bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of B‐cell activation by the B subunit of Escherichia coli enterotoxin: receptor interaction up‐regulates MHC class II, B7, CD40, CD25 and ICAM‐1
Authors: NASHAR T. O. , HIRST T. R. , WILLIAMS N. A.
Published: November 2003

The B subunits of cholera toxin (CtxB) and Escherichia coli heat‐labile enterotoxin (EtxB) are non‐toxic lectins that bind and cross‐link a ubiquitous cell glycolipid receptor, ganglioside GM1, and are recognized as potent mucosal and systemic immunogens. Here we examine the role of EtxB receptor occupancy in modulating the activation of B cells, in vitro, in primary lymphocyte cultures containing B and T cells. When 48‐hr spleen cell cultures containing EtxB were compared with those in the presence of a non‐receptor binding mutant, EtxB(G33D), a marked shift in the ratio of CD4+ T cells:B cells was noted. Evidence suggested that this was the result of either enhanced survival or proliferation of B cells associated with receptor occupancy by EtxB. Investigation revealed that EtxB induced only a minimal increase in proliferation above that of EtxB(G33D), in mixed cell cultures, and failed to induce any cell division of purified B cells or T cells. In contrast, receptor‐binding by EtxB markedly up‐regulated the expression of major histocompatability complex (MHC) class II, B7, intracellular adhesion molecule‐1 (ICAM‐1), CD40 and CD25 on the B‐cell surface. These results indicate that the polyclonal effects of EtxB on B cells are not associated with wide‐scale proliferation, but more likely with maintenance of B‐cell survival by activation of molecules essential for B‐cell differentiation. The findings also highlight the essential role of GM1‐interaction with EtxB in the regulation of lymphocyte responses.
Recruitment of multiple Vβ genes in the TCR repertoire against a single pathogenic thyroglobulin epitope
Authors: RAO V. P. , RUSSELL R. S. , CARAYANNIOTIS G.
Published: November 2003

In autoimmune thyroid disease, the question whether thyroid‐infiltrating, autoreactive T cells are derived from a polyclonal or oligoclonal subset has been the subject of considerable debate. In this report, we have examined the T‐cell receptor (TCR) Vβ profile of mouse clonal T cells responding to a single thyroiditogenic epitope, the As‐restricted, 9mer mouse thyroglobulin (MTg) peptide (2496‐04). In vitro recall assays based on lymph node cell (LNC) proliferation and cytokine release demonstrated that this peptide is a minimal T‐cell epitope inducing a T‐helper 1 (Th1) type of response in SJL hosts. A panel of cloned, interleukin‐2 (IL‐2)‐secreting hybridomas was generated from this Th1 subset and their TCR‐Vβ gene utilization was assessed by reverse transcription–polymerase chain reaction (RT–PCR). Ten clones derived from two independent fusions were found to utilize three Vβ gene families (Vβ 2, 4, and 17). To the extent that Tg or other thyroid autoantigens encompass multiple pathogenic epitopes it appears unlikely from these data that a restricted TCR‐Vβ chain usage will be a general characteristic of thyroiditogenic T cells.
Subunit composition of the pre‐T‐cell receptor complex analysed by monoclonal antibody against the pre‐T‐cell receptor α chain
Authors: KOSUGI A. , NODA S. , SAITOH S.‐I. , NARUMIYA S. , OGATA M. , HASHIMOTO Y. , TAKASE K. , SAITO T. , HAMAOKA T.
Published: November 2003

The pre‐T‐cell receptor (TCR) complex, which consists of a heterodimer of the TCRβ‐chain and the pre‐TCRα‐chain, is known to regulate early thymocyte development. The pre‐TCR complex contains CD3 subunits as a signal‐transducing molecule, but the exact subunit composition of the fully assembled pre‐TCR complex remains to be elucidated. In particular, the association of the CD3ζ‐chain with the pre‐TCR is controversial. In the present study, we have generated a monoclonal antibody against the cytoplasmic portion of the pre‐TCRα‐chain, and analysed a subunit composition of the pre‐TCR complex. We demonstrated that the CD3ζ‐chain is physically associated with the pre‐TCR in immature T cells. Thus, the result strongly supports the previous findings that CD3ζ contributes to signalling mediated through the pre‐TCR complex.
Unmethylated CpG‐containing oligodeoxynucleotides inhibit apoptosis in WEHI 231 B lymphocytes induced by several agents: evidence for blockade of apoptosis at a distal signalling step
Authors: MACFARLANE D. E. , MANZEL L. , KRIEG A. M.
Published: November 2003

Certain oligodeoxynucleotides (ODN) containing cytosine followed by guanosine (CpG) protect B cells from apoptosis, and induce B‐cell proliferation and cytokine production. We investigated the effect of phosphorothioate CpG‐containing ODNs (5′‐ATAATCGACGTTCAAGCAAG‐3′ or 5′‐TCCATGACGTTCCTGACGTT‐3′) and control ODNs (which did not contain CpG) on apoptosis and cell growth in WEHI 231 murine B lymphoma cells. Anti‐surface (α‐s)IgM antibody induces 40–60% DNA degradation and growth arrest of WEHI 231 cells in 24 h. Both of these effects were substantially reversed by 30 ng/ml CpG‐ODN added up to 8 hr after α‐sIgM. Control ODNs not containing the CpG motif were without effect. We explored various hypotheses to account for these effects. The phorbol ester, 12‐O‐tetradecanoyl phorbol‐13‐acetate, inhibits apoptosis induced by α‐sIgM, but the anti‐apoptotic effect of CpG‐ODN was not affected by inhibitors of protein kinase C, indicating that CpG‐ODN does not act via protein kinase C. CpG‐ODN inhibited apoptosis and growth arrest induced by C2‐ and C8‐ceramide, sphingomyelinase and an intracellular Ca2+ pump inhibitor thapsigargin, indicating that inhibition is not mediated via suppression of the ceramide cycle or suppression of Ca2+ mobilization. CpG‐ODN partially inhibited apoptosis induced by okadaic acid, a protein phosphatase inhibitor, and by menadione, a free radical generator. CpG‐ODN also inhibited apoptosis and growth arrest induced by ultraviolet‐irradiation, glucocorticoid, vinca alkaloids, and doxorubicin. CpG‐ODN significantly protected cells from DNA fragmentation induced by α‐sIgM in the presence of cycloheximide, but cycloheximide itself induces apoptosis which was unaffected by CpG‐ODN. These results suggest that CpG‐ODNs powerfully modulate the process by which immune cells are committed to death or proliferation by a mechanism acting on distal cell signalling events. CpG‐ODNs may be able to decrease immunosuppression in patients undergoing cancer chemotherapy.
Interferon‐γ induces the expression of immediate early genes c‐fos and c‐jun in astrocytes
Authors: RUBIO N.
Published: November 2003

The expression of the proto‐oncogenes, c‐fos and c‐jun, in cultured mouse astrocytes and its induction by the potent astrocyte activator interferon‐γ (IFN‐γ), were examined by Northern blot and flow cytometry. Both proto‐oncogenes were induced in a dose‐dependent manner, peaking around 100 U/ml of IFN‐γ. The kinetics of expression is very transient for c‐fos, reaching a maximum at 30 min and decreasing rapidly thereafter. The c‐jun remained high throughout the stages analysed. Cycloheximide superinduced c‐fos and c‐jun induction by IFN‐γ, thus indicating that both act as immediate early genes. The products of c‐fos and c‐jun, proteins FOS and JUN, that act in conjunction forming the regulatory factor AP‐1, were detected 1 hr after stimulation in virtually all cells, using flow cytometry. The induction in astrocytes of both proto‐oncogenes could be the first stage of immunological activation of these central nervous system cells by immune interferon.
Human cytomegalovirus infection up‐regulates interleukin‐8 gene expression and stimulates neutrophil transendothelial migration
Authors: CRAIGEN J. L. , YONG K. L. , JORDAN N. J. , MACCORMAC L. P. , WESTWICK J. , AKBAR A. N. , GRUNDY J. E.
Published: November 2003

Virus‐induced alterations in the cellular expression of chemokines may be important in directing the migration of specific leucocyte subsets to sites of infection, thereby playing a pivotal role in viral pathogenesis. We show here that cytomegalovirus (CMV) infection of human fibroblasts resulted in significantly increased expression of the C‐X‐C or α‐chemokine interleukin‐8 (IL‐8), at both the mRNA and protein levels. Increased IL‐8 production was seen following infection with the high passage laboratory CMV strains AD169, Towne, or Davis, as well as the low passage clinical CMV isolates Toledo or C1F. The increase in IL‐8 production had functional consequences, as demonstrated by the ability of supernatants from CMV‐infected fibroblasts to significantly enhance neutrophil transendothelial migration. The latter was independent of alterations in adhesion molecule expression on the endothelial cells, and was abrogated by neutralizing antibodies specific for IL‐8. Direct infection of endothelium with the endothelial cell‐tropic CMV strain C1FE, also resulted in enhanced neutrophil transendothelial migration. Neutrophils play an important role in the dissemination of CMV throughout the body, and thus CMV‐induced neutrophil recruitment would be expected to enhance CMV dissemination. Increased production of chemokines in response to CMV infection could also disrupt the fine balance between a beneficial and a destructive immune response, thereby potentially contributing to pathology.
Immunogenicity of bacterial carbohydrates: cholera toxin modulates the immune response against dextran B512
Authors: SVERREMARK E. , FERNANDEZ C.
Published: November 2003

Native dextran B512 is a T‐cell‐independent (TI) antigen. By conjugating low molecular weight (MW) dextran to protein, a T‐cell‐dependent (TD) response against dextran can be obtained. We have previously reported the effects of native dextran and two different protein–dextran conjugates on the immune system. While one type of conjugate induced an optimal TD response, the other conjugate (‘suboptimal’) evoked a response more similar to that induced by native dextran, i.e. with little immunoglobulin class switch and with a secondary response of similar magnitude to the primary response. In order to investigate if it was possible to augment the anti‐dextran response we examined the effects of cholera toxin (CT) in our dextran model system. CT is a potent mucosal, as well as systemic, adjuvant with effects on T cells, B cells and antigen‐presenting cells. We show that CT is a very efficient adjuvant for both the TD and TI forms of dextran. A major increase in IgM and IgG anti‐dextran antibody production was detected after administration of CT together with the conjugates compared with a conventional alum adjuvant. The effect was most pronounced for the suboptimal TD conjugate. CT was also able partially to abrogate the unresponsiveness to dextran in the TI type 2 (TI‐2) non‐responder strain CBA/N. CT was also found to be a very potent adjuvant for native dextran, secondary IgM levels were enhanced eightfold by the co‐administration of CT. Furthermore CTB–Dx, which is a conjugate of the non‐toxic part of CT and low MW, non‐immunogenic dextran, elicited an anti‐dextran response in nude mice. Collectively, our data show that it is possible to improve the immunogenicity of both TD and TI forms of a carbohydrate by co‐administration of CT. This is indicative of two components of the adjuvant effect, one could enhance antigen presentation and costimulation of T cells and the other could have a direct stimulatory effect on B cells.
Steroids inhibit uptake and/or processing but not presentation of antigen by airway dendritic cells
Authors: HOLT P. G. , THOMAS J. A.
Published: November 2003

Recent studies from our laboratory indicate that local and (particularly) systemic steroids can modulate the traffic of dendritic cells (DC) through resting and inflamed airway epithelial tissues. The present report focuses upon the T‐cell activating properties of DC, which are controlled by granulocyte–macrophage colony‐stimulating factor (GM‐CSF) signals, and in particular the question of whether the DC‐stimulating effects of GM‐CSF are susceptible to regulation by steroids. We present evidence that while dexamethasone inhibited GM‐CSF‐dependent uptake and/or processing of exogenous antigen by DC, it was ineffective in blocking the presentation of preprocessed self antigen to alloreactive T cells in a one‐way mixed lymphocyte reaction (MLR). Associated GM‐CSF‐induced up‐regulation of major histocompatability complex (MHC) class II and CTLA4 ligand expression by DC were also unaffected by dexamethasone phosphate (DX), reinforcing the view that the inhibitory effects of steroids on the T‐cell activating functions of DC are restricted to steps upstream from presentation of processed antigen to the T‐cell receptor (TCR). These findings have potentially important implications in relation to the use of topical steroids in the treatment of atopic asthma, a disease in which local T‐cell activation in airway tissue is a key pathogenic factor, and which furthermore is characterized by intense production of GM‐CSF within the airway epithelium.
A nitric oxide‐releasing reagent, S‐nitroso‐N‐acetylpenicillamine, enhances the expression of superoxide dismutases mRNA in the murine macrophage cell line RAW264‐7
Authors: SANO H. , HIRAI M. , SAITO H. , NAKASHIMA I. , ISOBE K.‐I.
Published: November 2003

Murine interferon‐&ggr; (IFN‐&ggr;) stimulates the murine macrophage tumour cell line RAW264‐7 to produce nitric oxide (NO). IFN‐&ggr; induces expression of inducible NO synthase (iNOS), manganese superoxide dismutase (Mn‐SOD) and copper zinc SOD (CuZn‐SOD) in these cells. To investigate the mechanism of induction of SOD expression, we added S‐nitroso‐N‐acetyl penicillamine (SNAP) to RAW264‐7 cells. SNAP enhanced the expression of Mn‐SOD and CuZn‐SOD. These results suggest that when producing NO, RAW264‐7 cells express SOD that might protect them from NO toxicity.
Identification of nicotinic acetylcholine receptors on lymphocytes in the periphery as well as thymus in mice
Authors: TOYABE S. , IIAI T. , FUKUDA M. , KAWAMURA T. , SUZUKI S. , UCHIYAMA M. , ABO T.
Published: November 2003

The existence of nicotinic acetylcholine receptors (nAChR) on lymphocytes remains controversial. We attempted to show the existence of nAChR on murine lymphocytes. The intraperitoneal injection of nicotine induced the lymphocytosis in the spleen on day 3. Although freshly isolated lymphocytes bound small quantities of fluorescein isothiocyanate (FITC)‐conjugated &agr;‐bungarotoxin (&agr;BuTx), they began to bind &agr;BuTx after incubation in medium. In contrast to granulocytes, various lymphocyte subsets obtained from various lymphoid organs were found to bind &agr;BuTx. Affinity purification of &agr;BuTx‐binding protein revealed that lymphocytes expressed the same nAChR molecules as those of muscle. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis showed that lymphocytes expressed the &agr;‐subunit mRNA of nAChR. These results suggest that lymphocytes carry nAChR on the surface and are stimulated directly via their nAChR by parasympathetic nerve stimuli.
Extracorporeal photochemotherapy restores Th1/Th2 imbalance in patients with early stage cutaneous T‐cell lymphoma
Authors: DI RENZO M. , RUBEGNI P. , DE ALOE G. , PAULESU L. , PASQUI A. L. , ANDREASSI L. , AUTERI A. , FIMIANI M.
Published: November 2003

Extracorporeal photochemotherapy (ECP) has been shown to be a potent activator of peripheral blood macrophages because it causes a marked release of macrophage‐dependent proinflammatory cytokines, and it is therefore currently considered to be a safe and non‐toxic immunomodulatory treatment. On this basis we studied the function of peripheral blood mononuclear cells (PBMC) in eight patients with early stage (Ib) cutaneous T‐cell lymphoma (CTCL), before and 1 year after ECP, together with their clinical and histological responses. In particular we evaluated in vitro phytohaemagglutinin (PHA)‐stimulated proliferation and production of interleukin‐4 (IL‐4) and interferon‐&ggr; (IFN‐&ggr;) as well as lipopolysaccharide (LPS)‐induced production of IL‐12. Before treatment we observed that PBMC of patients produced significantly higher levels of IL‐4 and lower levels of IFN‐&ggr; and IL‐12 than those of healthy control subjects. After 1 year of ECP, IL‐4, IFN‐&ggr; and IL‐12 production no longer differed from that of control subjects. Moreover, we observed a good clinical result matched by histological response. Our data confirm that early‐ stage CTCL patients show a predominantly type‐2 immune response that might be responsible for several immunological abnormalities found in this disease. We have demonstrated that ECP reverses the T‐helper type 1/T‐helper type 2 (Th1/Th2) imbalance and may therefore be considered an efficient biological response modifier.
Augmentation of monocyte chemotactic protein‐1 and mRNA transcript in chronic inflammatory states induced by potassium permanganate (KMnO4) in vivo
Authors: CONTI P. , REALE M. , FELICIANI C. , FRYDAS‡ S. , TRAKATELLIS§ M. , PLACIDO F. C. , CATALDO I. , DI GIOACCHINO M. , BARBACANE R. C.
Published: November 2003

Monocyte chemotactic protein‐1 (MCP‐1) is a proinflammatory cytokine that attracts and activates specific types of leucocytes. The purpose of this work was to analyse the generation of MCP‐1 and mRNA transcript in a model of chronic inflammation using a granulomatous tissue induced by potassium permanganate (KMnO4; water soluble crystals). The data presented here shows that MCP‐1 is generated in granuloma tissue and its level was strongly increased by i.p. injections of lipopolysaccharide (LPS) and inhibited in rats treated with injections of dexamethasone, 18 hr before the animals were killed. In histological studies LPS and dexamethasone increased and decreased, respectively, the recruitment of mononuclear cells in the granuloma tissue compared with the control granulomas from phosphate‐buffered saline (PBS)‐treated animals. Reverse transcriptase–polymerase chain reaction (RT–PCR) was used for mRNA extraction and cDNA synthesis. mRNA MCP‐1 was significasntly produced in the granuloma tissue of untreated animals, an effect increased by LPS and inhibited by dexamethasone, compared with the controls. Moreover, MCP‐1 protein was found in the supernatant from homogenized granuloma tissues and the levels of MCP‐1 were higher in the LPS‐treated animals, while they were lower in the dexamethasone group, compared with the granulomas from the PBS‐treated groups (control). The generation of MCP‐1 was also found in minced granuloma tissue incubated for 18 hr (overnight) from treated (LPS or dexamethasone) and untreated (PBS) rats. When LPS was added in vitro for 18 hr to the controls and treated animals the production of MCP‐1 was further increased except in the dexamethasone group (P > 0·05). Analysing blood serum from LPS, dexamethasone or PBS‐treated rats, we found that MCP‐1 was also present. The level was higher in the LPS group and lower in the dexamethasone group, compared with the control (PBS). In these studies we show for the first time that MCP‐1 transcript and translation is generated in chronic experimental inflammatory tissue, an effect inhibited by dexamethasone.
CD5+ B lymphocytes are the main source of antibodies reactive with non‐parasite antigens in Trypanosoma congolense‐infected cattle
Authors: BUZA J. , SILEGHEM* M. , GWAKISA† P. , NAESSENS J.
Published: November 2003

Mice infected with African trypanosomes produce exceptionally large amounts of serum IgM, a major part of which binds to non‐trypanosome antigens such as trinitrophenol and single‐strand DNA. In this paper, we describe that in cattle infected with Trypanosoma congolense and T. vivax, similar antibodies are found, although they bind mainly to protein antigens, such as &bgr;‐galactosidase, ovalbumin and ferritin. The parasite non‐specific IgM antibodies appear around the same time as the parasite‐specific antibodies, but their origin and function are not clear. We tested the hypothesis that CD5+ B cells (or B‐1 cells), which increase during trypanosome infections in cattle, are responsible for production of antibodies to non‐trypanosome antigens. Splenic CD5+ and CD5− B cells from infected cattle were sorted and tested in a single cell blot assay. The numbers of immunoglobulin‐secreting cells were similar in both B‐cell populations. However, antibodies with reactivity for non‐trypanosome antigens were significantly more prevalent in the CD5+ B‐cell fraction and were exclusively IgM. The preference for production of these antibodies by CD5+ B cells and the expansion of this subpopulation during infections in cattle, strongly suggest that CD5+ B cells are the main source of trypanosome non‐specific antibodies. We propose that these antibodies are natural, polyreactive antibodies that are predominantly secreted by CD5+ B cells. Since B‐1 cells are up‐regulated in many states of immune insufficiency, the immunosuppression associated with trypanosome infections may be responsible for the increase of this subset and the concomitant increase in trypanosome non‐specific antibodies.
T‐lymphocyte activation and the cellular form of the prion protein
Authors: MABBOTT N. A. , BROWN K. L. , MANSON J. , BRUCE M. E.
Published: November 2003

The transmissible spongiform encephalopathies are neurodegenerative disorders which include Creutzfeldt–Jakob disease in humans, and scrapie and bovine spongiform encephalopathy in animals. A major component of the infectious agent responsible for these diseases is considered to be a post‐translationally modified form of a host‐encoded glycoprotein PrPc, termed PrPSc. While PrPc is abundantly expressed in tissues of the central nervous system (CNS), little is known about its normal function. The expression of PrPc is not restricted to the CNS, as this protein can also be detected in the lymphoid tissues of mice and sheep. In this report we demonstrate that resting murine splenic lymphocytes express PrPc protein on their cell membranes. Furthermore, expression of PrPc was significantly enhanced following in vitro stimulation with the non‐specific T‐cell mitogen concanavalin A (Con A). Genetically engineered mice with an inactive PrPc gene (PrP−/− mice), were utilized to investigate the involvement of PrPc in lymphocyte activation. Experiments revealed that the Con A‐induced proliferation of lymphocytes from PrP−/− mice was significantly reduced to ≈50–80% that of wild‐type (PrP+/+) mice 48 hr post‐stimulation. These findings demonstrate an important role for PrPc in extra‐neuronal tissues and suggest that PrPc is a lymphocyte surface molecule that participates in T‐cell activation.
Detection of a novel 40 000 MW excretory Toxoplasma gondii antigen by murine Th1 clone which induces toxoplasmacidal activity when exposed to infected macrophages
Authors: REICHMANN G. , STACHELHAUS S. , MEISEL R. , MÉVÉLEC M.‐N. , DUBREMETZ J. F. , DLUGONSKA§ H. , FISCHER H.‐G.
Published: November 2003

To analyse target molecules of the CD4+ T‐cell response to toxoplasma infection, a panel of Toxoplasma gondii‐specific murine CD4+ T‐cell clones has been established. Clone 3Tx15, belonging to the T helper 1 (Th1) subtype, abolished intracellular parasite growth when co‐cultured with macrophages and live toxoplasma at a ratio of 2:2:1. This effect results from macrophage toxoplasmicidal activity induced upon parasite‐dependent cellular interaction, an irrelevant Th1 clone failed in this three‐party system. Clone 3Tx15 detects its corresponding antigen in the supernatant of infected cells and also reacts with a host cell‐free preparation of T. gondii‐excreted/secreted antigens. T‐cell blot analysis of two‐dimensionally separated toxoplasma lysate revealed a molecular weight of about 40 000 for the fractions stimulating clone 3Tx15. As checked in parallel enzyme‐linked immunosorbent assay, the 40 000 MW T‐cell antigen co‐migrates with the excretory protein GRA4, the sole 40 000 MW T. gondii antigen hitherto known to be recognized by T lymphocytes. Nevertheless, neither recombinant GRA4 nor immunoaffinity‐purified natural GRA4 was stimulatory for clone 3Tx15. Our findings thus demonstrate that Th1 clone 3Tx15 which induces toxoplasmicidal activity during antigenic interaction with infected macrophages defines a new 40 000 MW excretory T. gondii antigen.
Involvement of the membrane form of tumour necrosis factor‐&agr; in lipopolysaccharide‐induced priming of mouse peritoneal macrophages for enhanced nitric oxide response to lipopolysaccharide
Authors: ANCUTA P. , FAHMI H. , PONS J. ‐F. , LE BLAY K. , CHABY R.
Published: November 2003

We studied the pathways of macrophage response to lipopolysaccharide (LPS). When mouse macrophages pre‐exposed to LPS were restimulated with this agent, reduced tumour necrosis factor‐&agr; (TNF‐&agr;) responses (desensitization/endotoxin tolerance) were accompanied by increased (priming) nitric oxide (NO) responses. Priming was also inducible with recombinant interferon‐&bgr; (IFN‐&bgr;). The requirement of TNF‐&agr; biosynthesis in the LPS‐induced priming was also suggested by the observation that both anti‐TNF‐&agr; serum and pentoxifylline inhibited this effect. However, addition of mouse recombinant TNF‐&agr; (mrTNF‐&agr;) did not enhance the priming induced by LPS or IFN‐&bgr;, and preincubation with mrTNF‐&agr; alone, or in association with other cytokines produced by macrophages (interleukin‐1&bgr;, interleukin‐6, or leukaemia inhibitory factor), did not induce a priming effect. We found however, that pentoxifylline, which blocked the priming, also decreased the level of membrane‐bound TNF‐&agr;. Furthermore, exposure to compound BB‐3103 (a metalloproteinase inhibitor that blocks the processing of membrane‐bound TNF‐&agr; yielding to the secreted cytokine) enhanced the priming effect, the expression of membrane TNF‐&agr; and the specific binding of LPS. These observations suggest that the membrane form of TNF‐&agr; is involved in the interaction of LPS with a receptor required for LPS‐induced priming.
Role of spleen macrophages in innate and acquired immune responses against mouse hepatitis virus strain A59
Authors: WIJBURG O. L. C. , HEEMSKERK‡ M. H. M. , BOOG§ C. J. P. , VAN ROOIJEN N.
Published: November 2003

Owing to their scavenging and phagocytic functions, spleen macrophages are regarded to be important in the induction and maintenance of both innate and acquired immune defence mechanisms. In this study, we investigated the role of spleen macrophages in immunity against mouse hepatitis virus strain A59 (MHV‐A59). Previous studies showed that spleen and liver macrophages are the first target cells for infection by MHV‐A59 in vivo, suggesting that they could be involved in the induction of immune responses against MHV‐A59. We used a macrophage depletion technique to deplete macrophages in vivo and studied the induction of virus‐specific antibody and cytotoxic T‐cell (CTL) responses and non‐immune resistance against MHV‐A59 in normal and macrophage‐depleted mice. Virus titres in spleen and liver increased rapidly in macrophage‐depleted mice, resulting in death of mice within 4 days after infection. Elimination of macrophages before immunization with MHV‐A59 resulted in increased virus‐specific humoral and T‐cell proliferative responses. However, virus‐specific CTL responses were not altered in macrophage‐depleted mice. Our results show that spleen macrophages are of major importance as scavenger cells during MHV‐A59 infection and are involved in clearance of virus from the host. In addition, macrophages may be involved in the regulation of acquired immune responses. In the absence of macrophages, increased virus‐specific T‐cell and antibody responses are detectable, suggesting that macrophages suppress MHV‐A59‐specific T‐ and B‐cell responses and that other cells serve as antigen‐presenting cells.
The polymeric immunoglobulin receptor (secretory component) in a human intestinal epithelial cell line is up‐regulated by interleukin‐1
Authors: HAYASHI M. , TAKENOUCHI N. , ASANO M. , KATO M. , TSURUMACHI T. , SAITO T. , MORO I.
Published: November 2003

Secretory component (SC or polymeric immunoglobulin receptor) on mucosal epithelial cells mediates transcytosis of polymeric immunoglobulin into external fluids and functions as a receptor for polymeric immunoglobulin. SC expression in a human colonic adenocarcinoma cell line, HT‐29 has been reported to be up‐regulated by various cytokines, such as interferon‐&ggr;, tumour necrosis factor‐&agr; and interleukin‐4 (IL‐4). However, up‐regulation of SC by IL‐1 is controversial. In this study, we investigated the effect of human recombinant IL‐1 alone on SC expression in HT‐29 cells in detail. Immunocytochemistry and Northern blot analysis revealed that IL‐1&bgr; increased both the number of SC‐positive cells and SC mRNA expression. Enzyme‐linked immunosorbent assay revealed that IL‐1&bgr; enhanced secretion by HT‐29 cells in both time‐ and dose‐dependent manners. IL‐1&agr; had the same effects on HT‐29 cells. Northern blot analysis demonstrated that cycloheximide and actinomycin D abolished the effect of IL‐1. Moreover, we detected IL‐1 receptor (IL‐1R) type I mRNA in HT‐29 cells by polymerase chain reaction (PCR) and sequenced the PCR‐amplified product. We think that it reflects the possibility of the presence of IL‐1R in HT‐29 cells. From these data, we concluded that IL‐1&bgr; and IL‐1&agr; play regulatory roles in SC expression, and their effects depend on de novo protein synthesis and transcription.
Later development of Fas ligand‐mediated cytotoxicity as compared with granule‐mediated cytotoxicity during the maturation of natural killer cells
Authors: NAKAZAWA T. , AGEMATSU K. , YABUHARA A.
Published: November 2003

We classified CD56+ CD3− natural killer (NK) cells into CD2− CD56dim (CD2− NK), CD2+ CD56dim (CD2+ NK) and CD2+ CD56bright populations, and investigated mainly functional differences between the former two populations. CD2− and CD2+ NK cells were the same in their morphology and several surface molecules except for CD2. The percentages of CD2− NK cells in total NK cells were higher in the cord blood and bone marrow than in the peripheral blood of adults or children. Freshly isolated CD2− NK cells had CD2 in the cytoplasm, and gradually expressed it on the surface upon incubation with interleukin‐2 (IL‐2). These results demonstrated that CD2 is an antigen which appears on the surface during the maturation of NK cells. The granule‐mediated cytotoxicities, which are mainly performed by the perforin molecule, of CD2+ NK cells against K562 and Daudi cells were higher than those of CD2− NK cells, and they were inhibited to the levels of CD2− NK cells by the addition of a blocking anti‐CD2 monoclonal antibody (mAb). Fas ligand (FasL) mRNA was expressed in freshly isolated CD2+ NK cells but not in the CD2− NK cells. Neither freshly isolated NK populations showed FasL‐mediated cytotoxicity, and only CD2+ NK cells lysed Fas‐transfected targets after the 24‐hr incubation with IL‐2. Based on these results, CD2− NK cells have already developed granule‐mediated cytotoxicity equal to that of CD2+ NK cells except for the CD2‐associated activity, but they, unlike CD2+ NK cells, totally lack FasL‐mediated cytotoxicity. These findings suggest that FasL‐mediated cytotoxicity may be acquired at more mature stages of NK‐cell maturation than granule‐mediated cytotoxicity.
Interleukin‐10 rescues T cells from apoptotic cell death: association with an upregulation of Bcl‐2
Authors: COHEN S. B. A. , CRAWLEY J. B. , KAHAN M. C. , FELDMANN M. , FOXWELL B. M. J.
Published: November 2003

We demonstrate that interleukin‐10 (IL‐10) can inhibit T‐cell apoptosis. T cells, within a PBMC (peripheral blood mononuclear cell) population, were stimulated via the T‐cell receptor and grown in the presence of IL‐2. These cells had less apoptosis when in the continuous presence of IL‐10, compared with cells grown in the absence of IL–10. Conversely, when stimulated and grown in the presence of neutralizing antibody to IL‐10, there was an increase in T‐cell apoptosis. The in vitro rescue from apoptotic cell death of other lymphoid cells, such as germinal centre B cells, has been shown by others to involve a Bcl‐2 pathway. We therefore investigated whether IL‐10 might affect the Bcl‐2 expression on cultured T cells. By Western blotting we demonstrated that continuous exposure of IL‐10 to T cells (within a PBMC population) enhanced the expression of Bcl‐2. Furthermore, T cells protected from apoptotic cell death by IL‐10 were indistinguishable from viable untreated cells in their ability to proliferate to either immobilized anti‐CD3 or IL‐2. Thus, we have shown that continuous culture of T cells in the presence of IL‐10 will inhibit T‐cell apoptosis because of, at least in part, the upregulation of Bcl‐2, and this is associated with a normal proliferative function.
Cell‐specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression
Authors: TRAJKOVIĆ V. , BADOVINAC V. , POPADIĆ D. , HADŽIĆ O. , STOJKOVIĆ M. M.
Published: November 2003

Cytokine‐stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ‐specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon‐γ, IFN‐γ; interleukin‐1, IL‐1; tumour‐necrosis factor‐α, TNF‐α)‐induced NO production in both cell types, but in the opposite manner – enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.
Autoantibodies to evolutionarily conserved epitopes of enolase in a patient with discoid lupus erythematosus
Authors: GITLITS V. M. , SENTRY J. W. , MATTHEW M. L. S. M. , SMITH A. I. , TOH B.‐H.
Published: November 2003

Although the pathology of discoid lupus erythematosus is well documented the causative agents are not known. Here, we report the identity of the target antigen of an autoantibody present in high titre in the serum of a patient with discoid lupus erythematosus. We have demonstrated that the antigen is enolase; first, because it has properties consistent with this glycolytic enzyme (47 000 MW, cytosolic localization and ubiquitous tissue distribution). Secondly, limited amino acid sequence determination after trypsin digestion shows identity with α‐enolase. Finally, the autoimmune serum immunoblots rabbit and yeast enolase and predominantly one isoelectric form of enolase (pI∼6·1). These results indicate that the reactive autoepitopes are highly conserved from man to yeast. The results also suggest that the autoantibodies are most reactive to the α‐isoform of enolase, although it is possible that they may also be reactive with γ‐enolase, and have least reactivity to β‐enolase. The anti‐enolase autoantibodies belong to the immunoglobulin G1 (IgG1) isotype. This is the first report of IgG1 autoantibodies to evolutionarily conserved autoepitopes of enolase in the serum of a patient with discoid lupus erythematosus. Previous reports of autoantibodies to enolase have suggested associations with autoimmune polyglandular syndrome type I and cancer‐associated retinopathy. This report and an earlier report of what is likely to be enolase autoantibodies in two patients without systemic disease suggest that enolase autoantibodies have a broad association and are not restricted to any particular disease.
Mucosal tolerance is associated with, but independent of, up‐regulation Th2 responses
Authors: WOLVERS D. A. W. , VAN DER CAMMEN M. J. F. , KRAAL G.
Published: November 2003

Intranasal administration of protein antigen is an efficient way to induce mucosal tolerance. Suppressive mechanisms that might be involved in this phenomenon include down‐regulation of T‐helper type‐1 (Th1)‐mediated processes by Th2 cells. However, since Th2 responses can also be subjected to mucosal tolerance, we wanted to investigate whether suppression of a typical Th1 response, such as a delayed‐type hypersensitivity (DTH) reaction by intranasal tolerance induction, was causally related to up‐regulation of Th2 responses. We therefore treated mice either systemically or locally with anti‐interleukin‐4 (IL‐4) or anti‐IL‐10 antibodies before intranasal tolerance induction or before sensitization for DTH to see whether we could prevent or abrogate tolerance. Although the up‐regulation of antigen‐specific IgE levels in tolerant mice could be prevented by anti‐IL‐4 treatment, the extent of tolerance as measured by suppression of DTH was not affected. We therefore conclude that up‐regulation of Th2 responses observed after intranasal tolerance induction is an additional or consequential rather than a necessary reaction.
Lipids from Mycobacterium leprae cell wall suppress T‐cell activation in vivo and in vitro
Authors: MOURA A. C. N. , MARIANO M.
Published: November 2003

The influence of Mycobacterium leprae cell wall lipids on lymphocyte functions has been investigated in vivo (delayed‐type hypersensitivity) and in vitro. The inflammatory response has been earlier evaluated by the mouse footpad oedema model and the delipidated mycobacteria evoked a mild but significant inflammatory response. Herein a higher level of hypersensitivity reaction was observed with delipidated bacilli than with the intact mycobacteria. The lipids obtained from the extract of M. leprae external cell wall were used to prepare liposomes, which have not been shown to be toxic to lymphocytes. The method of lipidic extraction and the sodium dodecyl sulphate–polyacrylamide gel electrophoresis of the lipid fraction did not reveal any trace of proteins. Thin‐layer chromatography of this extract detected four different bands with an apolar character, suggestive of mycolic and fatty acids. These same M. leprae liposomes potently suppressed lymph node cells, as well CD4+ and CD8+ T‐cell lines, and an antigen‐specific T‐cell clone (T 4–9) proliferation, even under potent stimulus. Cholesterol‐choline liposomes, unrelated to M. leprae liposomes, used as a control in the biological assays showed no significant effect on lymphoblastic activity, which points to the specificity of M. leprae lipids. These data demonstrated that M. leprae cell wall lipids induce immune suppression in mice without causing any membrane alteration in T cells as assessed throughout kinetic studies in vitro. This fact is closely related to the down‐regulating effect induced by M. leprae lipids which we have previously observed in macrophage functions in vivo and in vitro. Although this lipidic fraction showed a suppressive action on T lymphocytes in vitro (proliferation) and in vivo (delayed‐type hypersensitivity), its possible significance in the establishment of a specific immune response to M. leprae must be further investigated.
Expression of HIS50 Ag: a rat homologue of mouse heat‐stable antigen and human CD24 on B lymphoid cells in the rat
Authors: HERMANS M. H. A. , DEENEN G. J. , DE BOER N. , BO WANG , KROESE F. G. M. , OPSTELTEN D.
Published: November 2003

Recently, a cDNA encoding a newly identified rat antigen (HIS50 Ag) that binds to monoclonal antibody (mAb) HIS50 was cloned and shown to be homologous to cDNA encoding murine heat‐stable antigen (HSA) and human CD24. Here we show that, like CD24 and HSA, at least part of HIS50 Ag is inserted into the plasma membrane by a glycosylphosphatidylinositol (GPI)–lipid linkage and we describe its expression in rat haemolymphopoietic tissues. HIS50 Ag expression was almost exclusively confined to B lymphoid cells, the vast majority of T lymphoid cells, erythroid and myeloid cells were HIS50−. Cell suspension analysis indicated that in bone marrow (BM) almost all Thy‐1+ cells, HIS24+ cells [
HIS24 recognizes the B‐cell form of leucocyte common antigen (LCA)], terminal deoxynucleotidyl transferase‐positive (TdT+) cells and (c+s)κ+ cells expressed HIS50 Ag, and all (c+s)μ+ cells. A presumably early population of B lymphoid cells, expressing HIS24 Ag without HIS50 Ag, TdT or immunoglobulin (HIS24+HIS50−TdT−Ig−), constituted 1·6% of BM nucleated cells. In blood, one‐fifth of mononuclear cells were HIS50+, and about 85% of these expressed μ and/or κ chains. In spleen, flow cytometry analysis and immunohistology demonstrated heterogeneous expression of HIS50 Ag: immunoglobulin M (IgM)bright cells (as found largely in red pulp and marginal zone) were HIS50bright, while IgMdull cells expressed low or undetectable levels of HIS50 Ag. Germinal centre B cells expressed high levels of HIS50 Ag. Germinal centres of lymph nodes and tonsil of man also bound HIS50. We conclude that HIS50 Ag expression in the haemolymphopoietic system of rat is virtually restricted to the B lineage.
Activation of neutral sphingomyelinase in human neutrophils by polyunsaturated‘qc fatty acids
Authors: ROBINSON B. S. , HII C. S. T. , POULOS A. , FERRANTE A.
Published: November 2003

Although unesterified polyunsaturated fatty acids (PUFA) have been shown to elicit marked changes in neutrophil function, the associated signal transduction processes require clarification. In this study we examined the effect of PUFA on the sphingomyelin (SM)‐signalling cycle in human neutrophils. Treatment of neutrophils with eicosatetraenoic acid [ arachidonic acid, 20:4(n‐6)] caused a decrease in the mass of cellular SM and an increase in the level of ceramide. 20:4(n‐6)‐stimulated neutral sphingomyelinase (SMase) activity of the leucocytes in a time‐ and concentration‐dependent manner. Other unsaturated fatty acids, docosahexaenoic [22:6(n‐3)], eicosapentaenoic [20:5(n‐3)], octadecenoic [oleic, 18:1(n‐9)] and octadecadienoic [linoleic, 18:2(n‐6)] acids also had the capacity to activate neutral SMase; however, certain 20:4(n‐6) derivatives {20:4(n‐6) methyl ester [20:4(n‐6)ME], 15‐hydroperoxyeicosatetraenoic (15‐HPETE) and 15‐hydroxyeicosatetraenoic (15‐HETE) acids}, very‐long‐chain PUFA {tetracosatetraenoic [24:4(n‐6)] and octacosatetraenoic [28:4(n‐6)] acids} and saturated fatty acids [octadecanoic (stearic, 18:0) and eicosanoic (arachidic, 20:0) acids] had no significant effect. Activation of neutral SMase by 20:4(n‐6) appeared to involve metabolism via 20:4(n‐6)CoA (arachidonoyl CoA) and was not dependent on prostaglandin and leukotriene synthesis. All of the fatty acids and derivatives tested failed to activate acidic SMase of neutrophils. Ceramide was found to inhibit 20:4(n‐6)‐induced superoxide generation by the cells. It is envisaged that the PUFA‐induced ceramide production in neutrophils plays a role in the regulation of biological responses.
An immunosuppressive agent, FTY720, increases intracellular concentration of calcium ion and induces apoptosis in HL‐60
Authors: SHINOMIYA T. , LI X.‐K. , AMEMIYA H. , SUZUKI S.
Published: November 2003

We previously reported that FTY720 is an efficient inducer of apoptosis in lymphocytes and cultured cell lines. In the present study, HL‐60 human promyerocytoma cells also induced apoptosis through in vitro treatment with the drug, demonstrating extensive DNA fragmentation 6 hr after incubation. The major target of FTY720 was the common signalling pathway of apoptosis, since a rapid (<1 min) increase in the intracellular Ca2+ concentration ([
Ca2+]i) was found in the cells treated with the drug. Calcium chelation in the culture medium with EGTA did not affect the [Ca2+]i mobilization. A phospholipase C inhibitor, U73122, inhibited the increase in [Ca2+]i as well as the fragmentation of the nuclear DNA, whereas U73343, a non‐effective analogue of U73122, had little effect. These results suggest that FTY720‐induced apoptosis is mediated through an activation of phospholipase C and the subsequent release of Ca2+ from intracellular calcium pools. In addition, the treatment of HL‐60 with pertussis toxin (PTX) did not inhibit Ca2+ mobilization or apoptosis, suggesting that the activation of phospholipase C is independent of PTX‐sensitive G‐proteins.
The role of interleukin‐4 in ultraviolet B light‐induced immunosuppression
Authors: EL‐GHORR A. A. , NORVAL M.
Published: November 2003

Prolonged exposure to ultraviolet light (UV) is known to lead to premature skin ageing, increased incidence of cataract and a high risk of developing skin cancers. UV‐B irradiation, even if given as a single suberythemal dose, suppresses some immune responses, possibly reducing the production of T helper (Th) 1 cytokines [ interleukin (IL)‐2 and interferon‐&ggr;] and augmenting Th2 cytokines (IL‐4, IL‐5 and IL‐10) in mice. We investigated the role of IL‐4 in UV‐B induced immunomodulation using IL‐4 knockout (IL‐4−/−) mice and the parent strain Bb129. Suberythemal UV‐B irradiation (1440 J/m2) led to a reduction in the density and antigen presenting ability of Langerhans’ cells in the epidermis of both normal and IL‐4−/− mice. Exposure also induced an accumulation of CD4+ and CD8+ lymphocytes as well as dendritic cells in the lymph nodes draining the irradiated site in both strains. The proliferation of lymph node cells in response to the mitogen concanavalin A was enhanced in the IL‐4−/− mice compared with the parent strain. Following UV‐B exposure, this proliferation was increased in lymph node cells of parent mice but was significantly suppressed in the IL‐4−/− mice. The contact hypersensitivity (CH) response to oxazolone was suppressed to the same extent by UV‐B irradiation in both strains. In the parent mice, infected with herpes simplex virus (HSV) following UV‐B exposure and challenged subsequently with inactivated virus, the delayed hypersensitivity (DH) response was suppressed by about 50% compared with unirradiated mice; no such suppression in DH occurred in irradiated IL‐4−/− mice infected with HSV. Thus, IL‐4 may be an important mediator of the UV‐B‐induced suppression in DH but not in CH, where other cytokines may be involved or may compensate for the lack of IL–4.
p56lckis not essential for the T‐cell response to allo‐MHC antigens
Authors: YAMADA H. , KONG Y.‐Y. , KISHIHARA K. , MAK T. W. , NOMOTO K.
Published: November 2003

In mice lacking the src family protein tyrosine kinase, p 56lck (lck−/−), a greatly reduced number of peripheral T cells is observed due to a profound blockage of the thymocyte development. The peripheral T cells in lck−/−mice exhibit proliferative response after T‐cell receptor (TCR)‐crosslinking, but can not respond to viral antigens. In this report, we examined the allo‐responses of peripheral T cells in the lck−/−mice and the following results were thus obtained. (1) After an intravenous injection of fully allogeneic [
allo‐major histocompatability complex (MHC)] spleen cells, an increase of interleukin (IL)‐2Rα+ cells was observed in both the CD4+ or CD8+ peripheral T cells of the lck−/− mice and the increase was similar to those in the lck+/+ littermate, with only a somewhat delayed and prolonged time kinetics observed in the CD4+ T cells of the lck−/− mice. (2) the lck−/− mice rejected the fully allogeneic trunk skin grafts several days later than the lck+/+ mice, but did not reject the minor allogeneic grafts. (3) The peripheral T cells of the graft‐rejected lck−/−mice exhibited a weaker but significantly proliferative response, while the cytotoxic T lymphocyte (CTL) activities to allo‐MHC antigens in vitro were comparable to those in lck+/+ mice. While the response to the minor allo‐antigens was shown by the peripheral T cells in the lck+/+ mice with minor allogeneic skin grafts but not by those in the lck−/− mice with the grafts. These results thus suggest that p56lck is not essential for peripheral T cells to both respond and exhibit effector functions to allo‐MHC antigens.