bluuebunny/crossref_metadata_2025 huggingface dataset:
Interference of natural mouse hepatitis virus infection with cytokine production and susceptibility to Trypanosoma cruzi
Authors: TORRECILHAS  , FAQUIM‐MAURO  , DA SILVA  , ABRAHAMSOHN
Published: March 2003

Mouse hepatitis virus (MHV) infection can have a pronounced impact on several investigation areas. Reports on natural MHV outbreaks are rare and most studies have been conducted by deliberately infecting mice with MHV laboratory strains that cause moderate to severe disturbances to the immune system. We have investigated the effects of a natural acute outbreak of MHV in our otherwise specific‐pathogen‐free (SPF) inbred mouse colonies, and of enzootic chronic MHV infection on cytokine production and resistance to the intracellular pathogen Trypanosoma cruzi. We found that BALB/c and/or C57BL/6 SPF mice that had been injected with T. cruzi blood trypomastigotes from recently MHV‐contaminated (MHV+) mice developed significantly higher parasite blood counts, accelerated death, and showed higher IL‐10 production by spleen cells than their counterparts whose T. cruzi inoculum was derived from MHV‐negative (MHV−) donors. Interferon‐γ (IFN‐γ) production by MHV+ and MHV− mice was not significantly different. In contrast, T. cruzi infection of chronically MHV‐infected mice did not result in major changes in the course of infection when compared with that observed in mice from MHV− colonies, although a trend to higher parasitaemia levels was observed in BALB/c MHV+ mice. Nevertheless, both BALB/c and C57BL/6 T. cruzi‐infected MHV+ mice had diminished IFN‐γ production to parasite‐antigen stimulation in comparison with similarly infected MHV− mice. Interleukin‐10 (IL‐10) production levels by spleen cells did not differ between chronic MHV+ and MHV− mice, but IFN‐γ neutralization by monoclonal antibody treatment of anti‐CD3‐stimulated spleen cell cultures showed higher levels of IL‐10 synthesis in MHV+ BALB/c mice.
A role for CD45RBlow CD38+ T cells and costimulatory pathways of T‐cell activation in protection of non‐obese diabetic (NOD) mice from diabetes
Authors: MARTINS  , ÁGUAS
Published: March 2003

Non‐obese diabetic (NOD) mice spontaneously develop autoimmune insulin‐dependent diabetes mellitus (IDDM). Infection of the animals with mycobacteria, or immunization with mycobacteria‐containing adjuvant, results in permanent protection of NOD mice from diabetes and we have recently reported that the phenomenon is associated with increased numbers of interferon‐γ‐producing T cells, possessing increased cytotoxic activity, and also with augmented numbers of activated immunoglobulin M‐positive (IgM+) B cells. Here, we have investigated whether protection of NOD mice from IDDM was associated with changes on costimulatory pathways of T and B cells, namely CD28/CTLA‐4–B7 and CD40–CD40 ligand (CD40L) and we also further characterized protective T helper (Th) cells with regards to the expression of the differentiation markers CD45RB and CD38. We report that Th cells involved in diabetes vaccination of NOD mice by mycobacterial infection seem to belong to CD45RBlo CD38+ phenotype. The protective effect of Mycobacterium avium infection is also associated with increased CD40L and CTLA‐4‐ expressing Th cells and with the generation of a CD40− IgG+ B cells. Our data are consistent with induction by mycobacterial infection of regulatory CD45RBlo CD38+ Th cells with the ability to trigger deletion or anergy of peripheral self‐reactive lymphocytes, with shutting down of IgG+ B‐cell response. They also implicate a role for IgG+ B cells in the autoimmune aggression of the endocrine pancreas of NOD mice.
Ganglioside GT1b suppresses immunoglobulin production by human peripheral blood mononuclear cells
Authors: KANDA  , TAMAKI
Published: March 2003

Gangliosides are sialic acid‐containing glycolipids and have various immunomodulatory effects. We previously reported that various gangliosides in vitro either inhibited or enhanced spontaneous immunoglobulin production by human peripheral blood mononuclear cells (PBMC). Among them, GT1b was the most inhibitory. In this study, we further examined the mechanism for the inhibitory effect of GT1b. The inhibitory effect of GT1b was apparent at 0·1 μm, increased dose dependently, and was maximal at 10 μm. In the presence of 10 μm GT1b, spontaneous production of immunoglobulin (Ig)G, IgM and IgA in human PBMC was reduced by 60%, 59·5% and 58%, respectively, compared with controls. GT1b did not affect the proliferation and viability of PBMC, and did not enhance their apoptosis. GT1b did not alter immunoglobulin production of B cells alone. Interleukin (IL)‐6 and IL‐10 each partially reversed the GT1b‐induced inhibition of immunoglobulin production by PBMC, and the presence of both cytokines completely reversed the inhibition. GT1b inhibited IL‐6 and IL‐10 production in monocytes, without affecting that in T or B cells. When monocytes were preincubated with GT1b, washed and then cultured with B and T cells, the immunoglobulin production was also suppressed. These results suggest that GT1b may indirectly suppress immunoglobulin production of B cells in whole PBMC via reducing the production of IL‐6 and IL‐10 in monocytes. It is thus indicated that GT1b may act as an important inhibitor for human humoral immune responses.
T‐cell anergy induced by clonotype‐specific antibodies: modulation of an autoreactive human T‐cell clone in vitro
Authors: STEENBAKKERS  , BOOTS  , RIJNDERS
Published: March 2003

Monoclonal antibodies (mAb) specific for the clonotype of an autoreactive T cell may be useful reagents in the modulation of autoimmune disease. We have previously reported the generation of a set of mAb specific for the clonotypic structure of a human T‐cell clone recognizing an epitope of human cartilage gp‐39. This glycoprotein was recently identified as a candidate autoantigen in rheumatoid arthritis. Here, we demonstrate for the first time that small amounts of immobilized anticlonotype mAb can induce anergy in the autoreactive clone. Following the anergic stimulus, T cells failed to proliferate upon restimulation as a result of a lack of interleukin‐2 (IL‐2) gene transcription. In addition, a diminished interferon‐γ (IFN‐γ) production was found. Our data indicate that anergy was not a result of T‐cell receptor (TCR) downmodulation or the absence of free TCR. The anergic state was induced independent of costimulation or the presence of IL‐2 and no protein synthesis was required for the induction of anergy. Anticlonotype mAb‐induced anergy was prevented by cyclosporin A, suggesting that active signalling via the calcium/calcineurin pathway was required for the induction of anergy. In coculture experiments, anergic T cells were found to suppress the response of reactive cells from the same clone. This bystander suppression led to 90% inhibition of peptide‐induced proliferation. Together, these findings suggest that mAb to the clonotypic structure of autoreactive T cells may be suitable reagents for the functional inactivation of these T cells in autoimmune diseases.
Induction of abortive and productive proliferation in resting human T lymphocytes via CD3 and CD28
Authors: Müller  , Wolf  , Wierenga  , Jung
Published: March 2003

How the T‐cell receptor (TCR)/CD3 complex mediates positive as well as negative signals for T‐cell regulation is not fully understood. We have previously described the induction of anergy in resting human T lymphocytes after mitogenic, high‐dose CD3 triggering with monoclonal antibodies. Here we report the concomitantly occurring cell death to be largely caused by apoptosis, which mainly affects the CD4+ T‐helper population. Because cell death becomes detectable after initial cell proliferation, it appears that a high‐dose CD3 stimulus constitutes a negative signal for resting T cells leading to an ‘abortive proliferation’ that is followed by anergy and/or apoptosis of the cells. In contrast, if initial proliferation is induced by a low dose of anti‐CD3 in the presence of an accessory signal via the CD28 receptor, anergy and cell death are markedly reduced and ‘productive proliferation’ may occur. Productive proliferation is characterized by an increased secretion of various cytokines measured (interleukin (IL)‐2, IL‐4, IL‐6, IL‐10, tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ)). A low‐dose submitogenic CD3 stimulus induced neither anergy nor cell death, supporting the view that negative CD3 signalling requires proliferation of resting cells.
Role of mitogen‐activated protein kinases in activation‐induced apoptosis of T cells
Authors: ZHU  , YU  , AKATSUKA  , COOPER  , ANASETTI
Published: March 2003

A member of the mitogen‐activated protein (MAP) kinase family, Jun N‐terminal kinase (JNK), has been implicated in regulating apoptosis in various cell types. We have investigated the requirement for another type of MAP kinase, extracellular signal‐regulated protein kinase (ERK) in activation‐induced cell death (AICD) of T cells. AICD is the process by which recently activated T cells undergo apoptosis when restimulated through the T‐cell antigen receptor. Here we show that both JNK and ERK are activated rapidly upon T‐cell receptor (TCR) ligation prior to the onset of AICD. A chemical inhibitor of ERK activation, PD 098059, inhibits ERK activation and apoptosis, while JNK activation is not inhibited. This suggests that JNK activation is not sufficient for apoptosis. TCR cross‐linking induces expression of the apoptosis‐inducing factor, Fas ligand (FasL), and its expression correlates with ERK activation. In addition, apoptosis induced by direct ligation of the Fas receptor by anti‐Fas antibody is not associated with ERK activation and is not inhibited by PD 098059. These data suggest that ERK activation is an early event during T‐cell apoptosis induced by antigen–receptor ligation, and is not involved in apoptosis per se but in the expression of FasL. MAP kinase family members may be similarly involved in inducing apoptosis signals in other cell types.
The incidence of Japanese cedar pollinosis and sensitization to the pollen allergens among Japanese monkeys in a troop
Authors: Sakaguchi  , Kobayashi  , Inouye  , Saito  , Hirahara  , Shiraishi  , Konaka  , Yamada  , Nigi
Published: March 2003

The natural occurrence of Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported in Japanese monkeys (Macaca fuscata), an appropriate animal model for developing antipollinosis therapies. However, there has been no study on the incidence of Japanese cedar pollinosis in monkeys. To evaluate the incidence of CJ pollinosis in Japanese monkeys, we investigated the presence of pollinosis symptoms among monkeys in a troop, and the response to CJ allergens in pollinosis monkeys. We examined the presence of pollinosis symptoms in 272 monkeys in a troop throughout the CJ pollination season (February to April). Of the 272 monkeys, 21 (7·7%) showed pollinosis symptoms during the CJ pollen season. Blood samples were taken from the 21 monkeys that showed pollinosis symptoms and were tested for the presence of immunoglobulin E (IgE) antibody for CJ allergens. All 21 monkeys with CJ pollinosis had anti‐CJ IgE. Of the 21 monkeys, peripheral blood mononuclear cells (PBMC) could be taken from 12, all of which showed CJ allergen‐specific PBMC proliferation. The incidence of CJ pollinosis in a troop was 7·7%. The monkeys with CJ pollinosis demonstrated specific IgE and PBMC proliferation for CJ allergens.
Distribution of membrane cofactor protein (MCP/CD46) on pig tissues. Relevance to xenotransplantation
Authors: Pérez De La Lastra  , Hanna  , Morgan
Published: March 2003

Membrane cofactor protein (MCP; CD46) is a 50–60 000 MW glycoprotein, expressed on a wide variety of cells and tissues in man, which plays an important role in regulating complement activation. Human MCP has also been shown to be the receptor for measles virus. We have recently identified the pig analogue of MCP and demonstrated that pig MCP has cofactor activity for factor I‐mediated cleavage of C3b when these components are derived either from pig or human. As a consequence, pig MCP is an efficient regulator of the classic and alternative pathways of human and pig complement. In order to define the potential importance of MCP in protecting against complement activation in the pig, we have conducted a comprehensive survey of its distribution in pig cells and organs. As in humans, MCP in the pig is broadly and abundantly distributed. Pig MCP is highly expressed on all circulating cells, including erythrocytes, in contrast to its absence on human erythrocytes. Multiple isoforms of MCP are found on cells and in tissues, probably representing products of alternative splicing analogous to those found in man. MCP is abundantly expressed throughout all tissues examined with particularly strong staining on the vascular endothelium. Connective tissue elements within liver and testis are also strongly stained by anti‐pig MCP antibodies. Pig MCP is expressed only weakly on skeletal muscle cells and expression is absent from smooth muscle cells in the lung and vessel walls, sites at which human MCP is expressed. Of particular note, MCP is not expressed in B‐cell areas of the germinal centres of lymph nodes.
Exogenous cytokine modulation or neutralization of interleukin‐10 enhance survival in lipopolysaccharide‐hyporesponsive C3H/HeJ mice with Klebsiella infection
Authors: Wang  , Jeng  , Ping
Published: March 2003

Klebsiella pneumoniae has been isolated from liver abscesses in patients with leukaemia or diabetes. The resistance of Klebsiella infection in lipopolysaccharide (LPS)‐hyporesponsive mice is unclear. Female C3H/HeJ and C3H/HeN mice, 6–8 weeks old, were intraperitoneally (i.p.) injected with K. pneumoniae. The results showed that C3H/HeJ mice were 24 times more susceptible [lethal dose 50% (LD50) 250 colony‐forming units] than C3H/HeN mice to K. pneumoniae infection. C3H/HeJ mice, uninfected or infected with K. pneumoniae, had higher liver interleukin (IL)‐10 levels and IL‐10 mRNA levels than C3H/HeN mice. Previously, pretreatment with IL‐1β and tumour necrosis factor‐α (TNF‐α) protected C3H/HeJ mice from lethal bacterial infection. Therefore the effects of pretreatment with IL‐1β and TNF‐α or antimurine IL‐10 antibody i.p. 1 hr before this infection in both strains of C3H mice were examined. Pretreatment with TNF‐α or anti‐IL‐10 antibody enhanced the survival of both strains of mice. TNF‐α, in combination with IL‐1β, enhanced the survival and bacterial clearance better than single pretreatment in C3H/HeJ mice. Anti‐IL‐10 antibody increased bacterial clearance and significantly reduced liver cytokine mRNA levels in C3H/HeJ mice more than it did in the controls during infection. These results indicate that exogenous cytokine modulation or neutralization of IL‐10 enhance the resistance of LD50 infection in C3H/HeJ mice.
Structural characterization of mouse CD97 and study of its specific interaction with the murine decay‐accelerating factor (DAF, CD55)
Authors: Qian  , Haino  , Kelly  , Song
Published: March 2003

CD97 is a newly identified, activation‐associated human leucocyte antigen with seven putative transmembrane domains. It has an extended extracellular segment containing several adhesion molecule structure motifs, and has been shown to interact with the human complement regulator, decay‐accelerating factor (DAF, CD55). To understand further the interaction between CD97 and DAF, as well as the structure and function of CD97 in general, we have cloned the mouse CD97 cDNA and studied the encoded protein for its membrane association property and ability to interact specifically with the murine decay‐accelerating factor. The full‐length mouse CD97 cDNA that we have cloned and characterized encodes a protein that is 60% identical to the three epidermal growth factor (EGF) domain‐containing form of human CD97 but does not contain the Arg‐Gly‐Asp (RGD) motif which is present in human CD97. Two other alternatively spliced forms of mouse CD97 were also identified. These forms differ by the number of EGF‐like sequence repeats present in the N‐terminal region. Northern blot analysis revealed that CD97 is expressed widely in mouse tissues and in resting as well as activated cultured mouse splenocytes. Transient transfection of human embryonic kidney (HEK) 293 cells with the mouse CD97 cDNA in a green‐fluorescence protein vector (pEGFP‐N1) showed plasma membrane targeting of the expressed protein. Western blot analysis confirmed its membrane association and identified the existence of a processed C‐terminal fragment, supporting the notion that CD97 on the cell membrane is composed of post‐translationally generated subunits. Adhesion studies demonstrated that normal, but not DAF knockout mouse erythrocytes and splenocytes adhered to mouse CD97‐transfected HEK cells. The interaction of CD97 and DAF was found to be species‐restrictive in that human erythrocytes were unable to bind to mouse CD97‐transfected HEK cells. These results indicate that the general structure, membrane association property and DAF‐binding ability of CD97 are conserved and that the adhesive interaction between CD97 and DAF is independent of the RGD motif. The finding that CD97 is distributed widely among various mouse tissues suggests that CD97 may have other roles beyond lymphocyte activation.
Both adhesion to immobilized vitronectin and FcɛRI cross‐linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells
Authors: Bhattacharyya S. P. , Mekori Y. A. , Hoh D. , Paolini R. , Metcalfe D. D. , Bianchine P. J.
Published: March 2003

Murine mast cells adhere spontaneously to plate‐bound vitronectin (VNPB) via αv‐containing integrins, and this adhesive interaction results in an augmented interleukin‐3 (IL‐3)‐dependent mast‐cell proliferation. In this report we demonstrate that the activation of murine mast cells through αv‐integrin, as well as through the high affinity immunoglobulin E (IgE) receptor (FcɛRI), results in enhanced tyrosine phosphorylation of focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase involved in mitogenic and oncogenic signal transduction. While mast cell adhesion to VNPB resulted in enhanced FAK phosphorylation, treatment with soluble vitronectin (VNSOL) failed to do so. Spontaneous mast cell adhesion to entactin (EN) did not induce tyrosine phosphorylation of FAK, demonstrating that not all adhesive interactions lead to the same sequence of biochemical events. Because FAK has intrinsic tyrosine kinase activity, we examined whether activating mast cells via αv‐integrins, or via FcɛRI‐cross‐linking stimulated the in vitro kinase activity of FAK. Both pathways were found independently to activate FAK in mast cells and together appeared additive. Protein kinase C depletion in mast cells and calcium depletion in the medium caused decreased tyrosine phosphorylation of FAK, indicating that optimal tyrosine phosphorylation of FAK is regulated by both pathways. These data are consistent with the conclusion that the tyrosine phosphorylation of FAK represents at least one example of a point of convergence in the intracellular tyrosine phosphorylation cascades induced by αv integrin‐and FcɛRI‐mediated signal transduction pathways in mast cells.
Induction of interactions between CD44 and hyaluronic acid by a short exposure of human T cells to diverse pro‐inflammatory mediators
Authors: Ariel A. , Lider, O. , Brill A. , Cahalon L. , Savion N. , Varon D. , Hershkoviz R.
Published: March 2003

Migration of T cells into extravascular sites of inflammation is mediated by cell–cell and cell–matrix adhesion receptors, including the hyaluronan‐binding glycoprotein, CD44. The biochemical nature of CD44 variants and the ligand specificity, function and the regulation of activation of CD44 expressed on various cell types have been extensively studied. However, little is still known about the short‐term influence of cytokines and chemokines on the activation of CD44 on human T cells. Therefore, we studied the role of inflammatory mediators in regulating the adhesion of T cells from human peripheral blood to immobilized hyaluronan under static or shear stress conditions. We found that the CD44‐dependent adhesion, under static and shear stress (i.e. relative gradual resistance to flow of 150 and 1500 s−1) conditions, of T cells to hyaluronan requires a T‐cell activation of 2–3 hr and is regulated by the cross‐linking of CD3, cytokines (e.g. interleukin‐2 and tumour necrosis factor‐α), and chemokines (e.g. MIP‐1β, interleukin‐8, and RANTES). This T‐cell adhesion was manifested by polarization, spreading and co‐localization of cell surface CD44 with a rearranged actin cytoskeleton in hyaluronan‐bound T cells. Thus, cytokines and chemokines present in the vicinities of blood vessel walls or present intravascularly in tissues where immune reactions take place, can rapidly activate the CD44 molecules expressed on T cells.
Signals sustaining human immunoglobulin V gene hypermutation in isolated germinal centre B cells
Authors: Dahlenborg K. , Pound J. D. , Gordon J. , Borrebaeck C. A. K. , Carlsson R.
Published: March 2003

Affinity maturation of antibody responses depends on somatic hypermutation of the immunoglobulin V genes. Hypermutation is initiated specifically in proliferating B cells in lymphoid germinal centres but the signals driving this process remain unknown. This study identifies signals that promote V gene mutation in human germinal centre (GC) B cells in vitro. Single GC B cells were cultured by limiting dilution to allow detection of mutations arising during proliferation in vitro. Cells were first cultured in the presence of CD32L cell transfectants and CD40 antibody (the ‘CD40 system’) supplemented with combinations of cytokines capable of supporting similar levels of CD40‐dependent GC B‐cell growth [interleukin (IL)‐10 + IL‐1β + IL‐2 and IL‐10 + IL‐7 + IL‐4]. Components of the ‘EL4 system’ were then added to drive differentiation, providing sufficient immunoglobulin mRNA for analysis. Analysis of VH3 genes from cultured cells by reverse transcription–polymerase chain reaction (RT–PCR)‐based single‐strand conformation polymorphism indicated that the combination IL‐10 + IL‐1β + IL‐2 promoted active V gene mutation whereas IL‐10 + IL‐7 + IL‐4 was ineffective. This was confirmed by sequencing which also revealed that the de novo generated mutations were located in framework and complementarity‐determining regions and shared characteristics with those arising in vivo. Somatic mutation in the target GC B‐cell population may therefore be actively cytokine driven and not simply a consequence of continued proliferation. The experimental approach we describe should facilitate further studies of the mechanisms underlying V gene hypermutation.
CD5‐positive and CD5‐negative human B cells converge to an indistinguishable population on signalling through B‐cell receptors and CD40
Authors: Gagro A. , Mccloskey N. , Challa A. , Holder M. , Grafton G. , Pound J. D. , Gordon J.
Published: March 2003

Whether CD5 on B cells marks a subset functionally distinct from the conventional CD5 negative (CD5neg) adult population or is more an indicator of activation, remains contentious. Here we have investigated whether CD5 positive (CD5pos) and CD5neg B cells can be distinguished in terms of their response to surrogate signals aimed to model, in vitro, T‐cell dependent (TD) and T‐independent (TI) encounters with antigen in vivo: the predominantly CD5pos B‐cell population found in cord blood, CD5 B cells positively selected from tonsils and their CD5neg counterparts, were compared. Neonatal B cells displayed a near‐identical phenotype to that of adult CD5pos B cells, being characterized by uniform immunoglobulin M (IgM), immunoglobulin D (IgD), CD23 and CD44 coexpression. When cultured with anti‐IgM maintained at high density on CD32‐tranfected mouse L cells to model TI responses or on CD40 ligand (CD40L)‐bearing L cells (with or without captured anti‐IgM) to model TD encounters, DNA synthesis was stimulated to a similar extent in all three populations. Focusing on CD5 and CD23, we found that – although the signals delivered promoted distinct profiles of expression – under each condition of activation, the phenotypes that emerged for adult CD5pos and CD5neg B cells were remarkably similar. Neonatal B cells displayed a greater diminution in CD5 expression than adult CD5pos B cells following CD40 signals but otherwise the two populations again behaved similarly. The inclusion of interleukin‐4 (IL‐4) to cultures where cells were costimulated via surface (s)IgM and CD40 resulted in a complete loss of CD5 expression and a corresponding hyperexpression of CD23, irrespective of the population studied. The near‐identical response of CD5pos and CD5neg B cells to surrogate TD or TI signals in vitro and their convergence to indistinguishable phenotypes is wholly supportive of CD5 being a fluctuating marker of activation rather than it delineating functionally distinct subsets.
A novel subset of murine B cells that expresses unmasked forms of CD22 is enriched in the bone marrow: implications for B‐cell homing to the bone marrow
Authors: Floyd H. , Nitschke L. , Crocker P. R.
Published: March 2003

CD22 is a B‐cell‐restricted transmembrane protein, which acts as a negative regulator of B‐cell signalling. CD22 also has lectin‐like adhesive properties. When expressed on transfected fibroblasts, it is capable of mediating adhesion to other cells via recognition of cell‐surface glycoconjugates terminating in α2,6‐linked sialic acids. In previous studies in the mouse, CD22 was implicated as a bone marrow homing receptor for recirculating immunoglobulin D+ (IgD+) B cells through recognition of sialylated ligands on marrow sinusoidal endothelium. As the adhesive function of CD22 can be masked when α2,6‐linked sialic acids are co‐expressed at the cell surface, the aim of the present study was to investigate whether recirculating B cells have unmasked forms of CD22 that could be involved in bone marrow homing. Using α2,6‐sialyllactose coupled to biotinylated polyacrylamide as a probe for detection of unmasked CD22, we showed that ≈ 2–5% of IgD+ murine B cells in the spleen and mesenteric lymph nodes were able to bind this synthetic ligand. In the bone marrow, however, the fraction of IgD+ B cells with unmasked CD22 was increased by two‐ to fivefold. B cells from CD22‐deficient mice were not stained with the polyacrylamide probe, confirming that staining of B cells in wild‐type mice was caused by CD22 and not by other potential sialic acid‐binding lectins. In conclusion, we have identified a new subset of mature B cells in the mouse with unmasked CD22. This subset of recirculating B cells may bind to CD22 ligands on bone marrow sinusoidal endothelium, leading to their selective homing and subsequent enrichment in this tissue.
Graft‐versus‐host‐disease‐associated donor cell engraftment in an F1 hybrid model is dependent upon the Fas pathway
Authors: Iwasaki T. , Hamano T. , Saheki K. , Kuroiwa T. , Kataoka Y. , Takemoto Y. , Ogata A. , Fujimoto J. , Kakishita E.
Published: March 2003

The graft‐versus‐host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell‐mediated attack on host lymphohaematopoietic populations, resulting in the reconstitution of the host with donor cells. We examined Fas–Fas ligand (FasL) interactions in donor and host haematopoietic cells over a prolonged period of parental‐induced GVHD. Fas expression on bone marrow cells of both donor and host origin increased at 2 weeks. Host cell incubation with anti‐Fas antibody induced apoptosis, and the number of haematopoietic progenitor cells decreased. Fas‐induced apoptosis by the repopulating donor cells, however, did not increase until 12 weeks, when more than 90% of the cells were donor cells. The expression of various cytokines, such as interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α), and FasL gene expression in the bone marrow increased concomitantly. To examine directly whether FasL has a major role in the development of donor cell engraftment, FasL‐deficient (gld) mice were used as donors. Injection of B6/gld spleen cells induced significantly less host lymphohaematopoietic depletion, resulting in a failure of donor cell engraftment. Furthermore, injection of IFN‐γ gene knockout (gko) B6 spleen cells failed to augment Fas and FasL expression in recipient mice, resulting in a failure of donor cell engraftment. This suggests that the induction of apoptosis by Fas–FasL interactions in host cells may contribute to a reconstitution of the host with donor cells and that donor‐derived IFN‐γ plays a significant role for Fas–FasL interactions in host cells during parental‐induced GVHD.
Bovine tuberculosis: immune responses in the peripheral blood and at the site of active disease
Authors: Rhodes S. G. , Buddle B. M. , Hewinson R. G. , Vordermeier H. M.
Published: March 2003

This report describes a comparison of immune responses in the peripheral blood and at the site of active disease in cattle 20 weeks after experimental infection with Mycobacterium bovis. Lymphocyte proliferation, and the production of interferon‐γ (IFN‐γ) and interleukin (IL)‐2 were measured in response to tuberculin and a number of mycobacterial antigens, including ESAT‐6, MPB64, MPB70, MPB83, hsp 16.1, hsp 65, hsp 70 and the 38 000 MW lipoprotein antigen. The level of transforming growth factor‐β (TGF‐β) was measured following stimulation of cells with tuberculin. Our results suggest little difference in the responses of peripheral blood and lymph node cells to most of the antigens used. However, tuberculin purified protein derivative (PPD) and ESAT‐6 elicited stronger responses in the peripheral blood compared with lymph node cells. Investigation of the responding T‐cell subpopulations in the peripheral blood showed that both CD4+ and, to a lesser extent, γδ T‐cell receptor‐positive (TCR+) T cells contributed to these responses. This is the first report to compare peripheral and local immune responses in bovine tuberculosis. Unlike cases of human tuberculosis where immune activity at the site of disease and anergy in the peripheral blood have been reported, our results suggest that for bovine tuberculosis immune responses occurring in the peripheral blood reflect those at the site of disease.
Neutralizing antibodies to granulocyte–macrophage colony‐stimulating factor, interleukin‐1α and interferon‐α but not other cytokines in human immunoglobulin preparations
Authors: Wadhwa M. , Meager A. , Dilger P. , Bird C. , Dolman C. , Das R. G. , Thorpe R.
Published: March 2003

Human immunoglobulin preparations are used therapeutically for various disorders. Such therapy is generally safe but adverse effects occasionally occur in recipients. It has been suggested that antibodies to cytokines present in clinical immunoglobulin products may contribute to undesirable effects in recipients. Therefore, we investigated intravenous and intramuscular immunoglobulin products for the presence of cytokine‐specific neutralizing antibodies. Using validated bioassays, we detected neutralizing activity against human granulocyte–macrophage colony‐stimulating factor (GM‐CSF), interferon‐α2a (IFN‐α2a) and interleukin‐1α (IL‐1α) in immunoglobulin products. We found no neutralization of granulocyte colony‐stimulating factor, macrophage colony‐stimulating factor, stem cell factor, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐6, IL‐9, IL‐10, IL‐12, tumour necrosis factor‐α, oncostatin M (OSM) and IFN‐γ. Most batches which neutralized IFN‐α2a activity also neutralized other IFN‐α subtypes, IFN‐ω and IFN‐β. Most products (94%) neutralized the biological activity of GM‐CSF. No correlation between batches and their ability to neutralize bioactivities of GM‐CSF, IFN‐α2a and IL‐1α was found. This neutralizing activity could be traced to plasma pools used for manufacture of immunoglobulins. The neutralization was mediated by specific cytokine antibodies contained within immunoglobulin products as it was present in specific immunoglobulin G (IgG) fractions eluted from cytokine affinity chromatography columns. Specific binding of such IgG fractions to cytokines in immunoblots and in enzyme‐linked immunosorbent assays (ELISAs) was observed. This contrasts with the broad non‐specific recognition of cytokine proteins observed using unfractionated immunoglobulins in ELISAs. This is the first comprehensive study showing the presence of neutralizing antibodies against GM‐CSF, IL‐1α, or IFN‐α2a in immunoglobulin products.
Characterization and role in experimental systemic lupus erythematosus of T‐cell lines specific to peptides based on complementarity‐determining region‐1 and complementarity‐determining region‐3 of a pathogenic anti‐DNA monoclonal antibody
Authors: Brosh N. , Eilat E. , Zinger H. , Mozes E.
Published: March 2003

Peptides based on the complementarity‐determining region 1 (CDR1) and CDR3 of an anti‐DNA monoclonal antibody (mAb) carrying the 16/6 idiotype (Id) were shown to induce experimental systemic lupus erythematosus (SLE) in susceptible mouse strains. In the present study, T‐cell lines specific to the pCDR1 and pCDR3 peptides were established in BALB/c and in SJL mice, respectively. The T‐cell lines were characterized and analysed for their pathogenicity upon administration to syngeneic mouse strains. Both T‐cell lines expressed the αβ T‐cell receptor (TCR) and the CD4+ CD8– phenotype. Additionally, both cell lines secreted interleukin (IL)‐4 and IL‐10 upon stimulation with their specific peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3‐specific T‐cell line induced experimental SLE in SJL mice. The animals produced high levels of autoimmune anti‐DNA and antinuclear protein antibodies, as well as anti‐16/6 Id antibodies (Abs). Furthermore, the mice developed clinical manifestations, including leukopenia, proteinuria and accumulation of immune complex deposits in their kidneys. The pCDR1‐specific T‐cell line failed to induce SLE when injected into BALB/c mice. It is thus suggested that pCDR3 is an immunodominant epitope in experimental SLE and that pCDR3‐specific T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.
Effective formation of major histocompatibility complex class II–peptide complexes from endogenous antigen by thyroid epithelial cells
Authors: Maile R. , Elsegood K. A. , Harding T. C. , Uney J. B. , Stewart C. E. H. , Banting G. , Dayan C. M.
Published: March 2003

In autoimmune thyroid disease, thyroid epithelial cells (TEC) express major histocompatibility complex (MHC) class II molecules, potentially enabling them to present thyroid self‐antigens to CD4‐positive T cells. However, despite this, TEC may fail to present endogenous antigen as a result of limited processing or MHC class II loading capacity, or inadequate MHC class II levels. We addressed these issues using the cloned rat TEC line, Fischer rat thyroid cell line (FRTL5), which was transfected using an adenoviral expression vector that expressed ovalbumin (OVA) as an integral membrane protein. OVA‐expressing FRTL5 cells very efficiently activated a panel of OVA‐specific, class II‐restricted T‐cell hybridomas. This response was dependent on induction of MHC class II molecules by interferon‐γ (IFN‐γ) and was blocked by anti‐MHC class II antibodies. Poor responses were seen to exogenously added OVA or OVA peptides. These results provide the most direct evidence to date that TEC can form MHC class II–peptide complexes derived from self‐antigen in sufficient quantities to activate T cells.
Structural and functional analysis of the human CD45 gene (PTPRC) upstream region: evidence for a functional promoter within the first intron of the gene
Authors: Timón M. , Beverley P. C. L.
Published: March 2003

Expression of the leucocyte common antigen (CD45) in mammals is restricted to the nucleated lineages of haematopoietic cells. It appears in early progenitors in the bone marrow and is expressed at the surface of these cells throughout their differentiation. However, at least in T cells, the pattern of expression switches between different isoforms during the successive stages of differentiation in the thymus and after activation in the periphery. In order to understand the mechanisms controlling the transcription of the human CD45 gene, 2·7 kbp of the 5′‐flanking region were sequenced and analysed for their ability to direct expression of a reporter gene. The only region with promoter activity was localized within the first intron of the gene. This promoter shows no tissue specificity but could be enhanced by a heterologous enhancer. Mobility shift assays showed complex but specific protein binding. The sequence in this region lacks similarity with known promoters or initiators but is highly conserved in evolution. No transcription initiation could be detected within or downstream of this region, suggesting that this might be a new type of RNA polymerase II promoter able to drive transcription from an upstream sequence. An additional exon was also found upstream of exon 1. The two exons 1 (1a and 1b) are mutually exclusive and both are spliced to exon 2. This makes the structure of the 5′ region of the human CD45 gene identical to its mouse homologue.
Polymorphism of the human α1 immunoglobulin gene 3′ enhancer hs1,2 and its relation to gene expression
Authors: Denizot Yves , Pinaud Eric , Aupetit Corinne , Le Morvan Caroline , Magnoux Emmanuelle , Aldigier Jean Claude , Cogné Michel
Published: March 2003

We studied the hs1,2 transcriptional enhancer identified downstream of the human α1 gene of the immunoglobulin H (IgH) locus, for which two different allelic configurations (a and b) were previously reported by Southern blotting. By using a polymerase chain reaction (PCR) method we amplified minisatellites within the hs1,2 core enhancer, with variable numbers of tandem repeats (VNTR) defining three ‘PCR alleles’α1A, α1B and α1C (including one, two and three repeats, respectively). Five different α1 h1,2 genotypes were encountered in a population of 513 donors, representing 13·8, 34·5, 49·7, 1·3 and 0·6% for the AA, BB, AB, AC and BC genotypes, respectively. Luciferase assays showed that increasing the number of minisatellites increased the transcriptional strength of the α1 hs1,2 enhancer. Simultaneous determination of Southern blot alleles and VNTR alleles only showed a partial linkage between both types of polymorphism, altogether defining at least six different allelic forms of the 3′α1 region. In conclusion, the present study further demonstrates the genetic instability of the 3′α region, for which multiple alleles have been generated through inversions and internal deletions and/or duplications. This study also strengthens the hypothesis that the polymorphism at the IgH 3′ regulatory region of the α1 gene could play a role in the outcome of diseases involving immunoglobulin secretion.
Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B‐cell receptor stimulation
Authors: Schmalzigaug Robert , Ye Qunrui , Berchtold Martin W.
Published: March 2003

Calmodulin (CaM) is the main intracellular Ca2+ sensor protein responsible for mediating Ca2+ triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the CaMII gene causes the intracellular CaM level to decrease by 60%. CaMII−/− cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon stimulation of the B‐cell receptor (BCR), the resting Ca2+ levels remain elevated after the initial transient in CaMII−/− cells. Despite higher Ca2+ resting levels, the CaMII−/− cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes.
The regulation of FasL expression during activation‐induced cell death (AICD)
Authors: Nguyen Thang , Russell John
Published: March 2003

Activation‐induced cell death (AICD), a Fas ligand (FasL)‐dependent pathway, is important for maintaining T‐cell homeostasis. Interleukin‐2 (IL‐2), an enhancer of AICD, can also enhance FasL expression. However, we show that the level of FasL or FLIP protein did not correlate with the susceptibility to AICD. Some T cells expressed high levels of FasL yet failed to undergo AICD, while others expressed little FasL and were sensitive. AICD susceptibility did not correlate with the kinetics of FasL up‐regulation or down‐regulation. The down‐regulation of FasL can be mediated by a metalloprotease. However, we describe an alternative mechanism for the loss of FasL by endocytosis. Endocytosis inhibitors such as cytochalasins, sodium azide, deoxyglucose, or low temperatures prevented the loss of FasL. KB8301, a metalloprotease inhibitor had no effect on the loss of FasL or AICD in the T cells. Enhancing FasL expression was not crucial for AICD and the down‐regulation of FasL proceeded via endocytosis.
Enhanced maturation and functional capacity of monocyte‐derived immature dendritic cells by the synthetic immunomodulator Murabutide
Authors: Vidal Vincent , Dewulf Joelle , Bahr George M.
Published: March 2003

Murabutide is a safe synthetic immunomodulator derived from muramyl dipeptide, the smallest bioactive unit of bacterial peptidoglycan. Although it is well known that muramyl peptides modulate the functions of monocytes/macrophages, their activity on dendritic cells is poorly documented. We thus investigated the effects of Murabutide on immunophenotype, endocytosis, T‐cell stimulatory capacity, and cytokine secretion of human monocyte‐derived immature dendritic cells (iDCs). We found that Murabutide triggers immunophenotypic changes as upon treatment, iDCs up‐regulate the surface expression of the major histocompatibility complex type II molecule human leucocyte antigen‐DR, the co‐stimulatory molecules CD80, CD86 and CD40 and the differentiation marker CD83, and down‐regulate the expression of the mannose receptor. These phenotypic changes are also mirrored by changes in their biological activity. Subsequent to treatment with the synthetic immunomodulator, DC have a decreased endocytic capacity but exhibit enhanced stimulatory capacity for both allogeneic and autologous T cells. In addition, Murabutide‐stimulated iDCs have a greater cytostatic activity toward the tumour cell line THP‐1. Furthermore, in the presence of Murabutide, DCs transiently increased the release of macrophage inhibitory protein‐1β, tumour necrosis factor‐α and interleukin‐10, whereas the enhanced production of macrophage‐colony stimulating factor was sustained over the 3‐day period analysed. In addition, Murabutide triggers the phosphorylation of the three classes of mitogen‐activated protein kinases in iDCs. Altogether our results demonstrate that Murabutide triggers the maturation and activation of monocyte‐derived iDCs. As this immunomodulator is approved for administration in humans, it could be a useful adjunct to boost the efficacy of DC‐based vaccines designed against tumours or virus‐infected cells.
Characterization of glycosylphosphatidylinositol‐anchored decay accelerating factor (GPI‐DAF) and transmembrane DAF gene expression in wild‐type and GPI‐DAF gene knockout mice using polyclonal and monoclonal antibodies with dual or single specificity
Authors: Miwa Takashi , Sun Xiujun , Ohta Rieko , Okada Noriko , Harris Claire L. , Morgan B. Paul , Song Wen‐Chao
Published: March 2003

Decay‐accelerating factor (DAF, CD55) is a glycosylphosphatidylinositol (GPI)‐linked membrane inhibitor of complement activation. While human and other mammalian species contain only one DAF gene, two distinct DAF genes, referred to as GPI‐DAF and transmembrane (TM)‐DAF, respectively, have been identified in the mouse. Using several independently generated monoclonal and polyclonal antibodies, either with dual or single specificity for GPI‐DAF and TM‐DAF gene products, we have examined the expression of the two DAF genes in tissues of the wild‐type and a strain of knockout mouse whose GPI‐DAF gene has been inactivated. By fluorescence‐activated cell sorting (FACS) analysis, we found that DAF protein is present on the wild‐type mouse erythrocytes and lymphocytes but no signal was detectable on the same cells of GPI‐DAF gene knockout mice. Both T and B lymphocytes and splenic macrophages express the GPI‐DAF gene but the expression level is higher on B lymphocytes than on T lymphocytes. Within the T cell population, both CD4+ and CD8+ T cells are positive. DAF protein was detected by immunohistochemistry at high levels on wild‐type mouse spermatids and mature sperm. In contrast, only mature sperm stained positive in the GPI‐DAF gene knockout mouse testis, suggesting that GPI‐DAF but not the TM‐DAF gene is expressed on spermatids. Examination of the fetoplacental unit at the day 7·5 stage revealed that GPI‐DAF but not the TM‐DAF gene is expressed in the maternal decidua cells surrounding the trophoectoderm of the embryo. No DAF expression was detected on trophoblast or the embryo proper. These findings suggest that although the TM‐DAF gene is irrelevant on mouse blood cells, the two DAF genes may have different roles in germ cell development and/or mature sperm function. Because complement receptor 1‐related gene/protein y (Crry) has been shown to be expressed on early mouse embryos, the complete lack of GPI‐DAF and TM‐DAF gene expression in early mouse development may explain the observed sensitivity of Crry‐deficient embryos to maternal complement attack.
Enhancement of stromal cell‐derived factor‐1α‐induced chemotaxis for CD4/8 double‐positive thymocytes by fibronectin and laminin in mice
Authors: Yanagawa Yoshiki , Iwabuchi Kazuya , Onoé Kazunori
Published: March 2003

Stromal cell‐derived factor‐1α (SDF‐1α) is a chemokine abundantly expressed in the thymus. However, a potential role of SDF‐1α in the thymus has been under consideration, since no appreciable difference was detected in the migratory responsiveness to the SDF‐1α between cortical and medullary thymocytes. In the present study, we examined the effects of extracellular matrix (ECM) on the responsiveness of murine thymocytes to several chemokines including SDF‐1α. In the absence of ECM, chemotactic activity of SDF‐1α for cortical (CD4/8 double‐positive) thymocytes was almost same as that for medullary (CD4 or CD8 single‐positive) thymocytes. In contrast, the chemotactic activity of SDF‐1α for cortical thymocytes was considerably (more than 10‐fold) enhanced by laminin or fibronectin as compared with that for medullary thymocytes. Chemotactic activities of macrophage‐derived chemokine and macrophage inflammatory protein‐3β for both cortical and medullary thymocytes were only slightly enhanced by fibronectin or laminin. Thus, fibronectin and laminin appear to enhance the chemotactic activity of SDF‐1α for cortical thymocytes selectively. Addition of a monoclonal antibody against CD29 showed no inhibitory effect on the enhanced chemotactic activity of SDF‐1α, suggesting that the other unknown receptor(s) is involved in this enhancement. Our present data demonstrate that SDF‐1α in the presence of fibronectin or laminin is involved in the distribution of developing thymocytes.