, fragments of them, or both, after the initial infection. Using transfected human monocytic U937 cell lines, we demonstrate that the expression of the HLA‐B27 antigen does not influence the uptake of S. enteritidis into U937 cells in vitro. Interestingly, HLA‐B27 remarkably impairs the elimination of S. enteritidis within the HLA‐B27 transfected U937 cells. The impaired elimination of ReA‐triggering microbes by HLA‐B27+ monocytes may offer an explanation for the persistence of ReA‐triggering microbes in susceptible HLA‐B27+ individuals. This modulation of the host/microbe interaction by HLA‐B27 may have an important role in the pathogenesis of ReA.
Direct cell/cell communication in the lymphoid germinal center: connexin43 gap junctions functionally couple follicular dendritic cells to each other and to B lymphocytes
Authors: Krenacs Tibor , van Dartel Maike , Lindhout Ernst , Rosendaal Martin
Published: March 2007

Direct cell/cell communication occurs through gap junctions (GJ). We mapped GJ expression in secondary lymphoid organs and found, for the first time, a high density of connexin43 (Cx43) GJ in follicular dendritic cells (FDC) in close association with lymphocytes (Krenacs T. and Rosendaal M., J. Histochem. Cytochem. 1995. 43: 1125–1137). In this work, we used a combination of ultrastructural, immunocytochemical, molecular methods, and functional dye transfer experiments to study which germinal center cells are involved in direct cell/cell communication and how GJ expression is regulated during antigen responses. One week after injecting the footpad of mice with 50 μg lysozyme, Cx43 GJ were detected on elongated cells in the paracortex of their popliteal lymph nodes. Repeated challenge led to the formation of secondary follicles with enlarged FDC meshwork full of Cx43 GJ. This positive correlation may reflect an importance for GJ in the pattern formation of FDC and lymphoid follicles. In human tonsil, the density of GJ and FDC was highest in the light zone of germinal centers where the fate of B cells is thought to be decided. Cx43 colocalized with CD21 and CD35 antigens in the vicinity of desmosomal junctions on FDC embracing lymphocytes. Freeze‐fracture hallmarks of GJ of 200–400 nm were also found on FDC in the vicinity of desmosomal plaques. Furthermore, Northern blot analysis showed the consistent presence of Cx43 mRNA in human tonsil and spleen. Most Cx43 message was localized in situ to cells with FDC morphology and some to a few germinal center lymphocytes. To investigate functional cell coupling, we set up FDC/B cell cultures from the low density cell fractions of human tonsils. Cx43 plaques associated with lymphocytes were detected both on elongated FDC processes in early cultures (up to 4 h) and in established FDC/B cell clusters (between 4 and 24 h). In early cultures, we injected FDC with Lucifer Yellow, a fluorescent dye which passes through GJ: the dye spread into adjacent FDC and occasionally from FDC into CD19+ B cells. Based on these results, we propose that direct cell/cell communication through Cx43 GJ is involved in FDC/FDC and in FDC/B cell interactions. The functionally coupled FDC meshwork may serve as a communication channel synchronizing germinal center events. FDC may also deliver crucial direct signals through GJ involved in the rescue of high‐affinity B cell clones from apoptotic cell death.
Excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class II molecules
Authors: Brazil Melanie I. , Weiß Siegfried , Stockinger Brigitta
Published: March 2007

The endogenous major histocompatibility complex (MHC) class II presentation pathway allows biosynthesized, intracellular antigens access for presentation to MHC class II‐restricted T cells. This pathway has been well documented in B cells and fibroblasts, but may not be universally available in all antigen‐presenting cell types. This study compares the ability of different antigen‐presenting cells, expressing endogenous C5 protein (fifth component of mouse complement) as a result of transfection, to present their biosynthesized C5 to MHC class II‐restricted T cells. B cells and fibroblasts expressing C5 were able to present several epitopes of this protein with MHC class II molecules, whereas macrophages were unable to do so, but readily presented C5 from an extracellular source. However, macrophage presentation of endogenous C5 could be achieved when they were treated with low doses of the lysosomotropic agent ammonium chloride. In the presence of an inhibitor of autophagy, presentation of endogenous C5 was abrogated, indicating that biosynthesized C5 is shuttled into lysosomal compartments for degradation before making contact with MHC class II molecules. Taken together, this suggests that proteolytic activity in lysosomes of macrophages may be excessive, compared with fibroblasts and B cells, and destroys epitopes of the C5 protein before they can gain access to MHC class II molecules. Thus, there are inherent differences in presentation pathways between antigen‐presenting cell types; this could reflect their specialized functions within the immune system with macrophages focussing preferentially on internalization, degradation, and presentation of extracellular material.
Differential expression of insulin‐dependent diabetes mellitus‐associated HLA‐DQA1 alleles in vivo
Authors: Maffei Antonella , Harris Paul E. , Reed Elaine F. , Pozzo Giovanna Del , Ciullo Marina , Suciu‐Foca Nicole , Guardiola John
Published: March 2007

The strong association of HLA‐DQ genes with insulin‐dependent diabetes mellitus (IDDM) susceptibility is persuasive evidence of their central role in the etiology of this autoimmune disease. Among other possibilities, it has been proposed that an unbalanced expression of IDDM‐associated DQA, and/or DQB alleles may lead to alterations in the composition of αβ heterodimers and preferential expression of a particular heterodimer on the antigen‐presenting cell surface, leading to self‐recognition. In this report, we demonstrate the differential expression of DQA1 alleles in vivo, in particular of the two diabetogenic alleles DQA1*0301 and DQA1*0501. Family studies suggest that unequal HLA‐DQA1 allele expression in heterozygous individuals is not associated in cis with the HLA‐DQA1 gene, but may be affected by trans‐acting determinant(s). We also discuss the segregation of this phenotype in IDDM‐affected members. Furthermore, we examined historical samples of PBL from an IDDM‐affected individual and an HLA‐identical unaffected sibling acting in a kidney transplant program as donor and recipient, respectively. This analysis allowed us to establish that unbalanced expression of DQA1*0301 and DQA1*0501 can be induced by microenvironmental conditions. Inducible differential expression of HLA‐DQA1 alleles may account for the discordance in the outcome of autoimmune disease in monozygotic twins and HLA‐identical siblings.
Antigen specificity of anti‐nuclear antibodies complexed to nucleosomes determines glomerular basement membrane binding in vivo
Authors: Van Bruggen Mieke C. J. , Walgreen Birgitte , Rijke Truus P. M. , Tamboer Wim , Kramers Kees , Smeenk Ruud J. T. , Monestier Marc , Fournie Gilbert J. , Berden Jo H. M.
Published: March 2007

Monoclonal anti‐nuclear antibodies which are complexed to nucleosomes are able to bind to the glomerular basement membrane (GBM) in vivo, whereas purified antibodies do not bind. The positively charged histone moieties in the nucleosome are responsible for the binding to anionic determinants in the GBM. We tested the hypothesis that the specificity of the autoantibodies complexed to the nucleosome influences the glomerular binding of the antibody‐nucleosome complex. We induced the formation of these immune complexes in vivo, by intraperitoneal inoculation of hybridomas producing monoclonal anti‐nuclear antibodies (four anti‐histone, three anti‐double stranded (ds)DNA and three anti‐nucleosome antibodies) into nude BALB/c mice. In ascites and plasma from the mice inoculated with these hybridomas, nucleosome/autoantibody complexes were detected in comparable amounts. Immunofluorescence of kidney sections revealed that about 60% of the mice inoculated with anti‐nucleosome or anti‐dsDNA hybridomas had immunoglobulin deposits in the GBM, whereas only 15% of the mice with anti‐histone hybridomas showed these deposits (p ≦ 0.04). In the Matrigel®‐ELISA (used as a GBM surrogate) ascites from anti‐nucleosome or anti‐DNA hybridomas displayed significantly higher titers (p ≦ 0.002) than ascites from anti‐histone hybridomas. In conclusion, nucleosome/immunoglobulin complexes comprising anti‐nucleosome or anti‐dsDNA auto‐antibodies do bind more frequently to the GBM in vivo than nucleosome/immunoglobulin complexes containing anti‐histone antibodies. It therefore appears that the specificity of the antibody bound to the nucleosome is a critical determinant for the nephritogenic potential of the nucleosome‐autoantibody complex.
Major histocompatibility complex‐controlled protective influences on experimental autoimmune encephalomyelitis are peptide specific
Authors: Issazadeh Shohreh , Kjellén Peter , Olsson Tomas , Mustafa Maha , Holmdahl Rikard
Published: March 2007

The myelin basic protein (MBP) peptide 63–88‐induced experimental autoimmune encephalomyelitis (EAE) and its associated T cell cytokine profile are influenced by the rat major histocompatibility complex (MHC). There is an allele‐specific protective influence of the MHC class I region, whereas the MHC class II region display either disease‐protective or ‐promoting effects. To investigate if the MHC‐associated protection is dependent on certain combinations of MBP peptide and MHC molecules, we have now used another peptide (MBP 89–101). A broader and different set of rat MHC alleles were associated with EAE induced with MBP 89–101 as compared to MBP 63–88. All EAE‐susceptible strains mounted peptide‐specific strong T helper (Th) 1‐like immune responses in vitro. Immunization of rats with an extended peptide (MBP 87–110) induced EAE associated with the same MHC haplotypes as the 89–101 peptide, except in LEW.1N (RT1n) rats which were relatively resistant. Only this strain responded with additional Th2‐like and transforming growth factor‐β responses to the peptide in vitro. In vivo depletion of CD8+ cells aggravated the disease in this strain. We conclude that both MHC‐controlled promoting and protective influences on EAE are dependent on certain MHC/MBP peptide combinations, and that the 87–110 region of MBP contains a major MHC‐associated encephalitogenic epitope in the rat.
Deletion of alloantigen‐reactive thymocytes as a mechanism of adult tolerance induction following intrathymic antigen administration
Authors: Jones Nick D. , Fluck Nick C. , Roelen Dave L. , Mellor Andrew L. , Morris Peter J. , Wood Kathryn J.
Published: March 2007

Direct injection of foreign antigen into the adult thymus is a potent route of antigen delivery for the induction of tolerance in vivo. In this report, we demonstrate that tolerance to C57BL/10 (H2b/BL10) alloantigens can be induced in CBA/Ca (H2k/CBA) mice by intrathymic (IT) administration of BL10 spleen leukocytes coincident with transient peripheral immunomodulation of CD4+ T cells using a depleting anti‐CD4 monoclonal antibody. T cell receptor (TCR) transgenic mice (BM3.6; H2k) expressing a CD8‐independent TCR specific for H2Kb were used as recipients to facilitate investigation of the mechanisms responsible for tolerance induction by allowing visualization of events in the thymus following IT injection. IT administration of 5 × 107 BL10 spleen leukocytes and concomitant transient peripheral T cell depletion in BM3.6 mice resulted in a substantial H2Kb‐specific deletion of transgenic‐TCR+ (tg‐TCR) thymocytes which was dependent on the level of tg‐TCR expression. IT deletion and the failure to export CD8+ T cells to the peripheral lymphoid organs correlated with the induction of tolerance to H2Kb; TCR transgenic mice that had received IT injection of BL10 splenocytes and peripheral T cell depletion accepted a H2Kb+ cardiac allograft indefinitely. Analysis of tolerant BM3.6 mice revealed that there were low numbers of CD8+ T cells in the periphery giving rise to a substantially reduced reactivity in vitro despite the fact that no donor cells or IT deletion were observed in the thymi of the majority of tolerant mice. These results demonstrate for the first time that IT injection of foreign alloantigen into an adult thymus results in the deletion of thymocytes expressing a TCR specific for the injected alloantigen and suggest that this is an important mechanism of tolerance induction following IT injection of alloantigen in vivo. Furthermore, analysis of tolerant TCR‐transgenic mice suggests that IT deletion is not required for the maintenance of tolerance, and that peripheral mechanisms enforce continued hyporesponsiveness to H2Kb following transplantation.
Ras activation leads to cell proliferation or apoptotic cell death upon interleukin‐2 stimulation or lymphokine deprivation, respectively
Authors: Gómez Javier , Martínez‐A. Carlos , Fernández Belén , García Alphonse , Rebollo Angelita
Published: March 2007

Lymphokine‐dependent cells undergo apoptosis upon lymphokine withdrawal. We describe that lymphokine deprivation of the interleukin (IL)‐2‐ or IL‐4‐dependent mouse T cell line TS1αβ induces Ras activation which plays a role in programed cell death, since blocking Ras activity reduces the induction of apoptosis. Induction of apoptosis by lymphokine deprivation can be prevented by expression of the Bcl‐2 protein. Rescue from cell death by IL‐2 also promotes Ras activation, but, in contrast to lymphokine withdrawal, stimulates Bcl‐2 expression. IL‐4‐induced cell survival is Ras‐ and Bcl‐2 independent. These results are compatible with a model in which cell proliferation requires the simultaneous induction of at least two pathways which act in combination to prevent cell death.
Normal B cells fail to secrete interleukin‐12
Authors: Guéry Jean‐Charles , Ria Francesco , Galbiati Francesca , Adorini Luciano
Published: March 2007

Interleukin‐12 is a key regulatory cytokine produced by antigen‐presenting cells (APC) which drives the development of interferon‐γ (IFN‐γ)‐producing cells and promotes cell‐mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL‐12 p75 upon antigen‐specific interaction with T cells. We have analyzed secretion of IL‐12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen‐specific CD4+ cells. These APC populations do not produce IL‐12 constitutively but, upon stimulation with heat‐fixed Staphylococcus aureus and IFN‐γ, IL‐12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL‐12. B cells also fail to secrete IL‐12 in response to stimulation with LPS and IFN‐γ. Co‐culture with CD4+ T hybridoma cells and antigen induces IL‐12 secretion by DC. Up‐regulation of IL‐12 secretion by interaction with antigen‐specific CD4+ T cells is abrogated by anti‐class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti‐CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR‐peptide/class II and by CD40‐CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4+ T cells. Yet, even upon interaction with antigen‐specific T cells, B cells fail to secrete IL‐12. The capacity of B cells to present antigen but not to secrete IL‐12 may explain their propensity to selectively drive T helper type 2 cell development.
Lack of correlation between rejection of tumor cells co‐expressing interleukin‐2 and B7.1 and vaccine efficiency
Authors: Cayeux Sophie , Richter Günther , Becker Christian , Beck Christian , Aicher Alexandra , Pezzutto Antonio , Dörken Bernd , Blankenstein Thomas
Published: March 2007

Genetically modifying tumor cells to express a variety of cytokines such as interleukin‐2 (IL‐2) or the co‐stimulatory molecule B7.1 leads to increased immunogenicity and reduced tumorigenicity of tumors in several models with T cells involved in the process. We have previously reported decreased tumorigenicity of the murine plasmacytoma J558L [major histocompatibility complex (MHC) class I+ and class II−] expressing IL‐2 or B7.1. When systemic immunity was analyzed, immunization with either J558‐IL2 or J558‐B7.1 cells generated moderate protection against unmodified J558L tumor cells, comparable to immunization with a tumor cells/adjuvant Corynebacterium parvum mixture. In this study, we asked whether the co‐expression of IL‐2 and B7.1 in tumor cells would augment vaccine potency, cytotoxic T lymphocyte (CTL) activity and protective immunity. Rejection of single IL‐2 or B7.1 or co‐transfected IL‐2/B7.1 cells occurred in most syngeneic animals but not in T cell‐deficient nude mice, thus confirming that T cells were required for tumor rejection. We knew from previous experiments that CD8+ T cells were responsible for rejection. Surprisingly, immunization with J558‐IL2/B7.1 cells followed by challenge with parental J558L caused a reduction in systemic protection as compared to J558‐B7.1 or J558‐IL2 alone. We examined the mechanism underlying this unexpected result: 6 days after injection of J558‐IL2/B7.1 cells, tumor were nearly completely destroyed and were almost devoid of CD8+ cells, while CD8+ cells were increased in both IL‐2‐ and B7.1‐transfected tumors. In addition, immunization with J558‐IL2/B7.1 tumors had an adverse effect on the generation of CTL. Mice immunized with J558‐B7.1 and to a lesser extent J558‐IL2 cells mounted a CTL response against J558L cells while, in contrast, no CTL activity could be detected in mice immunized with J558‐IL2/B7.1, thus showing a correlation between the absence of CTL activity and the lack of in vivo protection. We demonstrate that “hyperstimulation” of the immune response by genetically modified cancer vaccines can have adverse effects on tumor immunity, even though the mechanism is not yet completely understood.
A role for Th2 cytokines in the suppression of CD8+ T cell‐mediated graft rejection
Authors: Scully Ralph , Cobbold Stephen P. , Mellor Andrew L. , Wissing Martin , Arnold Bernd , Waldmann Herman
Published: March 2007

A major histocompatibility complex (MHC) class I‐specific T cell receptor (TCR)‐transgenic mouse was used to study classical‐type transplantation tolerance in the adult. Engraftment of MHC class I‐incompatible bone marrow and tolerance to donor‐type skin grafts were obtained using dimethylmyeleran (DMM) as a myeloablative agent and a non‐depleting anti‐CD8 monoclonal antibody (mAb) as the sole immunosuppressant. Surprisingly, bone marrow engraftment was facilitated by host CD4+ T cells, a subset normally considered unable to reject class I MHC‐incompatible grafts. A combination of mAb to interleukins (IL)−4 and −10 antagonized the “permissive” effects of host CD4+ T cells, indicating a possible role for Th2‐type immunoregulation that can act on CD8+ T cells in this form of transplantation tolerance. The fate of graft‐reactive T cells was monitored using anti‐clonotypic antibodies. It was observed that bone marrow engraftment then led to peripheral deletion of mAb‐blockaded, clonotype+ CD8+ T cells.
Complex CD44 splicing combinations in synovial fibroblasts from arthritic joints
Authors: Croft Daniel R. , Dull Peter , Davies Derek , Jackson David G. , McIntyre Peter , Kramer Ijsbrand M.
Published: March 2007

CD44 is a broadly expressed cell surface glycoprotein which is the major cell surface receptor for the glycosaminoglycan, hyaluronan. In humans, alternative splicing of up to 9 variant exons (v2–v10) into CD44 mRNA, together with post‐translational modification via glycosylation and chondroitin sulfate attachment has the potential of generating a large number of CD44 isoforms. Insertion of these various exons has the potential to change the functional capacities of the molecule and has implications in disease. We have analyzed CD44 splice variant expression in cultured VCAM‐1‐positive synovial fibroblasts isolated from patients with osteo‐ or rheumatoid arthritis and from normal synovium. Rheumatoid and osteoarthritic tissue express CD44 splice variants at the cell surface level. At the mRNA level exons v3, v6, v7, v8, v9 and v10 were detected in different splicing combinations. Rheumatoid tissue showed high expression, osteoarthritic tissues showed great variation. In contrast, non‐inflamed tissue showed no splicing events. Our results indicate that the nature of CD44 splice variant expression may be linked to the inflammatory state of the synovial joint.
Mouse macrophage development in the absence of the common γ chain: defining receptor complexes responsible for IL‐4 and IL‐13 signaling
Authors: Andersson Åsa , Grunewald Susanne M. , Duschl Albert , Fischer Alain , Disanto James P.
Published: March 2007

The common γ chain (γc) forms a critical component of the receptors for interleukins (IL)‐2, IL‐4, IL‐7, IL‐9, and IL‐15. We analyzed γc‐deficient mice to define a role for γc signaling in the development and function of the macrophage lineage. No major differences in absolute cell numbers, cell surface phenotype, or in vitro function of γ−c compared to γ+c macrophages were observed. We therefore conclude that signaling through the γc chain is not essential for the differentiation of mouse macrophages. Although B and T cells require γc for IL‐4 responses, IL‐4 up‐regulated major histocompatibility class II molecules and inhibited nitric oxide production from γ−c macrophages following stimulation with lipopolysaccharide and interferon‐γ. γ−c macrophages could also respond to IL‐13, consistent with the model of a type II IL‐4 receptor α/IL‐13R which can function in the absence of γc. Both IL‐4 and IL‐13 responses could be completely inhibited with the mouse IL‐4 antagonist QY, suggesting that all of the observed IL‐13 responses pass through the type II receptor, making it the primary signaling receptor complex for IL‐13 in mouse macrophages.
Th1 and Th2 subsets equally undergo Fas‐dependent and ‐independent activation‐induced cell death
Authors: Watanabe Norihiko , Arase Hisashi , Kurasawa Kazuhiro , Iwamoto Itsuo , Kayagaki Nobuhiko , Yagita Hideo , Okumura Ko , Miyatake Shoichiro , Saito Takashi
Published: March 2007

Stimulation of previously activated T cells results in apoptosis, termed activation‐induced cell death (AICD). Recent analysis revealed that the Fas/Fas ligand (FasL) interaction is predominantly involved in AICD of T cells. Furthermore, based on the analysis of various T cell clones and lines, it has been reported that FasL is expressed mainly in Th1 but not in Th2 cells. However, the exact expression pattern of FasL and its function in normal activated T cells has not been determined. In the present study, by utilizing completely differentiated Th1 and Th2 cell populations obtained from ovalbumin‐specific T cell receptor (TCR)‐transgenic mice, the FasL expression on Th1 and Th2 was determined. Furthermore, involvement of Fas‐FasL interaction in AICD of Th1 and Th2 cells was analyzed by two approaches: one was the inhibition of AICD by anti‐FasL monoclonal antibodies, and the other AICD of Th1/Th2 subsets from TCR‐transgenic mice backcrossed to lpr mice. We demonstrated that Th2 cells express FasL on the cell surface at a level similar to that expressed by Th1 cells, and that both subsets were equally susceptible to the Fas‐mediated AICD. These observations suggest not only that the expression of FasL is not always correlated with Th subsets as defined by the cytokine‐producing profile, but also that the responses of both Th1 and Th2 subsets are regulated by Fas‐mediated AICD. Finally, analysis of the kinetics of AICD revealed a novel Fas/FasL‐independent pathway in its initial stage. These findings revealed the precise function of Fas/FasL‐mediated as well as Fas/FasL‐independent AICD in the regulation of helper T cell responses.