bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide‐mediated immunosuppression following murine Echinococcus multilocularis infection
Authors: DAI  , GOTTSTEIN
Published: March 2003

In some parasitic infections immunosuppression is a prominent characteristic of the host–parasite interplay. We have used a murine alveolar echinococcosis (AE) model in susceptible C57BL/6 mice to document a suppressed splenocyte proliferative response to concanavalin A (Con A) at the early (1‐month) stage and to Echinococcus multilocularis‐crude antigen (Emc‐antigen) at the late (4–6‐month) stage of chronic infection. Despite proliferative suppression, splenic cytokine production [interleukin‐2 (IL‐2), IL‐4 and interferon‐γ (IFN‐γ)] in response to Con A or Emc‐antigen stimulation was not suppressed at 1 month postinfection (p.i.). Infection resulted in a strong Mac‐1+ cell infiltration of the peritoneal cavity and spleen. Peritoneal cells (PEC) from mice infected at the 1‐month stage were rich in macrophages and expressed significantly higher levels of transcripts for the inflammatory cytokine IL‐1β and for tumour necrosis factor‐α and inducible nitric oxide synthase (iNOS), when compared with PEC from non‐infected control mice. Conversely, the IL‐10 transcript level remained low and did not change during infection. Spleen cells supplemented with PEC from infected mice induced a marked increase in the levels of nitrite in response to Con A and Emc‐antigen stimulation, and also a complete suppression of splenic proliferation. The spleen cells from late‐stage infected mice expressed only background levels of IL‐10 but greatly increased levels of iNOS, when compared with normal spleen cells. This observation correlated with the immunosuppression demonstrated at the late stage of murine AE. Furthermore, the suppressed splenic proliferative responses observed at the early and late stage were reversed to a large extent by the addition of NG‐monomethyl‐l‐arginine and partially by anti‐IFN‐γ. Thus, our results demonstrated that the immunosuppression observed in chronic AE was not primarily dependent on IL‐10 but rather on nitric oxide production by macrophages from infected animals.
Infection‐stimulated infraosseus inflammation and bone destruction is increased in P‐/E‐selectin knockout mice
Authors: KAWASHIMA  , NIEDERMAN  , HYNES  , ULLMANN‐CULLERE  , STASHENKO
Published: March 2003

Infections of the dental pulp commonly result in infraosseus inflammation and bone destruction. However, the role of phagocytic leucocytes in the pathogenesis of pulpal infections has been uncertain. In this work we used P/E−/− selectin‐deficient mice, which lack rolling adhesion of leucocytes to endothelium and mimic the human syndrome, leucocyte adhesion deficiency II (LAD‐II), to test the hypothesis that phagocytic leucocytes protect against pulpal infection and subsequent periapical infraosseus bone resorption. P/E−/− mice and P/E+/+ wild‐type controls were subjected to surgical pulp exposure, and both groups were infected with a mixture of pulpal pathogens including Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus micros and Streptococcus intermedius. Animals were killed after 20 days, and the extent of infraosseus bone destruction was quantified by histomorphometry. In two separate experiments, P/E−/− mice had significantly greater bone resorption than P/E+/+ controls. The increased bone destruction correlated with a twofold decrease in polymorphonuclear (PMN) infiltration into periapical nflammatory tissues of P/E−/− mice. P/E−/− mice had higher tissue levels of the bone resorptive cytokine, interleukin (IL)‐1α. Tissue levels of IL‐2, IL‐4, IL‐10, tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) were all higher in P/E−/− mice, but the increases were not statistically significant. Only IL‐12 was higher in P/E+/+ mice, possibly reflecting a greater number of infiltrating monocytes in wild‐type mice. These findings demonstrate that phagocytic leucocytes are protective in this model, and suggest that elevated expression of inflammatory cytokines is responsible for the observed bone destruction.
Human mannan‐binding lectin inhibits the infection of influenza A virus without complement
Authors: Kase  , Suzuki  , Kawai  , Sakamoto  , Ohtani  , Eda  , Maeda  , Okuno  , Kurimura  , Wakamiya
Published: March 2003

Mannan‐binding lectin (MBL) is a C‐type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagen‐like sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro‐organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV‐A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti‐human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non‐damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.
FcεRI‐mediated antigen endocytosis turns interferon‐γ‐treated mouse mast cells from inefficient into potent antigen‐presenting cells
Authors: Tkaczyk  , Villa  , Peronet  , David  , Mécheri
Published: March 2003

Previous studies in our laboratory have shown that bone‐marrow‐derived mast cells (BMMC) could present immunogenic peptides, from soluble antigens endocytosed through fluid phase, only if they were subjected to a 48‐hr treatment with interleukin‐4 (IL‐4) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). In contrast to GM‐CSF, interferon‐γ (IFN‐γ) which highly upregulates major histocompatibility complex (MHC) class II expression, completely inhibits the generation of immunogenic peptides. We have used this model to study the role of FcεRI‐mediated antigen internalization in the regulation of the antigen‐presenting function of IFN‐γ‐treated mast cells. Here, we report that FcεRI can reverse the IFN‐γ‐treated mast cells from inefficient to highly efficient antigen‐presenting cells. Inhibition of the antigen presenting capacity by piceatannol, a protein tyrosine kinase (PTK) syk inhibitor, indicates that this is an active process resulting from immunoglobulin E (IgE)–antigen–FcεRI engagement which involves tyrosines found in the immunoreceptor tyrosine‐based activation motif (ITAM) embedded in the cytoplasmic tail of the FcεRI β and γ chains. Antigen‐presenting function was also shown to require the activation of phosphatidyl inositol 3 (PI3) kinase, downstream of PTK syk phosphorylation, since this activity was completely blocked by wortmannin, a PI3 kinase inhibitor. These data suggest that signalling generated by FcεRI provides mast cells with IgE‐mediated enhanced antigen presentation to T cells and emphasize a so far unknown immunoregulatory mast‐cell function that might take place in inflammatory sites.
Potent immunostimulatory dendritic cells can be cultured in bulk from progenitors in normal infant and adult myasthenic human thymus
Authors: Hill  , Ferguson  , Austyn  , Newsom‐Davis  , Willcox
Published: March 2003

Low density cells can readily be enriched from thymus tissue both of children undergoing cardiac surgery and of older patients with myasthenia gravis, and can be cryostored in bulk. When fresh or thawed cells are cultured with granulocyte–macrophage colony‐stimulating factor and stem cell factor with or without tumour necrosis factor‐α (TNF‐α), they generate numerous cells with the characteristic ultrastructural, phenotypic and functional properties of dendritic cells. These proved to be very potent, both as stimulators of primary mixed leucocyte responses and as costimulators in oxidative mitogenesis. Especially after exposure to TNF‐α, these dendritic cells also processed a natural epitope from a 437‐residue polypeptide and presented it efficiently to an autoimmune T‐cell clone (of T helper type 0 phenotype). Thus, immunostimulatory dendritic cells can be cultured in relative abundance from progenitors in infant and adult human thymus. Both are convenient sources of potent antigen‐presenting cells of identifiable origins, e.g. for use in selecting human T‐cell lines.
Macrophage–tumour cell interactions: identification of MUC1 on breast cancer cells as a potential counter‐receptor for the macrophage‐restricted receptor, sialoadhesin
Authors: Nath  , Hartnell*  , Happerfield  , Miles  , Burchell  , Taylor‐Papadimitriou  , Crocker*§
Published: March 2003

In many carcinomas, infiltrating macrophages are commonly found closely associated with tumour cells but little is known concerning the nature or significance of adhesion molecules involved in these cellular interactions. Here we demonstrate in primary human breast cancers that sialoadhesin (Sn), a macrophage‐restricted adhesion molecule, is frequently expressed on infiltrating cells that often make close contact with breast carcinoma cells. To determine whether Sn could act as a specific receptor for ligands on breast cancer cell lines, binding assays were performed with a recombinant form of the protein fused to the Fc portion of human immunoglobulin G1 (IgG1) (Sn‐Fc). Sn‐Fc was found to bind specifically and in a sialic acid‐dependent manner to the breast cancer cell lines MCF‐7, T47.D and BT‐20 both in solid‐ and solution‐phase binding assays. To investigate the nature of the sialoglycoproteins recognized by Sn on breast cancer cells, MCF‐7 cells were labelled with [6‐3H]glucosamine. Following precipitation with Sn‐Fc, a major band of ≈240 000 MW was revealed, which was shown in reprecipitation and Western blotting experiments to be the epithelial mucin, MUC1.
Interleukin‐10‐induced CD8 cell proliferation
Authors: Rowbottom  , Lepper  , Garland  , Cox  , Corley  , Oakhill  , Steward
Published: March 2003

Interleukin (IL)‐10, a product of T helper 2 (Th2) lymphocytes, has been shown to be an important regulator of lymphoid and myeloid cells, inhibiting mitogen, peptide and alloantigen‐induced T‐cell proliferation and IL‐2 production. The microenvironment at the time of cell activation, notably the presence or absence of cytokines such as IL‐10, interferon‐γ (IFN‐γ) and IL‐2, is believed to determine the lineage and magnitude of cell‐mediated responses. In this study, we show that recombinant human IL‐10 (rhIL‐10) exerts a dose‐dependent inhibitory effect on human peripheral blood mononuclear cells stimulated in vitro, when these cells have not previously been exposed to rhIL‐10. Furthermore, incubation of these cells with high doses of rhIL‐10, either before or at the time of activation, results in inhibition which is followed several days later by the emergence of a population of CD8 positive cells. This rhIL‐10‐responsive CD8, positive cell population still emerges even when the cells are washed following incubation with rhIL‐10 prior to cell activation. Using purified CD8 populations this was shown to be a direct action of rhIL‐10 on CD8 cells and not via CD4 positive cells and monocytes. This finding was only observed when cells were activated with a cross‐linking anti‐CD3 antibody and not when activated with phorbol‐12‐mystrate‐13‐acetate (PMA) and calcium ionophore (CaIon), suggesting that the effect is mediated through cell‐surface receptors. Analysis of CD8 positive clones reveal production of Tc2 patterns of cytokines and reduced cell cytotoxicity to allogeneic, natural killer and lymphokine activated cell targets.
Exploring the feasibility of an anti‐idiotypic cocaine vaccine: analysis of the specificity of anticocaine antibodies (Ab1) capable of inducing Ab2βanti‐idiotypic antibodies
Authors: Schabacker D. S. , Kirschbaum K. S. , Segre M.
Published: March 2003

Conventional vaccination with the cocaine molecule conjugated to a protein carrier is a new approach in the treatment of addiction. Experimentally, this strategy has been shown to alter the pharmacokinetics as well as the psychostimulant effect of a cocaine challenge. The purpose of this study was to investigate whether a more stable and less controversial molecule, an anti‐idiotypic antibody, which mimics the configuration of the cocaine molecule (Ab2β), could be successfully used instead of cocaine. Two cocaine conjugates that presented different areas of the cocaine molecule to the immune system were used to produce monoclonal antibodies specific for cocaine (Ab1). Several anti‐idiotypic antibodies were then produced. Four were identified as Ab2β, or internal images of the antigen; when injected into BALB/c mice, they elicited an anticocaine response. The anticocaine response elicited by one of the four Ab2β (K1–4c) was sufficient to significantly reduce the level of cocaine that targeted the brain following cocaine challenge, compared with the level of cocaine found in the brain of control animals immunized with irrelevant antibody. In conclusion, the possibility of an anti‐idiotypic vaccine seems to be worth pursuing.
Regulation of T‐cell apoptosis: a mixed lymphocyte reaction model
Authors: O'flaherty E. , Wong W.‐K. , Pettit S. J. , Seymour K. , Ali S. , Kirby J. A.
Published: March 2003

Despite the capacity for antigen‐specific activation and rapid clonal expansion, homeostatic mechanisms ensure that the mature immune system contains a relatively stable number of T cells. In recent years, it has become apparent that this stability is a consequence of apoptotic death of most of the specific T cells generated during an immune response. Clearly this process must be tightly regulated in order to retain sufficient T‐cell progeny to mediate an effective response, whilst allowing the rapid deletion of these cells at the end of the response to prevent lymphadenopathy and cross‐reactive autoimmunity. In this study, the factors that regulate the sensitivity of T cells to apoptosis were investigated in vitro after the induction of primary T‐cell activation within a mixed lymphocyte reaction (MLR). It was found that activated T cells rapidly acquire the expression of both Fas and Fas ligand (FasL) on their surface and contain high levels of the precursor form of the pro‐apoptotic enzyme, caspase 8 (FLICE). However, these T cells were resistant for up to 5 days to apoptosis following the stimulation of Fas; a maximal apoptotic response was observed after 7 days. This time point coincided with a marked reduction in expression of the FLICE inhibitory protein (FLIP) and maximal activity of caspase 8. At time points beyond day 7, the number of viable cells in the MLR decreased further despite a reduction in the expression of FasL. However, the expression of interleukin‐2 (IL‐2) at these late time points was low, resulting in a decrease in expression of the anti‐apoptotic protein Bcl‐2. This can produce apoptosis by allowing leakage of cytochrome‐c from mitochondria resulting in direct activation of the caspase cascade. In this study, it is shown that T cells are resistant to apoptosis for the first 5 days after activation as a consequence of insensitivity of the Fas pathway and the presence of intracellular Bcl‐2. After between 5 and 7 days, the cells become sensitive to Fas‐mediated apoptosis while retaining Bcl‐2 expression. At later time points, Fas ligation is reduced but the cells respond to a decreased availability of IL‐2 by reducing Bcl‐2 expression; this encourages further apoptosis by allowing the direct activation of caspase enzymes.
Mechanisms of pertussis toxin‐induced myelomonocytic cell adhesion: role of CD14 and urokinase receptor
Authors: Li H. , Wong W. S. F.
Published: March 2003

Pertussis toxin (PTX) has been shown previously to promote myelomonocytic cell adhesion in serum. The aim of the present study was to identify, using transforming growth factor‐β1 and 1,25‐(OH)2 vitamin D3 (TGF‐β1/D3)‐primed U937 cells, the PTX‐binding site(s) and the adhesion molecule(s) responsible for PTX‐induced myelomonocytic cell adhesion. Monoclonal antibodies (mAbs) directed against CD14, CD11b, CD18 or urokinase receptor (uPAR) significantly inhibited PTX‐induced primed U937 cell adhesion in serum in a concentration‐dependent manner. However, only anti‐CD14 and anti‐CD18 mAbs were able to prevent the myeloid cells from binding to PTX‐coated plates and significantly inhibited a PTX‐induced rise of [Ca2+]i in primed U937 cells. A receptor‐isolation study showed that biotinylated PTX recognized a 48 000‐molecular weight protein in primed U937 cell lysates, which could be specifically blocked by excess unlabelled PTX or by anti‐CD14 mAb. On the other hand, mAb directed against uPAR significantly blocked PTX‐induced myeloid cell adhesion to serum and to immobilized vitronectin, a major extracellular matrix protein in serum. Taken together, our data suggest that PTX may bind to cell‐surface CD14 to induce myelomonocytic cell adhesion to vitronectin in serum via uPAR activation, which may represent a pathogenetic mechanism for the respiratory tract infection induced by Bordetella pertussis.
Human and rodent decay‐accelerating factors (CD55) are not species restricted in their complement‐inhibiting activities
Authors: Harris C. L. , Spiller O. B. , Morgan B. P.
Published: March 2003

Homologous complement activation is restricted on cells by the complement regulators, decay‐accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. These proteins act in concert with other membrane structures to protect cells from homologous complement attack. In contrast, cells are usually sensitive to heterologous complement attack. It has been suggested that species‐specific restriction of complement activation can be attributed to the inability of regulators to inhibit across species. We have investigated the capacities of human, rat and mouse analogues of DAF to regulate homologous and heterologous complement. Cells transfected with cDNA encoding these analogues were protected from heterologous complement attack. C3b‐deposition experiments indicated that whilst cells were best protected by DAF from the same species, all three analogues inhibited human, rat and mouse complement. Comparable results were obtained in haemolysis assays using soluble, recombinant forms of the proteins. Inhibition of the classical pathway (CP) was best achieved with homologous DAF, although human DAF also inhibited rat complement, rat DAF also inhibited human complement and mouse DAF inhibited complement from all species. Human DAF was the best inhibitor of alternative pathway (AP)‐mediated attack, inhibiting complement from all species. Mouse DAF inhibited mouse and rat AP, whilst rat DAF inhibited only rat AP. These data indicate that human and rodent analogues of DAF are not species restricted and highlights interesting differences in the capacity to regulate AP and CP. This has implications in broader fields of research, such as xenotransplantation, where cross‐species regulation of complement is of paramount importance.
T lymphocytes from granulocyte colony‐stimulating factor–/– mice produce large quantities of interferon‐γ in a chronic infection model
Authors: Mannering S. I. , Zhan Y. , Gilbertson B. , Lieschke G. J. , Cheers C.
Published: March 2003

Little is known about the role of granulocyte colony‐stimulating factor (G‐CSF) in the response to chronic bacterial infections. To address this we infected G‐CSF knock out (G‐CSF–/–) mice with Mycobacterium avium. Infection was not exacerbated in G‐CSF–/– mice despite a deficiency in the total bone marrow cells, colony‐forming haemopoietic cells, granulocytes and monocyte precursors in the bone marrow. Peritoneal cells from G‐CSF–/– produced less nitric oxide (NO) upon culture in vitro with antigen than did wild‐type (WT) cells. Unexpectedly, T cells from infected G‐CSF–/– mice were able to produce significantly more interferon‐γ (IFN‐γ) than the wild type (WT) controls. T cells from G‐CSF–/– mice still produced more IFN‐γ even when in vitro NO production was inhibited, while enzyme‐linked immunospot assay (ELISPOT) assays showed more IFN‐γ‐producing cells in the G‐CSF–/– mice. This was confirmed by intracellular cytokine staining (ICCS), which showed that there were more IFN‐γ producing T cells in vivo in the G‐CSF–/– than the WT controls following M. avium infection. It is possible that a deficit of NO in vivo allows T cells to develop a higher IFN‐γ‐producing phenotype. Thus we show a novel relationship between G‐CSF and IFN‐γ production by T cells revealed in this chronic bacterial infection model.
Reversal of ultraviolet radiation‐induced immune suppression by recombinant interleukin‐12: suppression of cytokine production
Authors: Schmitt D. A. , Walterscheid J. P. , Ullrich S. E.
Published: March 2003

Exposure to ultraviolet (UV) radiation, a complete carcinogen, suppresses the immune response. Data from a number of laboratories have indicated that one consequence of UV exposure is suppressed T helper type 1 (Th1) cell function with normal Th2 cell activation, resulting in a shift to a Th2‐like phenotype. The reversal of UV‐induced immune suppression and tolerance induction by recombinant interleukin‐12 (rIL‐12) supports this observation. The focus of this study was to determine the mechanism(s) by which rIL‐12 reverses UV‐induced immune suppression. Two possibilities were considered: up‐regulation of interferon‐γ (IFN‐γ) secretion by rIL‐12 and suppression of UV‐induced cytokine secretion by rIL‐12. To our surprise we found that the ability of rIL‐12 to overcome UV‐induced immune suppression was independent of its ability to up‐regulate IFN‐γ secretion. Rather, rIL‐12 suppressed the production of cytokines that are known to be important in UV‐induced immune suppression. Injecting UV‐irradiated mice with rIL‐12, or adding rIL‐12 to UV‐irradiated keratinocyte cultures suppressed IL‐10 secretion, in part by affecting the transcription of the IL‐10 gene. Furthermore, we found that rIL‐12 suppressed UV‐induced tumour necrosis factor‐α (TNF‐α) production. Because IL‐10 is involved in the UV‐induced suppression of delayed‐type hypersensitivity and TNF‐α in the UV‐induced suppression of contact allergy, these findings provide a mechanism to explain how rIL‐12 overcomes UV‐induced immune suppression in these related but different immune reactions. In addition, they suggest a novel mechanism by which rIL‐12 alters immune reactivity, direct suppression of cytokine secretion induced by UV radiation.
Interleukin‐4 and RANTES expression in maturing eosinophils derived from human cord blood CD34+ progenitors
Authors: Velazquez J. R. , Lacy P. , Mahmudi‐Azer S. , Bablitz B. , Milne C. D. , Denburg J. A. , Moqbel R.
Published: March 2003

Eosinophils elaborate a number of proinflammatory mediators, including immunoregulatory cytokines and chemokines. Interleukin (IL)‐4 and RANTES are important cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL‐4 and RANTES occurs in nascent eosinophils, leading to storage of newly produced proteins in crystalloid granule‐like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from purified cord blood CD34+ cells and generated in semisolid media (methylcellulose) in the presence of recombinant human (rh)IL‐3 and rhIL‐5. Cytokine mRNA profiles were analysed by the reverse transcription–polymerase chain reaction (RT–PCR) to determine transcription of IL‐4 and RANTES in cells on days 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine products and granule major basic protein (MBP) was confirmed, from day 23 onwards, for colonies cultured in semisolid media, by immunofluorescent labelling and confocal laser‐scanning microscopy (CLSM). We found that mRNA sequences encoding IL‐4 and RANTES were expressed in freshly prepared, non‐differentiated CD34+ cells. Furthermore, RANTES mRNA localized to carbol chromotrope 2R‐positive colony cells, as assessed using in situ RT–PCR on day 21 of culture in semisolid media, and was found to gradually decrease (relative to β2‐microglobulin) in rhIL‐3‐ and rhIL‐5‐treated colony cells (comprising > 90% eosinophil‐like cells) up to day 28. Immunoreactivity for IL‐4 and RANTES co‐localized with MBP in maturing colony eosinophils on day 23 of culture in semisolid media, as judged by CLSM. These results suggest that synthesis and storage of immunoregulatory cytokines, essential for processes associated with adaptive immunity, occurs in nascent eosinophils during their growth and differentiation.
Cellular interactions in bovine tuberculosis: release of active mycobacteria from infected macrophages by antigen‐stimulated T cells
Authors: Liébana E. , Aranaz A. , Aldwell F. E. , Mcnair J. , Neill S. D. , Smyth A. J. , Pollock J. M.
Published: March 2003

The outcome of Mycobacterium bovis infections depends on the interactions of infected macrophages with T lymphocytes. Several studies in humans and in mouse models have suggested an important role for cytotoxicity in the protective immune response to mycobacterial infections, and both CD4+ and CD8+ T cells have been shown to elicit appropriate cytolytic activity. The present study investigated in vitro interactions of T cells with M. bovis‐infected macrophages in bovine tuberculosis. The results showed that following interaction with antigen‐stimulated peripheral blood mononuclear cells (PBMC) from infected cattle, there was an increased presence of M. bovis in the extracellular compartment of infected macrophage cultures, as measured by incorporation of [3H]uracil into mycobacterial RNA. Furthermore, out of a panel of T‐cell clones from infected cattle, it was found that a higher proportion of CD8+ clones produced an increase in the number of metabolically active extracellular M. bovis organisms compared with CD4+ clones. Finally, a positive correlation between percentage of antigen‐dependent release of mycobacteria and total uracil uptake by M. bovis within culture systems was detected. This could be regarded as an indication of preferential intracellular control of mycobacteria by activated macrophages.
Activation of bovine B cells via surface immunoglobulin M cross‐linking or CD40 ligation results in different B‐cell phenotypes
Authors: Haas K. M. , Estes D. M.
Published: March 2003

Experiments reported herein demonstrate that activation of bovine B cells via surface immunoglobulin M (sIgM) cross‐linking, analogous to T‐cell independent (TI‐2) antigenic stimulation, results in the expression of CD5. Interestingly, in the presence of CD40 ligand, sIgM‐mediated induction of CD5 on B cells was inhibited. These findings indicate that activation of bovine B cells via B‐cell receptor (BCR) cross‐linking results in a CD5+B‐cell phenotype and that CD40 signalling is inhibitory to this process. Analysis of cytokine mRNA indicates that bovine B cells constitutively express tumour necrosis factor‐α (TNF‐α) and interleukin (IL)‐1β transcriptsin vitro, while IL‐10 mRNA expression is induced following sIgM cross‐linking. IL‐12 p40 transcripts were produced by B cells activated by CD40, but not by BCR, ligation. Analysis of cytokine receptor mRNA indicates that activation through CD40, in the presence or absence of IgM cross‐linking, results in increased IL‐4 receptor‐α (IL‐4Rα), IL‐13Rα1 and interferon‐α receptor 1 (IFN‐αR1) mRNA levels. Overall, these findings suggest that activation of bovine B cells through BCR cross‐linking yields an activation phenotype that differs substantially from that of B cells activated through CD40.
Monoclonal anti‐double‐stranded DNA autoantibody stimulates the expression and release of IL‐1β, IL‐6, IL‐8, IL‐10 and TNF‐α from normal human mononuclear cells involving in the lupus pathogenesis
Authors: Sun K.‐H. , Yu C.‐L. , Tang S.‐J. , Sun G.‐H.
Published: March 2003

In our previous reports, we found polyclonal anti‐double‐stranded DNA antibodies (anti‐dsDNA) purified from patients with active systemic lupus erythematosus (SLE) exerted inhibitory effect on [3H]thymidine incorporation of human mononuclear cells (MNC). However, the other immunological effects of anti‐dsDNA on the functions of MNC have not yet been reported. In this study, two monoclonal antibodies, 12B3 and 9D7, with different anti‐dsDNA activity were evaluated for their effects on the expression and release of different cytokines from human MNC. We confirmed absence of endotoxin in the two monoclonal antibody preparations and the used medium as detected by Limulus amoebocyte lysate test. The mRNA expression and release of different cytokines including interleukin (IL)‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) were measured. We found the two monoclonal anti‐dsDNA not only dose‐responsively suppressed the phytohaemagglutinin (PHA)‐induced thymidine uptake of human MNC but stimulated the mRNA expression of IL‐1β, IL‐6 and IL‐8 in normal human MNC detected by reverse transcription–polymerase chain reaction (RT–PCR). Enzyme‐linked immunosorbent assay (ELISA) measurement of cytokines in MNC culture supernatants revealed that anti‐dsDNA enhanced IL‐1β, IL‐8, TNF‐α and IL‐10 release from resting MNC. These effects of anti‐dsDNA antibodies were not affected by polymyxin B, a potent binder and neutralizer of lipopolysaccharide (LPS). These in vitro studies suggest that anti‐dsDNA possess a dual effect on normal human MNC: (a) to enhance the release of proinflammatory cytokines (IL‐1β, IL‐8 and TNF‐α) from MNC to augment inflammatory reaction; and (b) to polarize the immune reaction towards the T helper 2 (Th2) (increased IL‐10 production) pathway. This unique effect of anti‐dsDNA may play a role in lupus pathogenesis by augmenting inflammatory reactions and autoantibody production which are commonly found in patients with active SLE.
Evidence for multiple CD95–CD95 ligand interactions in anteriorchamber‐associated immune deviation induced by soluble protein antigen
Authors: Kezuka T. , Streilein J. W.
Published: March 2003

We have investigated whether CD95–CD95 ligand interactions are important in anterior chamber‐associated immune deviation (ACAID) induced by soluble protein antigen, and if so, to identify the participating cells on which these molecules are expressed. Peritoneal exudate cells as antigen‐presenting cells (APC) obtained from B6.lpr/lpr, B6.gld/gld and C57BL/6 mice were cultured with ovalbumin (OVA) and transforming growth factor‐β2 (TGF‐β2) overnight, then injected intravenously into C57BL/6 or B6.lpr/lpr recipients. Some B6.lpr/lpr mice were reconstituted with naive T cells from wild‐type C57BL/6 donors. In other experiments, B6.lpr/lpr and B6.gld/gld mice received an anterior chamber injection of OVA followed 7 days later by subcutaneous immunization with OVA plus adjuvant. Delayed hypersensitivity (DH) was assessed with an ear swelling assay. T cells activated in vitro with OVA‐pulsed, TGF‐β‐treated APC were tested in vivo for their capacity to suppress DH expression in a local adoptive transfer assay. The results indicate that when ACAID was induced by in‐vitro generated ACAID‐inducing cells, the APC expressed CD95L, and recipient T cells expressed CD95. The capacity of in vitro generated regulatory T cells to suppress DH expression to OVA in vivo was not governed by CD95–CD95L interactions. When OVA was injected into the anterior chamber of naive mice, CD95 expression was required for ACAID induction, although ACAID was readily induced in CD95L‐deficient mice. We conclude that CD95–CD95L interactions are required in ACAID for the initial stage of APC presentation of eye‐derived antigens to T cells, and that CD95–CD95L interactions participate at one or more additional step in the process by which ACAID is induced by soluble protein antigens.
Analysis of histamine‐producing cells at the late phase of allergic inflammationin rats
Authors: Shiraishi M. , Hirasawa N. , Oikawa S. , Kobayashi Y. , Ohuchi K.
Published: March 2003

To identify histamine‐producing cells at the late phase of allergic inflammation, the expression of l‐histidine decarboxylase (HDC) was examined in the infiltrating leucocytes in the inflammatory locus. HDC activity and HDC mRNA levels in the infiltrating leucocytes in the pouch fluid of the immunized rats (that were injected with the antigen solution into the air pouch) were increased compared with those in the infiltrating leucocytes of the non‐immunized rats. When infiltrating leucocytes collected 8 hr after antigen injection were cultured, histamine production by the cells from the immunized rats was higher than that from the non‐immunized rats. In situ hybridization of HDC mRNA revealed that almost all the infiltrating leucocytes of the immunized rats, 4 hr after injection of the antigen, expressed HDC mRNA with high intensity, while those of the non‐immunized rats showed only a weak intensity of HDC mRNA. In the immunized rats, ≈ 90% of leucocytes infiltrating in the pouch fluid at 4 hr were neutrophils and 8% were monocytes/macrophages. Neither mast cells nor basophils were detected in the infiltrating leucocytes. When rat peritoneal neutrophils were incubated in the presence of 12‐O‐tetradecanoylphorbol 13‐acetate, histamine production was significantly increased. These findings suggest that the leucocytes, mainly neutrophils, infiltrating at the inflammatory locus are responsible for histamine production at the late phase of allergic inflammation.
Adjuvant effects of CpG oligodeoxynucleotides on responses against T‐independent type 2 antigens
Authors: Kovarik J. , Bozzotti P. , Tougne C. , Davis H. L. , Lambert P.‐H. , Krieg A. M. , Siegrist C.‐A.
Published: March 2003

Oligodeoxynucleotides containing CpG motifs (CpG‐ODN) are potent in vitro B‐cell activators and they have been successfully used to increase in vivo antibody responses to T‐dependent peptide and protein antigens. In contrast, the use of CpG‐ODN to enhance in vivo antibody responses to various T‐independent type 2 (TI‐2) antigens has recently generated contradictory results. In this study, we compared the CpG‐ODN stimulatory effect on antibody responses of adult and young BALB/c mice to trinitrophenylaminoethyl‐carboxymethyl (TNP) ‐Ficoll and to polysaccharides (PS) from several distinct serotypes of Streptococcus pneumoniae (SPn). CpG‐ODN co‐administration significantly enhanced antigen‐specific immunoglobulin M (IgM), IgG, IgG1 and IgG2a titres to TNP‐Ficoll. The depletion of CD4+ cells by monclonal antibodies (GK1.5) identified their essential role in CpG‐ODN‐mediated enhancement of antibody responses. In contrast to TNP‐Ficoll, CpG‐ODN failed to enhance IgM and IgG responses to any of the 18 SPnPS serotypes tested. Providing T‐cell epitopes by the conjugation of SPnPS to the carrier protein tetanus toxoid again allowed CpG‐ODN to mediate enhancement of IgG, IgG2a and IgG3 responses to most SPnPS serotypes. Thus, antigen‐presenting cell/T‐cell interaction appears to largely mediate the in vivo influence of CpG‐ODN on antibody responses to TI‐2 antigens. In early life, additional factors limit CpG‐ODN modulation of antibody responses to TI‐2 antigens.
The immune dysregulatory compound mercuric chloride induces integrin‐mediated T‐lymphocyte adhesion
Authors: Roos A. , Neeft M. , Engelen L. , Schilder‐Tol E. J. M. , Kunzendorf U. , Weening J. J. , Aten J.
Published: March 2003

Exposure of Brown Norway rats to mercuric chloride induces systemic autoimmunity, involving T‐ and B‐lymphocyte activation, (auto‐)antibody production and multiorgan inflammation. Several divalent metal ions, such as Mg2+ and Mn2+, can activate binding of integrins to their ligands, thus causing lymphocyte adhesion. To test the hypothesis that Hg2+ acts in a similar way, we studied the effect of HgCl2 on integrin‐mediated T‐cell adhesion. HgCl2 induced cell–cell aggregation of human T lymphoblasts. Exposure of a human T‐cell clone to HgCl2 for 1 hr enhanced, in a dose‐dependent way, cell binding to fibronectin (FN) and to intercellular adhesion molecules (ICAM) ‐1, ‐2 and ‐3. Furthermore, HgCl2 induced strong binding of Jurkat T cells to FN. These effects of HgCl2 were of similar magnitude as the effects of phorbol 12‐myristate 13‐acetate (PMA) or MnCl2. Studies using blocking antibodies indicated the involvement of CD11a in binding to ICAMs, and of CD49d, CD49e, and CD29 in binding to FN. Adhesion to FN induced by HgCl2 or by PMA, but not by MnCl2, was dependent on temperature and on extracellular Ca2+ or Mg2+. Addition of cytochalasin B enhanced synergistically the FN adhesion induced by MnCl2, whereas the effects of PMA and HgCl2 were not modified. These results indicate that Hg2+ is a potent activator of T‐cell adhesion, mediated by several integrins and ligands. In contrast to the effect of MnCl2, HgCl2‐induced cell adhesion probably involves an intracellular pathway. Activation of integrins by HgCl2 may play an important role in activation and migration of leucocytes involved in HgCl2‐induced immune dysregulation in vivo.
Zymosan enhances the immune response to DNA vaccine for human immunodeficiency virus type‐1 through the activation of complement system
Authors: Ara Yuki , Saito Tomoyuki , Takagi Toshitaka , Hagiwara Eri , Miyagi Yohei , Sugiyama Mitsugu , Kawamoto Susumu , Ishii Norihisa , Yoshida Takuji , Hanashi Daisuke , Koshino Tumihisa , Okada Hidechika , Okuda Kenji
Published: March 2003

In the present study, the adjuvant effect of zymosan on human immunodeficiency virus type‐1 (HIV‐1)‐specific DNA vaccine and the mechanism of this enhancement were studied in a murine model. We coinoculated zymosan with our candidate HIV‐1 specific DNA vaccine (pCMV160IIIB) into skeletal muscles of BALB/c mice. Higher levels of both humoral immune response and HIV‐specific delayed‐type hypersensitivity (DTH) response were observed when zymosan was coinoculated with pCMV160IIIB compared with that obtained using pCMV160IIIB alone. HIV‐specific cytotoxic T lymphocyte (CTL) activity was also enhanced. This enhancing activity was suppressed when coinoculated to the fifth complement (C5)‐deficient DDD and AKR mice. The enhanced activity was also suppressed when anti‐C3 antibody was inoculated to mice intramuscularly. There was significant induction of immunoglobulin G2a (IgG2a) and interferon‐γ (IFN‐γ) in pCMV160IIIB vaccine with zymosan. These results suggest that zymosan‐mediated DNA vaccination enhances helper T cell (Th) 1‐mediated immunity. The effect is suggested to be based on the consequences of its recruitment and activation of macrophages, dendritic cells or antigen‐presenting cells (APC) through complement activation, especially through the alternative pathway. Taken together, these results suggest that zymosan can be an effective immunological adjuvant in DNA vaccination against HIV‐1.
Tumours can act as adjuvants for humoral immunity
Authors: Brown D. M. , Fisher T. L. , Wei C. , Frelinger J. G. , Lord E. M.
Published: March 2003

Tumour cells transfected with cDNAs encoding non‐self proteins were used to investigate the ability of the immune system to respond to immunogenic antigens expressed by tumours. Secreted, intracellular and surface proteins were used as model antigens, as these reflect the potential forms of tumour antigens. Syngeneic BALB/c mice injected with viable line 1 lung carcinoma or EMT6 mammary tumour cells secreting ovalbumin (OVA) or prostate‐specific antigen (PSA) produced very high immunoglobulin G (IgG) antibody titres, equivalent to those of mice injected with protein in Freund's complete adjuvant (FCA). Secretion of the antigens was not necessary as tumour cells expressing a cell‐surface antigen (HER‐2/Neu) or an intracellular antigen – green fluorescence protein (GFP) – also generated high‐titre antigen‐specific IgG antibodies. In interleukin‐4 (IL‐4)‐deficient mice, both IgG1 and IgG2a were produced in response to OVA administered in FCA, whereas in response to tumour‐produced antigen, the antibodies switched from predominantly IgG1 to IgG2a, indicating that the mechanisms responsible for antibody induction differed between these forms of immunization. In contrast to the line 1 and EMT6 tumours, which are of BALB/c origin, OVA‐ or PSA‐producing B16 melanoma cells, which are of C57BL/6 origin, failed to elicit antibody production. This was not the result of strain differences, as a similar finding was observed when the tumours were grown in (BALB/c × C57BL/6)F1 mice, but appeared to be caused by intrinsic differences in the tumours. Furthermore, co‐injection of both B16/OVA and line 1 tumours resulted in production of anti‐OVA antibody, indicating that B16 tumours were not immunosuppressive, but instead line 1 tumours appear to exert an adjuvant effect.
Glycyrrhizin enhances interleukin‐12 production in peritoneal macrophages
Authors: Dai Ji Hong , Iwatani Yasumasa , Ishida Takaomi , Terunuma Hiroshi , Kasai Hirotake , Iwakula Yoichiro , Fujiwara Hiromi , Ito Masahiko
Published: March 2003

Interleukin‐12 (IL‐12) is a monocyte/macrophage‐derived cytokine that plays a prominent role in the development of T helper type 1 (Th1) cell‐mediated immune responses. Glycyrrhizin (GL), an aqueous extract of liquorice root, used as Chinese medicine, is known to have various immunomodulating activities. In this study, GL showed a dose‐dependent priming effect on lipopolysaccharide (LPS)‐induced IL‐12 p40 and IL‐12 p70 (heterodimer of p40 and p35) protein production by peritoneal macrophages (PM). The maximal effect was observed when GL was intraperitoneally administered 12 hr before the PM were harvested and stimulated in vitro with LPS. The increases in IL‐12 p70 and p40 protein production were primarily due to up‐regulated transcription of IL‐12 p35 and p40 messenger RNAs (mRNAs), as demonstrated by RNase protection assay. The augmentation of IL‐12 p40 mRNA expression induced by GL pretreatment was associated with increased NF‐κB activation. Moreover, GL exhibited the same priming effect on IL‐12 production in interferon‐γ knockout (IFN‐γ–/–) mice. The production of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was not induced at any time point after GL pretreatment. These findings demonstrated the ability of GL to enhance LPS‐induced IL‐12 production by peritoneal macrophages, and indicated that the priming effect of GL on IL‐12 production was independent of both IFN‐γ and GM‐CSF.
Lipopolysaccharide and phorbol 12‐myristate 13‐acetate both impair monocyte differentiation, relating cellular function to virus susceptibility
Authors: Basta Sameh , Knoetig Sonja , Summerfield Artur , McCullough Kenneth C.
Published: March 2003

Both lipopolysaccharide (LPS) and phorbol 12‐myristate 13‐acetate (PMA) impeded monocyte to macrophage differentiation with respect to typical phenotypic modulation and certain phagocyte‐related processes. The down‐regulation of the porcine monocyte marker SWC1, and up‐regulation of the SWC9 macrophage marker were retarded, but not inhibited, as was the differentiation‐associated down‐regulation of p53 and myeloperoxidase. Despite this clear impairment of macrophage differentiation, not all cellular functions were equally susceptible. Both agents inhibited phagocytosis, but not low‐density lipoprotein receptor‐associated endocytosis. Only LPS inhibited tartrate‐resistant acid phosphatase up‐regulation. In contrast, increase of vacuolar acidification rates was more susceptible to PMA. The activity of certain endosomal/lysosomal enzymes – esterase, nucleotidase, peroxidase and cathepsins – was generally enhanced by both LPS and PMA. This contrasted with autophagosomal activity, detected through the induction of an antiviral state. Disruption of autophagosomes and lysosomes (methionine‐O‐methyl ester), but not lysosomes alone (glycyl‐l‐phenylalanine) reversed LPS‐induced inhibition of virus replication, without influencing the PMA‐induced antiviral effect. Thus, PMA is similar to LPS in inhibiting monocyte to macrophage differentiation, when primary blood monocytes are employed, but not all pathways are equally susceptible. The analyses demonstrate that the pathways modulated during monocyte differentiation function somewhat independently. Moreover, certain functions of monocytic cells are more important with respect to the outcome of virus infection, with autophagosomal activities in particular favouring cell survival.
Enumeration of hepatitis B surface antigen‐specific B lymphocytes in responder and non‐responder normal individuals vaccinated with recombinant hepatitis B surface antigen
Authors: Shokrgozar Mohammad A. , Shokri Fazel
Published: March 2003

Determination of the frequency of specific B lymphocytes has important implications for investigation of the immune response to different antigens and pathogens. Unresponsiveness to some viruses and antigens, such as hepatitis B virus (HBV) and its surface antigen (HBsAg), has been attributed to lack or insufficient production of the specific B‐cell repertoire. In this study, peripheral blood B lymphocytes of 45 adult normal individuals vaccinated with recombinant hepatitis B vaccine were transformed with Epstein–Barr virus (EBV) and cultured at different dilutions on human fetal fibroblasts as a feeder layer. The vaccinees were classified into good, poor and non‐responder groups. Following 2 to 3 weeks of incubation, culture supernatants were collected from wells containing transformed and proliferating B lymphocytes. The supernatants were subsequently screened for the presence of total immunoglobulin and antibody to HBsAg (anti‐HBs) by enzyme‐linked immunosorbent assay (ELISA). Accordingly, positive and negative wells were enumerated in each plate and the frequency of B lymphocytes producing anti‐HBs antibody was estimated based on the Poisson statistical analysis. The total number of CD19+ B lymphocytes were counted in the peripheral blood of all subjects by flow cytometry. Our results demonstrated a similar precursor frequency of specific B lymphocytes in all subject groups before vaccine administration (< 2 × 10−5). Following vaccination, however, a significant increase in the number of specific B lymphocytes was observed in good‐responder (1·5 × 10−4) and to a lesser extent poor‐responder (3·5 × 10−5) individuals, but not in non‐responders. These findings suggest a defect in either the primary B‐cell repertoire or helper T‐cell function in non‐responder individuals.