bluuebunny/crossref_metadata_2025 huggingface dataset:
T‐helper 1 dominated responses to erythrocyte Band 3 in NZB mice
Authors: SHEN C.‐R. , MAZZA G. , PERRY F. E. , BEECH J. T. , THOMPSON S. J. , CORATO A. , NEWTON S. , BARKER R. N. , Elson C. J.
Published: November 2003

Band 3, the red blood cell (RBC) anion channel protein, is the target autoantigen for the pathogenic RBC autoantibodies and T‐helper (Th) cells in New Zealand Black (NZB) mice with autoimmune haemolytic anaemia (AIHA). To determine the subpopulation of these Th cells, they were stimulated with Band 3 and the profile of the cytokines elaborated by the responding cells was measured. NZB T cells stimulated with Band 3 produced high levels of the Th1 cytokine, interferon‐γ (IFN‐γ), but little or no interleukin‐4 (IL‐4), IL‐5 or IL‐10. Similar patterns were produced by NZB T cells responding to a spectrin preparation from the RBC membrane skeleton, or to mycobacterial heat‐shock protein (hsp) 65 following immunization of mice with hsp  65 in incomplete adjuvant. By contrast, T cells from CBA mice similarly immunized with hsp  65 produced high levels of IL‐4 and IL‐5 in response to hsp  65. Examination of the isotype of the RBC‐bound immunoglobulins in NZB mice revealed that immunoglobulin G2a (IgG2a) autoantibodies were the first to be detected in most mice and that later in the disease, IgG3 autoantibodies were often prominent. It is concluded that, contrary to expectation, the development of RBC autoantibodies in NZB mice is associated with Th1 cytokine‐dominated responses.
Legionella pneumophila heat‐shock protein‐induced increase of interleukin‐1β mRNA involves protein kinase C signalling in macrophages
Authors: RETZLAFF C. , YAMAMOTO Y. , OKUBO S. , HOFFMAN P. S. , FRIEDMAN H. , KLEIN T. W.
Published: November 2003

Heat‐shock proteins (hsp) are chaperon molecules important in protein folding and assembly. Furthermore, they may have functions in immunoregulatory processes, like T‐cell stimulation and antigen presentation, which are not yet fully understood. It has been shown that several hsp of various species and family derivations modulate functions in macrophage immunity by directly increasing cytokine production. In the present study we showed that the 60 000 MW hsp of Legionella pneumophila (Lp‐hsp 60) increased cellular steady‐state levels of interleukin‐1β (IL‐1β) mRNA measured by quantitative reverse transcription–polymerase chain reaction and Northern blotting as well as IL‐1 secretion, when added to cultures of thioglycollate‐elicited mouse peritoneal macrophages in vitro. The level of mRNA increased in a dose‐dependent manner with a minimum effective concentration of 0.5 μg/ml and peaked 3 hr after stimulation. Lp‐hsp 60‐coated latex beads also increased IL‐1β mRNA levels in the presence of cytochalasin D, which inhibits bead uptake but permits binding, indicating that binding to the macrophage surface was sufficient for induction. Accumulation of IL‐1β mRNA was completely blocked by pretreatment with the protein kinase C (PKC) inhibitor, H7, but not decreased by prior treatment with cycloheximide. The cell lysates of macrophages stimulated with Lp‐hsp 60 showed an increased PKC activity measured by phosphorylation of PKC pseudosubstrate. The IL‐1 bioactivity in culture supernatants after 24 hr of stimulation with Lp‐hsp 60 was increased in a dose‐dependent manner but at hsp concentrations in excess of those needed to increase mRNA. Thus, the present study demonstrates that Lp‐hsp 60 rapidly increases the steady‐state level of IL‐1β mRNA, possibly through a cell surface receptor system involving a PKC‐dependent signalling pathway.
Genetic stability of gene targeted immunoglobulin loci. I. Heavy chain isotype exchange induced by a universal gene replacement vector
Authors: KARDINAL C. , SELMAYR M. , MOCIKAT R.
Published: November 2003

Gene targeting at the immunoglobulin loci of B cells is an efficient tool for studying immunoglobulin expression or generating chimeric antibodies. We have shown that vector integration induced by human immunoglobulin G1 (IgG1) insertion vectors results in subsequent vector excision mediated by the duplicated target sequence, whereas replacement events which could be induced by the same constructs remain stable. We could demonstrate that the distribution of the vector homology strongly influences the genetic stability obtained. To this end we developed a novel type of a heavy chain replacement vector making use of the heavy chain class switch recombination sequence. Despite the presence of a two‐sided homology this construct is universally applicable irrespective of the constant gene region utilized by the B cell. In comparison to an integration vector the frequency of stable incorporation was strongly increased, but we still observed vector excision, although at a markedly reduced rate. The latter events even occurred with circular constructs. Linearization of the construct at various sites and the comparison with an integration vector that carries the identical homology sequence, but differs in the distribution of homology, revealed the following features of homologous recombination of immunoglobulin genes: (i) the integration frequency is only determined by the length of the homology flank where the cross‐over takes place; (ii) a 5′ flank that does not meet the minimum requirement of homology length cannot be complemented by a sufficient 3′ flank; (iii) free vector ends play a role for integration as well as for replacement targeting; (iv) truncating recombination events are suppressed in the presence of two flanks. Furthermore, we show that the switch region that was used as 3′ flank is non‐functional in an inverted orientation.
The strain difference in the effect of mercuric chloride on antigen‐triggered serotonin release from rat mast cells is not mediated via interferon‐γ
Authors: HODSON D. , OLIVEIRA D. B. G.
Published: November 2003

Previous work has shown that in vitro exposure of Brown–Norway (BN) rat peritoneal mast cells to mercuric chloride (HgCl2) causes enhancement of subsequent mediator release induced by cross‐linking of surface immunoglobulin E (IgE). This enhancing effect is seen significantly less often with peritoneal cells from Lewis rats. In addition HgCl2 has been shown to suppress interferon (IFN)‐γ production by BN but not Lewis splenocytes. Given that IFN‐γ is known to inhibit mediator release by mast cells, we hypothesized that the strain difference in the effect of HgCl2 on mediator release was mediated via a differential effect on IFN‐γ release from T cells in the mixed peritoneal cell population: IFN‐γ release would be suppressed in the case of the BN rat, releasing the mast cells from inhibition and resulting in the enhancing effect of HgCl2. The aim of the study was to test two predictions of this hypothesis. Exposure of BN rat mast cells to IFN‐γ inhibited subsequent antigen‐induced mediator release but did not significantly reduce HgCl2‐mediated enhancement of this release. Exposure of Lewis rat mast cells to blocking concentrations of anti‐IFN‐γ did not reveal any HgCl2‐mediated enhancement of mediator release. These observations provide strong evidence against the hypothesis that the differential effects of HgCl2 on BN and Lewis rat mast cells are mediated via IFN‐γ. In addition the results revealed that BN rat mast cells are significantly more sensitive than Lewis rat mast cells to the inhibitory effects of IFN‐γ on antigen‐induced mediator release.
Completely allogeneic spleen cells induced cytolytic neonatal tolerance to alloantigens, but failed to establish allo‐helper interactions with host T cells
Authors: RAMOS A. , GONZÁLEZ M. , LÓPEZ‐HOYOS M. , CARRIÓ R. , MERINO J.
Published: November 2003

The injection of spleen cells from F1 mice into newborns from a parental strain results in the establishment of cytolytic tolerance to donor alloantigens and the development of a lupus‐like disease. This syndrome is the consequence of the recognition by alloreactive host CD4+ T cells of discordant major histocompatibility complex (MHC) class II antigens on semi‐allogeneic donor B cells. We have analysed whether completely allogeneic spleen cells are as able as semi‐allogeneic spleen cells to induce cytolytic tolerance to donor alloantigens and to co‐operate with alloreactive T cells for autoantibody production. BALB/c mice were injected at birth with Thy‐1‐depleted spleen cells from (C57BL/6 × BALB/c)F1 or C57BL/6 mice, either alone or in combination. Cytolytic tolerance was always induced, as manifested by persistence of chimerism and acceptance of skin allografts. However, only F1 semi‐allogeneic B cells were activated by alloreactive host T cells to produce anti‐DNA IgG antibody. The deficient co‐operation between BALB/c CD4+ T cells and completely allogeneic C57BL/6 B cells was confirmed after neonatal injection of (C57BL/6 × BALB/c)F1 (Igha) spleen cells together with C57BL/6 (Ighb) spleen cells. These mice developed anti‐DNA antibodies bearing only the Igha allotype. Similar results were observed in experiments of allogeneic interaction in vitro, in which BALB/c CD4+ T cells were cocultured with either (C57BL/6 × BALB/c)F1 or C57BL/6 B cells. The present results demonstrate that completely allogeneic spleen cells efficiently induced cytolytic unresponsiveness to donor alloantigens, but B cells contained in this spleen cell population were unable to establish allo‐helper interactions with alloreactive CD4+ T cells, suggesting that cytolytic and helper T‐cell interactions involved in alloreactivity may be different.
Role of prolactin in the in vitro development of interleukin‐2‐driven anti‐tumoural lymphokine‐activated killer cells
Authors: MATERA L. , BELLONE G. , LEBRUN J.‐J. , KELLY P. A. , HOOGHE PETERS E. L. , FRANCIA DI CELLE P. , FOA R. , CONTARINI M. , AVANZI G. , ASNAGHI V.
Published: November 2003

Exogenous prolactin (PRL) has been shown to synergize with low‐dose interleukin‐2 (IL‐2) and induce the proliferation and lymphokine‐activated killer (LAK) maturation of natural killer (NK) cells. PRL itself can also generate LAK activity. Here we show that its local production occurs during, and is necessary for, LAK development. IL‐2‐stimulated peripheral blood mononuclear cells (PBMC) and purified NK cells were exposed to anti‐human (h)PRL antiserum, and residual LAK activity was measured on day 7 against the promyelocytic leukaemia cell line HL‐60. Inhibition of LAK activity was much more evident in PBMC compared with NK cell cultures (47% decrease, P=0.013 and 18.5% decrease, P=0.048, respectively). Up‐modulation of a 32S‐methionine‐labelled 27000MW protein was detected in the lysates and supernatants of IL‐2‐stimulated PBMC immunoprecipitated with an anti‐PRL antiserum. By contrast, the cytoplasmic PRL immunoreactivity observed in freshly isolated NK cells and in IL‐2‐stimulated, but not unstimulated, NK cell cultures was not associated with PRL gene activation, and can thus be referred to internalized PRL. Preferential re‐uptake of externally derived PRL by IL‐2‐stimulated NK cells was also indicated by up‐modulation of the PRL receptor. These data, as a whole, indicate that the PRL promotion of LAK differentiation is mainly mediated by paracrine secretion, with a minor contribution from internalized PRL.
Control of major histocompatibility complex class II expression on human monocytes by interleukin‐4: regulatory effect of lipopolysaccharide
Authors: HART P. H. , BONDER C. S. , JONES C. A. , FINLAY‐JONES J. J.
Published: November 2003

Interleukin‐4 (IL‐4), like interferon‐γ (IFN‐γ), stimulates monocyte major histocompatibility complex (MHC) class II expression and thus, by increasing antigen presentation, has the potential to increase immune reactivity. In this study, activation of human monocytes by lipopolysaccharide (LPS) prevented concomitant IL‐4 stimulation of MHC class II expression. However, this was not a general observation for activated monocytes because although the high levels of MHC class II antigen expressed by monocytes stimulated in vitro with IFN‐γ were not further regulated by IL‐4, the stimulatory effects of IL‐4 persisted on cells activated with granulocyte–macrophage colony‐stimulating factor and tumour necrosis factor‐α for enhanced MHC class II expression. MHC class II expression by monocytes cultured for 7 days with macrophage colony‐stimulating factor was regulated by IL‐4 and LPS in a manner very similar to that detected for freshly isolated monocytes. The inhibitory effect of LPS was not due to LPS‐induced production of IL‐10 or regulatory prostanoids. Furthermore, IFN‐γ‐increased MHC class II expression was suppressed by LPS, suggesting that the regulation was at the level of MHC class II expression per se. This study suggests that during Gram‐negative bacterial infections, IL‐4 and IFN‐γ may not be able to signal enhanced MHC class II expression and thus, enhanced immune reactivity.
3‐deazaadenosine analogues inhibit the production of tumour necrosis factor‐α in RAW264.7 cells stimulated with lipopolysaccharide
Authors: JEONG S.‐Y. , LEE J.‐H. , KIM H.‐S. , HONG S.‐H. , CHEONG C.‐H. , KIM I.‐K.
Published: November 2003

The effects of 3‐deazaadenosine (DZA), 3‐deaza(±)‐ aristeromycin (DZAri) and 3‐deazaneplanocin (DZNep) on tumour necrosis factor‐α (TNF‐α) production were examined in the mouse macrophage cell line, RAW264.7, stimulated with lipopolysaccharide (LPS). The 3‐deazaadenosine analogues inhibited the TNF‐α production and the inhibition was dependent upon the concentration of the analogue. DZA reduced the level of TNF‐α mRNA suggesting that DZA acts at a transcriptional step. In contrast, DZAri and DZNep had little effect on mRNA levels for TNF‐α, implying that these compounds inhibit a post‐transcriptional or translational biosynthetic step of TNF‐α synthesis. The observation that homocysteine (Hcy) potentiated the DZA inhibition of TNF‐α production and of TNF‐α mRNA levels suggests that the inhibition of TNF‐α production may be caused by elevated levels of 3‐deazaadenosylhomocysteine (DZAHcy). The results show that the 3‐deazaadenosine analogues are potent inhibitors of TNF‐α production in the RAW264.7 cell line stimulated with LPS and suggest that these analogues may be effective agents for the treatment of diseases in which TNF‐α plays an important pathogenic role.
Release of preformed Fas ligand in soluble form is the major factor for activation‐induced death of Jurkat T cells
Authors: MARTíNEZ‐LORENZO M. J. , ALAVA M. A. , ANEL A. , PIÑEIRO A. , NAVAL J.
Published: November 2003

Interaction of Fas/APO‐1 (CD95) and its ligand (FasL) plays an important role in the activation‐induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T‐cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non‐activated Jurkat cells, and also of L1210Fas, but not that of Fas‐negative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non‐activated counterparts. Time–course analysis revealed that PHA‐stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti‐Fas monoclonal antibody, suggesting that PHA‐stimulated Jurkat cells exert Fas‐based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non‐activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti‐FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of preformed FasL in soluble form upon stimulation.
Differential expression and function of CD80 (B7‐1) and CD86 (B7‐2) on human peripheral blood monocytes
Authors: FLEISCHER J. , SOETH E. , REILING N. , GRAGE‐GRIEBENOW E. , FLAD H.‐D. , ERNST M.
Published: November 2003

The interaction of CD28 with its ligands is important for T‐cell activation. Recent studies demonstrated the existence of at least two ligands on accessory cells, CD80 (B7‐1) and CD86 (B7‐2). In this study we demonstrate that, although CD80 and CD86 are both expressed on monocytes, they seem to have different functions. Freshly isolated monocytes express CD86 but are CD80‐negative. CD80 expression is weakly induced after 6–8 hr of in vitro culture and is enhanced by stimulation. CD86 expression is enhanced faster than CD80 expression and reaches the peak level after 4–6 hr in stimulated cells. Reverse transcription–polymerase chain reaction studies demonstrate that freshly isolated monocytes contain no CD80‐mRNA. The mRNA of CD80 is induced after 4–6 hr of culture, which matches with the expression of the protein. Inhibition studies using different antibodies against both molecules and the fusion protein CTLA4Ig show that only anti‐CD80 and CTLA4Ig could partially inhibit antigen‐specific (tuberculin) and polyclonal (anti‐CD3) lymphoproliferation and interferon‐γ (IFN‐γ) secretion of T cells cocultured with autologous monocytes. IFN‐γ secretion was more sensitive to blocking costimulation than proliferation. The antibody BB‐1 did not inhibit proliferation and cytokine secretion, nor did the anti‐CD86 clone IT2.2. CTLA4Ig, which binds both CD80 and CD86, has the same inhibitory capacity as the anti‐CD80 antibody tested. From those findings we conclude that human monocytes use CD80 as a costimulatory ligand for CD28 and utilize other costimulatory mechanisms besides those mediated via molecules of the B7 family.
MUCOSAL IMMUNOADJUVANT ACTIVITY OF LIPOSOMES: ROLE OF ALVEOLAR MACROPHAGES
Authors: DE HAAN A. , GROEN G. , PROP J. , VAN ROOIJEN N. , WILSHUT J.
Published: November 2003

Previously, we have reported on a liposomal adjuvant system for stimulation of both systemic IgG and mucosal s‐IgA responses against viral antigens (influenza virus subunit antigen or whole inactivated measles virus) administered intranasally to mice. Immune stimulation is observed with negatively charged, but not with zwitterionic, liposomes and is independent of a physical association of the antigen with the liposomes. Furthermore, liposome‐mediated immune stimulation requires deposition of the liposomes and the antigen in the lower respiratory tract. In the present study, it is shown that alveolar macrophages (AM) are the main target cells for negatively charged liposomes administered to the lungs of mice. AM isolated from animals, to which negatively charged liposomes were administered beforehand, showed large intracellular vacuoles, suggestive of massive liposome uptake. Under ex vivo conditions, both AM and RAW 264 cells exhibited a high capacity to take up negatively charged liposomes. The deposition of negatively charged liposomes, but not zwitterionic, liposomes in the lung reduced the phagocytic and migratory behaviour of AM, as assessed on the basis of transport of carbon particles to the draining lymph nodes of the lungs. Depletion of AM in vivo with dichloromethylene diphosphonate, facilitated an enhanced systemic and local antibody response against influenza subunit antigen deposited subsequently to the lower respiratory tract. In conclusion, these data provide support for the hypothesis that uptake of negatively charged liposomes blocks the immunosuppressive activity of AM, thereby facilitating local and systemic antibody responses.
A human CD4+ T‐cell line expresses functional CD64 (FcγRI), CD32 (FcγRII), and CD16 (FcγRIII) receptors but these do not enhance the infectivity of HIV‐1‐IgG complexes
Authors: MCLAIN L. , DIMMOCK N. J.
Published: November 2003

T cells do not generally express Fc receptors (FcRs). However, we report here that C8166 cells, a human CD4+ T lymphoblastoid cell line, widely used in research into the human immunodeficiency virus type 1 (HIV‐1), expressed CD64 (FcγRI), CD32 (FcγRII), and CD16 (FcγRIII) on the plasma membrane as shown by immunostaining with specific monoclonal antibody fragments. Another human CD4+ T lymphoblastoid cell line, H9, expressed none of these FcRs. C8166 cells bound monomeric normal rat serum IgG in a dose‐dependent manner, and when saturated bound heat‐complexed immunoglobulin G (IgG) also dose dependently. These observations are consistent with the presence on the C8166 T‐cell line of both high‐ and low‐affinity FcγRs. FcγRs are putative receptors for virus–IgG complexes, but in this study did not enhance infectivity of HIV‐1 complexed with a human neutralizing mAb or three rat neutralizing mAbs. Virus complexed with a non‐neutralizing mouse mAb was unable to infect cells using FcγRs as receptors after CD4 was blocked with a specific anti‐CD4 mAb.
HIV‐1‐specific cell‐mediated immunity is enhanced by co‐inoculation of TCA3 expression plasmid with DNA vaccine
Authors: TSUJI T. , FUKUSHIMA J. , HAMAJIMA K. , ISHII N. , AOKI I. , BUKAWA H. , ISHIGATSUBO Y. , TANI K. , OKUBO T. , DORF M. E. , OKUDA K.
Published: November 2003

We developed a candidate DNA vaccine designated pCMV160IIIB with pcREV (pCMV160IIIB/REV) that encodes gp160 of human immunodeficiency virus (HIV)‐1IIIB and Rev driven by the cytomegalovirus (CMV) promotor. This vaccine induced both HIV‐1‐specific antibodies and cytotoxic T lymphocyte (CTL) activity. In the present study, we inoculated the TCA3 expression plasmid into mouse skeletal muscle with pCMV160IIIB/REV to determine whether this cytokine expression plasmid was able to modify the immune response. Results of a delayed‐type hypersensitivity (DTH) assay using footpad swelling as well as those of a CTL assay clearly demonstrated that cell‐mediated immunity (CMI) elicited by co‐inoculation of pCMV160IIIB/REV with the TCA3 expression plasmid was markedly enhanced compared with that obtained using pCMV160IIIB/REV alone. When TCA3 expression plasmid was inoculated with anti‐TCA3 antibody, enhancement of the DTH response was suppressed below the level of that obtained with pCMV160IIIB/REV alone. The titre of HIV‐1‐specific IgG2a was slightly high when pCMV160IIIB/REV was co‐inoculated with this plasmid, suggesting that T‐helper 1 (Th1) response was predominant in TCA3‐inoculated mice. Infiltration of mononuclear cells was seen in the muscles at sites where TCA3 expression plasmid had been inoculated. Our present data suggest that TCA3 expression plasmid has potent adjuvant activity that results in an augmented CMI response.
Recovery from retrovirus‐induced immune suppression in BDP/J mice: dominance of the ‘regressor’ phenotype
Authors: GILMORE G. L.
Published: November 2003

Murine acquired immune deficiency syndrome (MAIDS) is an immunosuppressive disease of mice induced by infection with the LP‐BM5 murine leukemia virus (MuLV) retrovirus isolate. Certain inbred strains of mice are resistant to disease, but F1 crosses between sensitive and resistant strains are predominantly sensitive to MAIDS. One inbred strain, BDP, demonstrates a novel disease phenotype, recovery of immune function after a period of profound immune suppression. This trait is genetically dominant in crosses between BDP and either sensitive or resistant strains. The ‘regressor’ phenotype reveals the existence of a mechanism for recovery from immunosuppressive retrovirus infections, which may be of import in developing therapies for AIDS patients.
Induction of murine CD5 expression by v‐H‐ras
Authors: WEICHERT T. R. , SCHWARTZ R. C.
Published: November 2003

The murine CD5 surface antigen is a frequent marker on B‐lineage cell lines produced from bone marrow infected with retroviruses expressing v‐H‐ras. Since CD5+ B cells cannot be detected in adult murine bone marrow, either the viral targets of transformation are a minor contaminating population of CD5+ B‐lineage cells or the v‐H‐ras oncogene is inducing the expression of CD5 on B‐lineage cells not previously expressing this marker. We have found that v‐H‐ras can induce the expression of CD5 on two CD5− pre‐B‐cell lines established from murine bone marrow. This induction correlates with increased steady‐state levels of CD5 mRNA. These results present the possibility that CD5 expression may be modulated by specific signalling as well as early lineage commitment.
Isoproterenol regulates tumour necrosis factor, interleukin‐10, interleukin‐6 and nitric oxide production and protects against the development of vascular hyporeactivity in endotoxaemia
Authors: SZABÓ C. , HASKÓ G. , ZINGARELLI B. , NÉMETH Z. H. , SALZMAN A. L. , KVETAN V. , MCCARTHY PASTORES S. , VIZI E. S.
Published: November 2003

Pro‐inflammatory cytokines, such as tumour necrosis factor (TNF) and free radicals, such as nitric oxide (NO), are mediators of endotoxaemia. Catecholamines are in clinical use to treat the haemodynamic consequences of severe septic shock. Beta‐adrenergic agonists exert many of their effects by elevation of intracellular cyclic AMP (cAMP) concentration. Cyclic AMP can modulate endotoxin‐induced cytokine and NO production. Here we investigate the effect of isoproterenol pretreatment on the cytokine and NO production induced by bacterial lipopolysaccharide (LPS, 4–10 mg/kg). Pretreatment with isoproterenol (10 mg/kg) blunted the LPS‐induced TNF response, increased the LPS‐induced formation of interleukin‐10 and interleukin‐6 and reduced the LPS‐induced production of NO in conscious mice. In anaesthetized rats, pretreatment with isoproterenol prevented the LPS‐induced suppression of vascular contractility to norepinephrine in the thoracic aorta ex vivo. The hyporeactivity is due to expression of the inducible isoform of NO synthase (iNOS) and was restored by in vitro administration of NG‐methyl‐l‐arginine (l‐NMA), an inhibitor of NO synthase. However, l‐NMA did not alter vascular contractility in control vessels or in rings taken from the LPS‐treated rats pretreated with isoproterenol. Our findings suggest that, in addition to its haemodynamic actions, isoproterenol may also exert beneficial effects by modulating the endotoxin‐induced inflammatory response.
Human dendritic cells handling of binding, uptake and degradation of free and IgG‐immune complexed dinitrophenylated human serum albumin in vitro
Authors: LARSSON M. , BERGE J. , JOHANSSON A. G. , FORSUM U.
Published: November 2003

The handling of free and IgG‐complexed dinitrophenylated human serum albumin (DNP‐HSA) by human dendritic cells (DC) cultured with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) was studied. It has been shown that the amount of uncomplexed or IgG‐complexed antigen required by DC to start an immune response is low compared with other antigen‐presenting cells. We therefore examined whether such efficient presentation of immune complexes is due to an enhanced FcγRII‐mediated endocytosis or to a specialized and efficient antigen handling, i.e. macropinocytosis. The FcγRII expression was found to be heterogeneous on the GM‐CSF‐ and IL‐4‐cultured DC, i.e. it ranges from low to high expression. The handling of antigen and immune complexes revealed, that the level of binding and uptake of IgG–DNP‐HSA complexes by in vitro expanded DC is low compared with free antigen. Uncomplexed DNP‐HSA is probably handled either by endocytosis via receptors being more abundant and/or efficient than the FcγRII or via non‐receptor‐mediated endocytosis. The binding and uptake of IgG‐complexed DNP‐HSA was blocked by anti‐FcγRII antibody, indicating the specificity of the interaction.
Increase of heat‐shock protein and induction of γ/δ T cells in peritoneal exudate of mice after injection of live Fusobacterium nucleatum
Authors: SAITO K. , KATSURAGI H. , MIKAMI M. , KATO C. , MIYAMARU M. , NAGASO K.
Published: November 2003

Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans are Gram‐negative rod periodontal pathogens. The peritoneal cavity of Institute of Cancer Research (ICR) mice was used as the local infection model. In vivo production of heat‐shock proteins (hsp) was studied by injection of 1/10 minimum lethal dose (MLD) of each live bacteria into mice. Heat‐shock proteins 70 and 60 were examined in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with either F. nucleatum or A. actinomycetemcomitans by using sodium dodecylsulphate–polyacrylamide gel electrophoresis and immunoblotting analysis. Although hsp are present in PEC without injection of the bacteria, both hsp increased and reached a peak on day 3 after F. nucleatum injection but not after A. actinomycetemcomitans. Kinetic study of γ/δ T cells in PEC after injection of bacteria showed that the increase of γ/δ T cells was observed only in the PEC from mice injected with F. nucleatum but not A. actinomycetemcomitans. The γ/δ T cells in PEC were either CD3+ and CD4+ or CD3+ and CD8+. The differential cell count of PEC suggested that γ/δ T‐cell induction is related to the expansion of the macrophage population. The phagocytic and chemiluminescence responses of macrophages against the same bacteria were compared after intensive immunization with live F. nucleatum and A. actinomycetemcomitans. Elevations of chemiluminescence response and phagocytic function by immunization were observed in the macrophages of mice immunized with F. nucleatum. These results suggest the sequential appearance of hsp, γ/δ T cells and macrophage activation after fusobacterial infection.
Monocyte‐bound monoclonal antibodies inhibit the FcγRI‐mediated phagocytosis of sensitized red cells: the efficiency and mechanism of inhibition are determined by the nature of the antigen
Authors: SHEPARD S. L. , HADLEY A. G.
Published: November 2003

Monocyte‐binding monoclonal antibodies (mAbs) inhibited the Fcγ receptor I (FcγRI)‐mediated phagocytosis of red cells sensitized with human monoclonal immunoglobulin G (IgG) anti‐D (E‐IgG) via three distinct mechanisms depending on their specificity. First, all monocyte‐binding mAbs tested inhibited the adherence (and hence the phagocytosis) of E‐IgG. They also inhibited the binding of fluorescein isothiocyanate (FITC) conjugated IgG anti‐D. This inhibition of ligand binding was more efficiently promoted by murine (m) IgG2a than mIgG1 mAbs and presumably involved receptor blockade via the formation of antigen (Ag)–mAb–FcγRI complexes on the monocyte membrane. Monocytes passively sensitized with human monoclonal anti‐D (M‐IgG) were used in experiments to distinguish between inhibition of ligand binding and inhibition of phagocytosis. In this way, it was shown that mAbs to transmembrane molecules (CD11b/CD18, CD44, and HLA) inhibited the phagocytosis of red cells adherent to M‐IgG. Under the same conditions, mAbs to glycosylphosphatidylinositol (GPI) linked molecules (CD14, CD55 and CD59) did not inhibit phagocytosis. These data suggested a second mechanism of inhibition of FcγRI‐mediated phagocytosis that involved the cross‐linking of a proportion of FcγRI (i.e. those not ligated with IgG anti‐D) to molecules which are relatively constrained in the cell membrane. A third mechanism of inhibition was revealed by the use of F(ab′)2 fragments of mAb to CD11b which inhibited FcγRI‐mediated interactions with E‐IgG in a manner that did not involve IgG (Fc) crosslinking or blockade of FcγRI. In this respect, FcγRI‐mediated phagocytosis was more susceptible to inhibition than receptor‐mediated adherence.
Spontaneous priming for anti‐viral envelope cytotoxic T lymphocytes in mice transgenic for a murine leukaemia virus envelope gene (Fv4)
Authors: NIHRANE A. , SILVER J.
Published: November 2003

Compared with non‐transgenic controls, mice bearing an Fv4 murine retroviral env transgene resist infection and do not become immunosuppressed when inoculated with Friend virus (FV). When immunized with FV antigens in the absence of infectious virus, they make antibodies and cytotoxic lymphocytes (CTL) to FV comparably to non‐transgenic controls. Unimmunized transgenic mice were found to have CTL precursors, which could be activated by in vitro stimulation, specific for viral (and self) envelope protein (Env). This ‘spontaneous priming’ for antiviral CTL is surprising because the transgene Env is present on the surface of thymocytes and in serum from before birth. Our experiments demonstrate that T cells reactive with self‐thymic and serum antigens sometimes avoid clonal elimination or inactivation.
Environmental regulation of T‐cell function in mice: group housing of males affects accessory cell function
Authors: GREWAL* I. S. , HEILIG M. , MILLER A. , SERCARZ E. E.
Published: November 2003

The number of mice housed in a cage was found to exert a major impact on immune function in male mice. Lymph node cells from hen lysozyme (HEL)‐primed C57BL/6 male mice, housed in groups of 6/cage or individually, were tested for T‐cell proliferative response. Group‐housed males showed markedly lower responses than age‐matched females. However, in males housed singly for 4–15 weeks, responses were considerably higher, approaching those of female controls. To examine the cellular site of action of the housing effect, the efficiency of splenic antigen‐presenting cells (APC) was examined. APC from grouped males were considerably less efficient than APC from females, whereas males housed singly had increased APC function, reaching female levels. Our results demonstrate that environmental manipulation can profoundly modulate cellular immunity, and provide a first mechanistic indication that APC function is a major target for this modulation.
CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes
Authors: ONG C. J. , DUTZ J. P. , CHUI D. , TEH H.‐S. , MARTH J. D.
Published: November 2003

T‐cell development is arrested at the CD4+ CD8+ (DP; double‐positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T‐cell receptor (TCR) specific for the male antigen presented by H‐2Db (H‐Y TCR), the up‐regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)‐restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H‐Y TCR transgenic mice. It was found that in female H‐Y TCR transgenic mice, MHC‐restricted positive selection of CD4–CD8+ H‐Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4–CD8+ thymocytes that express H‐Y TCR.
Characterization of the mucosal cell‐mediated immune response in IL‐2 knockout mice before and after the onset of colitis
Authors: MCDONALD S. A. C. , PALMEN M. J. H. J. , VAN REES E. P. , MACDONALD T. T.
Published: November 2003

One of the major advances in the understanding of inflammatory bowel disease has been the observation that mice with immunoregulatory defects, such as interleukin‐2 knockout (IL‐2 –/–) mice, develop spontaneous gut inflammation. Here we have characterized the immune response in the ileum, caecum and colon of these mice before and after the onset of colitis by examining the cellular infiltrate, the cytokines produced by these cells and the mucosal vascular addressin MAdCAM‐1. IL‐2 –/– mice developed colitis after 35 days of age and before this the mice were apparently healthy. IL‐2 –/– mice aged over 35 days with colitis had large numbers of CD4+, CD8+, αβ T‐cell receptor (TCR)+ and γδ TCR+ T cells, macrophages, dendritic cells and MAdCAM‐1+ endothelial cells in the caecum and colon. This was associated with an increase in the number of interferon‐γ (IFN‐γ), IL‐1 and tumour necrosis factor‐α (TNF‐α) transcripts and a decrease in IL‐4 and IL‐10 transcripts. Treatment of IL‐2 –/– mice with cyclosporin A significantly delayed mortality. Interestingly, IL‐2 –/– mice under 35 days, although healthy, did show some subtle immunological signs of preclinical disease. There was a significant increase in the number of macrophages and dendritic cells in the colonic lamina propria and increased mRNA for IL‐1 and TNF‐α. There were also increased numbers of MAdCAM‐1+ endothelial cells, but IFN‐γ transcripts were not elevated. These results suggest that T‐cell‐mediated colitis in IL‐2 –/– mice may be secondary to an initial non‐specific inflammation.
Self‐reactive forbidden clones are confined to pathways of intermediate T‐cell receptor cell differentiation even under immunosuppressive conditions
Authors: MORODA T. , KAWACHI Y. , IIAI T. , TSUKAHARA A. , SUZUKI S. , TADA T. , WATANABE H. , HATAKEYAMA K. , ABO T.
Published: November 2003

It is believed that self‐reactive forbidden T‐cell clones are generated by ‘failure’ of the pathway of T‐cell differentiation in the thymus, if it is disturbed. We examined how such forbidden clones are generated under immunosuppressive conditions. Mice were treated with an injection of deoxyspergualin, FK506, or cycloporin A. From day 3, the number of cells yielded by various organs decreased. Because of the resistance of intermediate (int) T‐cell receptor (TCR) cells (i.e. TCRint cells), they became more prominent in proportion than TCRhigh cells. TCRhigh cells are conventional T cells generated through the mainstream in the thymus, whereas TCRint cells are primordial T cells generated by the extrathymic pathway or an alternative intrathymic pathway. Similar to untreated mice, forbidden Vβ3+ and Vβ11+ clones in C3H/He (Mls‐1b2a) mice were confined to TCRint cells after treatment; there was no leakage of forbidden clones into TCRhigh cells in the thymus and periphery. In parallel with the increase in the proportion of TCRint cells, the proportion of forbidden clones also increased under immunosuppressive states, especially in the liver. Liver mononuclear cells isolated from treated mice still had the potential to mediate autologous killing. The present results suggest that the generation of self‐reactive clones is highly restricted to the pathways of TCRint cell differentiation even under immunosuppressive conditions.
The roles of tumour necrosis factor‐α, interleukin‐1 and interleukin‐12 in murine cytomegalovirus infection
Authors: YERKOVICH S. T. , OLVER S. D. , LENZO J. C. , PEACOCK C. D. , PRICE P.
Published: November 2003

The roles of the inflammatory cytokines tumour necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1) and IL‐12, in murine cytomegalovirus (MCMV) disease were investigated in susceptible BALB/c and resistant C57BL/6 mice. MCMV infection induced IL‐1 and TNF‐α production by peritoneal cells from BALB/c mice, as demonstrated previously in C57BL/6 mice. Overt ill‐health and viral replication in the spleens of BALB/c mice were increased by in vivo treatment with soluble TNF‐α receptors to inhibit the activity of this cytokine, whilst antibodies to IL‐12 had a similar but more restricted effect. C57BL/6 mice were not affected by either treatment, suggesting TNF‐α and IL‐12 are not critical for natural killer cell‐mediated restriction of viral replication in the spleen. Soluble TNF‐α receptors and antibodies to IL‐12 also enhanced MCMV titres and numbers of viral antigen‐positive cells in the livers of BALB/c mice and TNF‐α receptors have similar effects in C57BL/6 livers. In contrast, IL‐1 receptors improved the health of MCMV‐infected BALB/c mice and reduced viral replication and hepatitis at some time‐points. Mechanisms which may underlie these changes are discussed.
Inhibitory effects of interleukin‐10 on synovial cells of rheumatoid arthritis
Authors: KAWAKAMI A. , EGUCHI K. , MATSUOKA N. , TSUBOI M. , URAYAMA S. , KAWABE Y. , AOYAGI T. , MAEDA K. , NAGATAKI S.
Published: November 2003

This paper describes the immunoregulatory effects of interleukin‐10 (IL‐10) on synovial cells in vitro. Synovial cells were cultured with IL‐10 in the presence or absence of various cytokines. Following incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analysed by an indirect immunofluorescence method and enzyme‐linked immunosorbent assay, respectively. We also examined the effect of IL‐10 on the function of synovial cells as antigen‐presenting cells (APC). Synovial cells spontaneously express several kinds of costimulatory molecule and produce various kinds of cytokines. Stimulation of synovial cells with interferon‐γ (IFN‐γ), IL‐1β, or 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) markedly enhanced the expression of costimulatory molecules and cytokine production of these cells. Both spontaneous and up‐regulated costimulatory molecule expression and cytokine production were significantly suppressed by the addition of IL‐10. Autologous T‐cell proliferation was stimulated by purified protein derivative (PPD) in IFN‐γ‐treated synovial cells and treatment of these synovial cells with IL‐10 also suppressed T‐cell proliferation. Our results suggest that IL‐10 has an inhibitory effect on synovial cells and is an important immunoregulatory component of the cytokine network in rheumatoid arthritis.
The anti‐erythrocyte autoimmune response of NZB mice. Identification of two‘qc distinct autoantigens
Authors: DIIULIO N. A. , FAIRCHILD R. L. , CAULFIELD M. J.
Published: November 2003

With age, New Zealand black (NZB) mice spontaneously develop anti‐mouse red blood cell (RBC) autoantibodies resulting in the development of autoimmune haemolytic anemia (AIHA). Previously, we characterized a panel of monoclonal autoantibodies derived from unimmunized, adult NZB mice. One of these antibodies (G8) was shown to be pathogenic, inducing AIHA in a non‐autoimmune‐prone mouse strain (BALB/c). Using G8, and two other antibodies from our panel, we have characterized two distinct autoantigens on the surface of mouse RBCs. The autoantigen, historically referred to as antigen X (AgX), was found to be partially hidden on the surface of the mouse RBC because glycosidase treatment or mild digestion with proteinase K resulted in increased reactivity with autoantibodies. One of the monoclonal antibodies (3H5G1) was found to immunoprecipitate a 110 000 MW protein identified as the erythrocyte anion transporter (band 3) whereas the pathogenic antibody (G8) as well as a third monoclonal antibody (2E6m) were shown to immunoprecipitate a 60 000 MW protein that was not reactive with the anti‐band 3 serum. Finally, we show that the autoantigen recognized by G8 is expressed on differentiated mouse erythroleukaemia (MEL) cells. The results suggest that a protein distinct from band 3 can serve as a target for AIHA in NZB mice.
Neutrophils from the synovial fluid of patients with rheumatoid arthritis express the high affinity immunoglobulin G receptor, FcγRI (CD64): role of immune complexes and cytokines in induction of receptor expression
Authors: A QUAYLE J. , WATSON F. , BUCKNALL R. C. , EDWARDS S. W.
Published: November 2003

Neutrophils isolated from the synovial fluid of 16/24 patients with rheumatoid arthritis expressed FcγRI (CD64), the high‐affinity receptor for monomeric immunoglobulin G (IgG), on their cell surface. Receptor expression ranged from 17% to 168% of the level of expression obtained after incubation of control blood neutrophils with 100 U/ml interferon‐γ (IFN‐γ) for 24 hr in vitro. Similarly, mRNA for FcγRI was detected in synovial fluid neutrophils from 12/15 patients and transcript levels ranged from 5% to 200% of the values obtained after treatment of blood neutrophils with IFN‐γ for 4 hr in vitro. No surface expression nor mRNA were detected in freshly isolated blood neutrophils from either patients or from healthy controls. Addition of cell‐free synovial fluid to control blood neutrophils induced both mRNA and surface expression of FcγRI to levels that were comparable to those achieved after addition of IFN‐γ. Neither soluble nor insoluble immune complexes appeared to be involved in induction of FcγRI expression in spite of the ability of these complexes to induce protein biosynthesis. Synovial fluid‐induced expression of FcγRI was partially blocked by incubation with neutralizing IFN‐γ antibodies, whilst neutralizing interleukin (IL)‐6 antibodies had little effect. Levels of IFN‐γ measured within these synovial fluids ranged from 0 to 2·7 U/ml, well within the range known to induce neutrophil FcγRI expression. These data thus indicate that gene expression in synovial fluid neutrophils is selectively activated as the cells enter the diseased joint. Furthermore, these data indicate that induced expression of FcγRI may alter the ability of infiltrating neutrophils to respond to IgG‐containing immune complexes present in these joints.
Pattern of cytokine receptors expressed by human dendritic cells migrated‘qc from dermal explants
Authors: LARREGINA A. T. , MORELLI A. E. , KOLKOWSKI E. , SANJUAN N. , BARBOZA M. E. , FAINBOIM L.
Published: November 2003

Different reasons account for the lack of information about the expression of cytokine receptors on human dendritic cells (DC): (a) DC are a trace population; (b) the proteolytic treatment used to isolate DC may alter enzyme‐sensitive epitopes; and (c) low numbers of receptors per cell. In the present work the expression of cytokine receptors was analysed by flow cytometry on the population of dermal DC (DDC) that spontaneously migrate from short‐term culture dermal explants. DDC obtained after dermal culture were CD1alow, CD1b+, CD1c+, human leucocyte antigen (HLA)‐DR+, CD11chigh, CD11b+ and CD32+. The DC lineage was confirmed by ultrastructural analysis. DDC expressed interleukin (IL)‐1R type 1 (monoclonal antibody (mAb) hIL‐1R1‐M1; and 6B5); IL‐1R type 2 (mAb hIL‐1R2‐M22); IL‐2R α chain (mAb anti‐Tac; and hIL‐2R‐M1) and IL‐2R γ chain (mAb 3B5; and AG14C). DDC did not stain for IL‐2R β chain using four mAbs recognizing two different epitopes of IL‐2Rβ (mAb 2R‐B; Mik‐β1; and CF1; Mik‐β3, respectively). DDC were also positive for the cytokine binding chains (α chains) of IL‐3R (mAb 9F5); IL‐4R (mAb hIL‐4R‐M57; and S456C9); and IL‐7R (mAb hIL‐7R‐M20; and R3434). DDC showed low levels of IL‐6R α chain (mAb B‐F19; B‐R6; and B‐E23) and its signal transducer gp130 (mAb A2; and B1). DDC strongly expressed interferon‐γ receptor (IFN‐γR) (mAb GIR‐208) and were negative for IL‐8R (mAb B‐G20; and B‐F25). All DDC were highly positive for granulocyte–macrophage colony‐stimulating factor receptor (GM‐CSFR) α chain (mAb hGM‐CSFR‐M1; SC06; SC04, and 8G6) and to a lesser extent for the common β chain of GM‐CSFR, IL‐3R and IL‐5R (mAb 3D7). On the other hand, reactivity was not found for granulocyte colony‐stimulating factor receptor (G‐CSFR) (mAb hGCSFR‐M1) nor macrophage colony‐stimulating factor receptor (M‐CSFR) (mAb 7‐7A3‐17) confirming the DC lineage of DDC. As previously reported for lymphoid DC, DDC expressed tumour necrosis factor receptort (TNFR) 75 000 MW (mAb utr‐1; hTNFR‐M1; and MR2‐1) but lacked TNFR 55 000 MW (mAb htr‐9; MR1‐1; and MR1‐2). In summary, DDC express receptors for a broad panel of cytokines, even receptors for cytokines whose effects on DC are still unknown (i.e. IL‐2Rαγ; IL‐6Rα/gp130; IL‐7Rαγ).