bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific inhibition of immunoglobulin E‐mediated allergic reaction using antisense FcεRIα oligodeoxynucleotides
Authors: KIM  , KIM  , LEE
Published: March 2003

We have investigated the ability of an antisense immunoglobulin E (IgE) receptor α‐subunit oligodeoxynucleotide (FcεRIα ODN) specifically to inhibit IgE‐mediated allergic reactions in the mouse. Synthetic antisense FcεRIα ODN dose‐dependently inhibited passive cutaneous anaphylaxis and histamine release from the mouse peritoneal mast cells (MPMC) activated by anti‐dinitrophenyl (DNP) IgE. Northern blot analysis showed that the mast cells treated with antisense FcεRIα ODN exhibited no detectable levels of l‐histidine decarboxylase mRNA after anti‐DNP IgE stimulation, whereas the cells treated with sense FcεRIα ODN possessed significant amounts of this mRNA. Examination of the elevation of cAMP levels in MPMC following the activation with anti‐DNP IgE demonstrated a significant rise in activated cells, but not in the antisense FcεRIα ODN‐treated cells. Moreover, antisense FcεRIα ODN had a significant inhibitory effect on anti‐DNP IgE‐induced tumour necrosis factor‐α production. Our results demonstrated that antisense FcεRIα ODN inhibited the IgE‐mediated allergic reaction in vivo and in vitro.
Characterization of a monoclonal antibody that recognizes a lymphocyte surface antigen for the cetacean homologue to CD45R
Authors: De Guise S. , Erickson K. , Blanchard M. , Dimolfetto L. , Lepper H. , Wang J. , Stott J. L. , Ferrick D. A.
Published: March 2003

As part of our current efforts to develop assays and reagents to study the immune system of marine mammals, and in view of the effort currently made to develop monoclonal antibodies to cell surface proteins of lymphocyte subsets in different species, the present paper reports on the characterization of a monoclonal antibody against the homologue of CD45R on cetacean lymphocytes. The specificity of this antibody has been characterized on the basis of immunoprecipitation of the antigen it recognized, immunoperoxidase staining on cetacean lymph node and thymus sections, as well as one and two‐colour flow cytometric analysis of cetacean peripheral blood mononuclear cells and single‐cell suspensions of thymus, lymph node and spleen. Anti‐cetacean CD45R (F21.H) immunoprecipitated proteins of 180, 200 and 220×103 MW, with the 180×103 MW form being predominantly expressed on T cells and the 220×103 MW form expressed predominantly on B cells and thymocytes. F21.H labelled all B cells and a proportion of T cells on single‐cell suspensions of spleen cells. CD45R− killer whale peripheral blood lymphocytes expressed a higher density of CD2 than CD45R+, a characteristic of memory T cells. Killer whale T lymphocytes also lost the expression of CD45R upon activation with concanavalin A (Con A) and phytohaemagglutinin (PHA). This is the first report of a monoclonal antibody to CD45R in cetaceans, and this antibody is foreseen as a possible valuable diagnostic and research tool to assess immune functions of captive and wild cetaceans as part of the evaluation of their health status.
Novel mode of action of the calcium antagonist mibefradil (Ro 40‐5967): potent immunosuppression by inhibition of T‐cell infiltration through allogeneic endothelium
Authors: Blaheta R. A. , Hailer N. P. , Brude N. , Wittig B. , Oppermann E. , Leckel K. , Harder S. , Scholz M. , Weber S. , Encke A. , Markus B. H.
Published: March 2003

Cyclosporin A reduces the mitotic activity of allosensitized lymphocytes, but fails to limit emigration of these cells into the donor organ. However, the modulation of both lymphocyte proliferation and infiltration are desirable characteristics of immunosuppressive therapy. The calcium‐channel blocker, verapamil, has recently been shown to effectively prevent the transmigration of CD4+ and CD8+ T cells through allogeneic endothelium. Mibefradil (Ro 40‐5967) represents a new generation of calcium antagonists with high potency and long‐term activity. To evaluate the immunosuppressive potential of this drug, the influence of mibefradil on lymphocyte adhesion to, horizontal locomotion along, and penetration through allogeneic endothelium (HUVEC) was performed. When lymphocytes were prestimulated for 24 hr with mibefradil, adhesion and penetration were dose‐dependently reduced. The adhesion ID50 values were 3·4 μm (CD4+ T cells) versus 9·2 μm (CD8+ T cells) and 2·1 μm (CD4+ T cells) versus 3·9 μm (CD8+ T cells) with regard to penetration. Mibefradil also effectively blocked horizontal locomotion. Specific down‐regulation of T‐cell binding to the P‐selectin receptor (ID50: CD4+ T cells, 0·8 μm; CD8+ T cells, 1·2 μm) and to the intracellular adhesion molecule‐1 (ICAM‐1) receptor (ID50: CD4+ T cells, 1·9 μm; CD8+ T cells, 1·5 μm) by mibefradil seems to be responsible for the decreased adhesion and penetration rates. Reduction of intracellular F‐actin in T lymphocytes could diminish cell locomotion. In conclusion, the potent suppressive properties of mibefradil support its use as a co‐medication in cyclosporin A‐based immunosuppressive therapy.

CsA, cyclosporin A HUVEC, human umbilical vein endothelial cells ICAM‐1, intercellular adhesion molecule‐1 LFA‐1, leukocyte function‐associated antigen‐1 PBL, peripheral blood lymphocytes PSGL‐1, P‐selectin glycoprotein ligand‐1.
A comparison of two techniques for the molecular tracking of specific T‐cell responses; CD4+ human T‐cell clones persist in a stable hierarchy but at a lower frequency than clones in the CD8+ population
Authors: MAINI  , WEDDERBURN  , HALL  , WACK  , CASORATI  , BEVERLEY¶
Published: March 2003

Oligoclonal or clonal T‐cell expansions, presumed to be antigen driven, are frequently sought and followed for diagnostic and prognostic purposes, as well as to understand more about their natural history. Techniques based on conservation of T‐cell receptor CDR3 length are increasingly widely used, often without assessment of sensitivity or specificity. We present a comparative evaluation of a novel modified heteroduplex technique and a CDR3‐length‐based assay. Dilution of a known clone in a mixed T‐cell population shows that in our hands the heteroduplex technique is at least 10‐fold more sensitive than the CDR3‐length‐based assay. However, even with this level of sensitivity, we do not detect clonal expansions in unstimulated CD4+ T cells. This contrasts with the frequent detection of CD8+ clones in fresh samples and suggests different mechanisms of clonal homeostasis in the two subsets. We show that both techniques detect functional expansions after in vitro stimulation with a recall antigen. The distinct molecular footprint seen with the heteroduplex technique allows reproducible follow up of specific clonal expansions. We have exploited this to demonstrate that the repertoire of clones expanded by in vitro tetanus toxoid stimulation shows stability within an individual, implying long‐term maintenance of multiple CD4+ clones.
Lymphoid hyperplasia in transgenic mice over‐expressing a secreted form of the human interleukin‐1β gene product
Authors: BJÖRKDAHL  , ÅKERBLAD  , GJÖRLOFF‐WINGREN  , LEANDERSON  , DOHLSTEN
Published: March 2003

To evaluate the biological effects of over‐expression of interleukin‐1β (IL‐1β) on the immune system we have generated transgenic mice, expressing the IL‐1β gene fused to a heterologous signal sequence under the control of the mouse immunoglobulin enhancer (Eμ). A prominent hyperplasia and a disturbed microarchitecture of lymphoid tissues were observed in the transgenic mice. The CD4+ T cells in the hyperplastic lymphoid organs seemed to invade the majority of the lymphoid organs including B‐cell restricted areas. Analysis of lymph node cells revealed an increased frequency of CD4+ CD44high CD62L− T cells and local secretion of IL‐2 and IL‐4, compatible with an elevated number of activated T cells. Furthermore, significant levels of human IL‐1β in sera and high concentrations of serum immunoglobulin G (IgG) were observed in the transgenic mice. The data suggest a role for IL‐1β in controlling lymphoid microarchitecture and, when over‐expressed, breaking the threshold in T‐helper–B‐cell interaction.
The protective role of T‐cell receptor Vγ1+ T cells in primary infection with Listeria monocytogenes
Authors: NAKAMURA  , MATSUZAKI  , NOMOTO
Published: March 2003

We have previously reported that the T‐cell receptor (TCR) γδ+ T cells increase in mice infected with an intracellular bacteria Listeria monocytogenes, and the cells predominantly express Vδ6 and Vγ1 genes. In this study, we used a monoclonal antibody (mAb) specific to TCR Vγ1 to estimate the frequency of Vγ1+ T cells and we discuss their significance in protection against L. monocytogenes. The spleen, liver and peritoneal exudate cells from mice intraperitoneally infected with L. monocytogenes were analysed by flow cytometry. In all the organs investigated, Vγ1+ cells increased predominantly among TCR γδ+ T cells at an early phase (day 5–7) of the infection. To elucidate the significance of the Vγ1+ T cells in the protection against L. monocytogenes, mice were depleted of TCR Vγ1+γδ T cells or all TCR γδ+ T cells by intraperitoneal inoculation of anti‐Vγ1 mAb or anti‐pan TCR γδ mAb, respectively, before infection with L. monocytogenes. The bacterial growth in the spleen and the liver examined on day 5 after the infection increased significantly by the depletion of TCR Vγ1+ T cells. The numbers of L. monocytogenes in TCR Vγ1+ T‐cell‐depleted mice were nearly the same as in mice depleted of all TCR γδ+ T cells. These results demonstrated that Vγ1+ T cells are the predominant population of γδ T cells in protection against L. monocytogenes at the early phase of the primary infection.
Dietary lipids modify the cytokine response to bacterial lipopolysaccharide in mice
Authors: SADEGHI  , WALLACE  , CALDER
Published: March 2003

To investigate the effect of dietary lipids with different fatty acid compositions upon the in vivo cytokine response to bacterial lipopolysaccharide (LPS), mice were fed for 5 weeks on a low‐fat diet or on one of four high‐fat diets that contained 20%, by weight, of coconut oil (CO), olive oil (OO), safflower oil (SO) or fish oil (FO). The mice were injected intraperitoneally with a non‐lethal dose of Escherichia coli LPS (100 μg/20 g body weight) and killed 90 or 180 min later. Plasma tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐1α, IL‐6 and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay (ELISA). Plasma TNF‐α and IL‐10 concentrations were higher 90 min postinjection than after 180 min, whereas plasma IL‐1β and IL‐6 concentrations were higher 180 min postinjection than after 90 min. Peak plasma TNF‐α, IL‐1β and IL‐6 concentrations were lower in the CO‐ and FO‐fed mice than in those fed the SO diet. Peak plasma IL‐10 concentrations were higher in CO‐fed mice than in those fed some of the other diets. These observations suggest that, relative to the n‐6 polyunsaturated fatty acid‐rich SO diet, CO and FO diminish production of proinflammatory cytokines in vivo. This indicates that these fatty acids might be useful therapies in acute and chronic inflammatory diseases. The enhanced production of IL‐10 following CO feeding appears to be an additional antiinflammatory effect of this oil, which could give added benefit in various clinical conditions.
Kinetics and intracellular pathways required for major histocompatibility complex II–peptide loading and surface expression of a fluorescent hapten‐protein conjugate in murine macrophage
Authors: WEAVER JR  , VOSS JR
Published: March 2003

A fluorescent antigen, FITC10BSA, that is sensitive to several of the biochemical processes involved in antigen processing was constructed. In combination with both flow cytometry and subcellular fractionation, the unique probe provided new details regarding the kinetics and intracellular pathways involved in antigen processing in murine macrophage. These studies suggested that macrophage utilized multiple vesicles as opposed to a few specific organelles for major histocompatibility complex (MHC) type II–peptide loading and transport. Although newly formed MHC II–peptide complexes were detected in cathepsin D‐positive, lysosomal associated membrane glycoprotein (LAMP‐1)‐positive lysosomes, MHC II–peptide loading also occurred in transferrin receptor‐positive endosomes. Interestingly, MHC II‐fluoresceinated complexes were only observed in transferrin receptor‐positive organelles as opposed to MHC II‐unlabelled peptide complexes which were detected in traditional early lysosomal compartments. More importantly, MHC II–peptide complexes were monitored in light transferrin receptor‐positive fractions following their initial appearance in dense endosomal/lysosomal fractions. Control experiments suggested that these complexes represented intermediates in the process of migrating to the cell surface through a retrograde pathway within the macrophage.
A peptide derived from a polyreactive monoclonal anti‐DNA natural antibody can modulate lupus development in (NZB×NZW)F1 mice
Authors: JOUANNE  , AVRAMEAS  , PAYELLE‐BROGARD
Published: March 2003

In lupus‐prone (NZB×NZW)F1 (B/W) mice, elevated levels of polyreactive autoantibodies bearing the D23 idiotype (Id), characteristic of natural antibodies, were detected before and after the appearance of pathological anti‐DNA antibodies. While these D23 Id+ antibodies were able to regulate anti‐DNA antibodies in the early stage of the disease, we found that during disease evolution they had lost their normal ability to regulate anti‐DNA antibodies and furthermore could participate in the lupus‐like syndrome. To explore further the role of the D23 Id+ antibodies, we injected young B/W mice with a peptide corresponding to the VH CDR3 region of the D23 monoclonal natural antibody (mNAb). High levels of monospecific antipeptide, as well as polyreactive antibodies, were induced. Among them, the most markedly enhanced antibody population was DNA‐reactive immunoglobulin G1 (IgG1). Compared with controls, these immunized mice had a delayed 50% survival rate and proteinuria developed later. Furthermore, IgG1 able to react with IgG2a anti‐DNA monoclonal antibodies derived from B/W mice were also produced after peptide immunization. Thus, a peptide corresponding to the CDR3 of the D23 mNAb antibody might play a role in the regulation of murine lupus.
Local effects of recombinant rat interleukin‐6 on the peripheral nervous system
Authors: Deretzi  , H Pelidou  , Zou L. P. , Quiding C. , Zhu
Published: March 2003

Interleukin‐6 (IL‐6) is a multifunctional cytokine with a broad range of activities and can affect a variety of target cells or systems in multiple ways. However, there is currently no consensus on how IL‐6 directly affects the peripheral nervous tissue. We performed histopathological and immunohistochemical analyses to investigate the direct effects of recombinant rat IL‐6 (rrIL‐6) following its intraneural injection into the sciatic nerve of adult Lewis rats. One day after injection, a large number of macrophages, major histocompatibility complex (MHC) class II positive cells, and CD4+ and CD8+ T cells appeared within the perineurium and endoneurium. From day 4 to day 7 after injection, we observed a gradual increase of inflammation and demyelination. On day 7, demyelination affected more than 80% of nerve fibres. In contrast, in the sterile phosphate‐buffered saline (PBS)‐injected control group, lower inflammation and fewer demyelinating nerve fibres were observed on days 4 and 7. Thus, intraneural injection of rrIL‐6 into the sciatic nerve induces high inflammation and severe demyelination. This study improves our understanding of the effector mechanisms underlying inflammation and demyelination and identifies IL‐6 as an essential mediator of inflammation and demyelination in the peripheral nervous system after local administration.
Analysis of mouse dendritic cell migration in vivo upon subcutaneous and intravenous injection
Authors: Lappin  , Weiss  , Delattre  , Mai  , Dittmar  , Maier  , Manke  , Grabbe  , Martin  , Simon
Published: March 2003

Dendritic cells (DC) have an increasingly important role in vaccination therapy; therefore, this study sought to determine the migratory capacity and immunogenic function of murine bone‐marrow (BM)‐derived DC following subcutaneous (s.c.) and intravenous (i.v.) injection in vivo. DC were enriched from BM cultures using metrizamide. Following centrifugation, the low‐buoyant density cells, referred to throughout as DC, were CD11chigh, Iab high, B7‐1high and B7‐2high and potently activated alloreactive T cells in mixed lymphocyte reactions (MLR). In contrast, the high‐density cells expressed low levels of the above markers, comprised mostly of granulocytes based on GR1 expression, and were poor stimulators in MLR. Following s.c. injection of fluorescently labelled cells into syngeneic recipient mice, DC but not granulocytes migrated to the T‐dependent areas of draining lymph nodes (LN). DC numbers in LN were quantified by flow‐cytometric analysis, on 1, 2, 3, 5 and 7 days following DC transfer. Peak numbers of around 90 DC per draining LN were found at 2 days. There was very little migration of DC to non‐draining LN, thymus or spleen at any of the time‐points studied. In contrast, following i.v. injection, DC accumulated mainly in the spleen, liver and lungs of recipient mice but were largely absent from peripheral LN and thymus. The ability of DC to induce T‐cell‐mediated immune responses was examined using trinitrobenzenesulphate (TNBS)‐derivatized DC (TNBS‐DC) to sensitize for contact hypersensitivity responses (CHS) in naive syngeneic recipients. Following s.c. injection, as few as 105 TNBS‐DC, but not TNBS‐granulocytes, sensitized for CHS responses. However, the same number of TNBS‐DC failed to induce CHS following i.v. injection. In summary, this study provides new and quantitative data on the organ specific migration of murine BM‐derived DC following s.c. and i.v. injection. The demonstration that the route of DC administration determines the potency of CHS induction, strongly suggests that the route of immunization should be considered in the design of vaccine protocols using DC.
CD137‐induced apoptosis is independent of CD95
Authors: Michel  , Pauly  , Langstein  , Krammer  , Schwarz
Published: March 2003

CD95 (APO‐1/Fas) and CD137 (ILA/4‐1BB) are members of the tumour necrosis factor receptor family, and both are involved in induction of apoptosis in lymphocytes. Contrary to the case of CD95, apoptosis by CD137 is caused by cross‐linking of the respective ligand rather than the receptor. Nothing is known so far about the mechanism of CD137‐induced cell death. Here, we show that immobilized CD137 protein induces expression of CD95 in resting primary T and B lymphocytes. However, induction of apoptosis by CD137 is independent of CD95, because: (1) antagonistic anti‐CD95 antibody fragments do not block CD137‐induced apoptosis; and (2) CD137, but not anti‐CD95, can induce apoptosis in resting lymphocytes.
The preventive effects of incomplete Freund’s adjuvant and other vehicles on the development of adjuvant‐induced arthritis in Lewis rats
Authors: Zhang  , Mia  , Zheng  , Hossain  , Yamasaki  , Tokunaga  , Kohashi
Published: March 2003

The present study showed a novel finding that the development of adjuvant‐induced arthritis (AA) in Lewis rats was completely prevented by incomplete Freund’s adjuvant (IFA) injected 21 or 28 days before complete Freund’s adjuvant (CFA) challenge. Hexadecane also completely prevented AA and squalane, methyl oleate and pristane moderately prevented AA, though pristane by itself induced mild arthritis in two out of five rats. Concanavalin A‐stimulated lymph node cells (LNCs) isolated from AA rats were able to adoptively transfer the severe polyarthritis to all the naive recipients or even to the IFA pretreated recipients with earlier onset and more rapid progression than those of AA. The LNCs from the donors who had been pretreated with IFA and subsequently challenged with CFA could induce mild arthritis in only two out of eight naive recipients, whereas all the recipients who were challenged with CFA immediately after intravenous injection of these LNCs developed significantly less severe arthritis. However, the LNCs from IFA‐pretreated donors failed to prevent AA. According to the T helper type 1 (Th1)/Th2 paradigm, it was suggested that the adjuvant‐active vehicles such as IFA, hexadecane, squalane, methyl oleate and pristane, can affect and deviate the Th1/Th2 balance of immune responses in host. CFA could promote the propagation of Th2 cells rather than Th1 cells in these vehicle‐pretreated rats through as yet undetermined mechanisms, eventually resulting in the prevention of AA. Finally, we discussed a regulatory role of adjuvant vehicles for induction and suppression of AA.
Schistosomal egg antigen‐responsive CD8 T‐cell population in Schistosoma mansoni‐infected BALB/c mice
Authors: Pancré V. , Delacre M. , Herno J. , Auriault C.
Published: March 2003

We demonstrated here that schistosomal egg antigen (SEA) is able to stimulate an antigen‐specific, cytotoxic CD8+ T‐cell response in mice. Indeed, a single i.p. immunization with SEA resulted in the in vivo induction of significant cytotoxic T lymphocyte (CTL) activity in the spleen within 20 days. Effector cells were classic class I major histocompatibility complex (MHC)‐restricted CD8+ lymphocytes producing interferon‐γ (IFN‐γ) and interleukin‐2 (IL‐2), suggesting a type 1 response to SEA. We therefore investigated the relevance of these observations in the context of the Schistosoma mansoni parasite infection. CTL activity against SEA‐pulsed target cells was evidenced throughout the infection after in vitro stimulation of recovered splenic cells with SEA demonstrating that SEA‐specific CD8+ T cells with cytotoxic potentialities are present during infection. This activity was strongly increased after immunization of mice with SEA like the production of IFN‐γ in the sera. A marked reduction in the number of granulomas and of fibrosis with the presence of cells producing IFN‐γ in the liver was also observed leading to the survival of SEA‐immunized mice.
Pregnancies modulate B lymphopoiesis and myelopoiesis during murine ageing
Authors: Barrat F. S. , Lesourd B. M. , Louise A. S. , Boulouis H.‐J. , Thibault D. J. , Neway T. , Pilet C. A.
Published: March 2003

We recently reported that pregnancy affects age‐related changes in the distribution of lymphoid and macrophage populations in the spleen of C57Bl/6 mice. In the present study, we examined the influence of pregnancies on the generation of various developmental B‐cell subsets and granulocyte/macrophage lineage cells during murine ageing. Using flow cytometry, changes in lymphoid (mature and early B‐cell precursors: B220high, B220low, surface immunoglobulin M (sIgM) µ chain +/−) and myeloid (monocyte/macrophage Mac‐1/CD11b, granulocyte Gr‐1/Ly‐6G) compartments were monitored in the bone marrow of young (2 months) and 15‐ and 23‐month‐old mice including male, multiparous and virgin female mice. Pregnancies delayed the age‐related decline in murine B lymphopoiesis and maintained B‐cell reserve capacity during ageing. We also found an increased production of myeloid cells induced by pregnancies at middle (15 months) and advanced (23 months) ages. This comparative study provides new information on changes in marrow lymphopoiesis and myelopoiesis with age. Our data emphasizes that the onset, magnitude and kinetics of age‐related changes in the haematopoietic marrow are parity dependent. These changes could influence the incidence of age‐related diseases and may account for the greater longevity of females.
Contrasting effects of myeloid dendritic cells transduced with an adenoviral vector encoding interleukin‐10 on organ allograft and tumour rejection
Authors: Lee, W.‐C. , Qiani, S. , Wan Y. , Li, W. , Xing, Z. , Gauldie, J. , Fung, J. J. , Thomson A. W. , Lu L.
Published: March 2003

Mouse bone marrow‐derived myeloid dendritic cells (DC) propagated in granulocyte–macrophage colony‐stimulating factor and transforming growth factor‐β1 (TGF‐β1) (so‐called ‘TGF‐β DC’) are phenotypically immature, and prolong allograft survival. Interleukin‐10 (IL‐10) has been shown to inhibit the maturation of DC by down‐regulating surface major histocompatibility complex (MHC) class II, co‐stimulatory and adhesion molecule expression. Genetic engineering of TGF‐β DC to overexpress IL‐10 might enhance their tolerogenic potential. In this study, adenoviral (Ad) vectors encoding the mouse IL‐10 gene were transduced into B10 (H2b) TGF‐β DC. Transduction with Ad‐IL‐10 at a multiplicity of infection (MOI) of 50–100 resulted in a modest reduction in the incidence of DC expressing surface MHC class II, CD40, CD80 and CD86. Paradoxically, Ad‐IL‐10 transduction enhanced the allostimulatory activity of DC in mixed leucocyte reactions and cytotoxic T lymphocyte assays, and increased their natural killer cell stimulatory activity. Systemic injection of normal C3H recipients with Ad‐IL‐10‐transduced B10‐DC 7 days before organ transplantation, exacerbated heart graft rejection and augmented circulating anti‐donor alloantibody titres. Contrasting effects were observed in relation to tumour growth. All mice preimmunized with Ad‐IL‐10‐transduced, tumour antigen (B16F10)‐pulsed DC developed palpable tumours, associated with significant inhibition of splenic anti‐tumour cytotoxic T lymphocyte generation. Animals pretreated with control Ad‐LacZ‐transduced, B16F10‐pulsed DC however, remained tumour free. These findings are consistent with the multifunctional immunomodulatory properties of mammalian IL‐10.
The lipid A region of lipopolysaccharides from Rhizobiaceae activates bone marrow granulocytes from lipopolysaccharide‐hyporesponsive C3H/HeJ andC57BL/10ScCr mice
Authors: Pedron T. , Girard R. , Jeyaretnam B. , Carlson R. W. , Chaby R.
Published: March 2003

We established in previous studies that the binding of Salmonella lipopolysaccharide (LPS) to constitutive receptors of low affinity triggers the expression of the inducible LPS‐binding molecule CD14 in bone marrow cells (BMC) of C3H/HeOU mice, but not in BMC from C3H/HeJ mice. We show in this study that BMC from C3H/HeJ and C57BL/10ScCr mice do not express CD14 after exposure to LPSs from Salmonella enterica and Bordetella pertussis, but do express this marker when treated with several LPSs from Rhizobiaceae, or their lipid A fragments. This shows that the constitutive LPS receptor in BMC from C3H/HeJ and C57BL/10ScCr mice is fully able to trigger a complete signalling cascade. Results of cross‐inhibition of the binding of radiolabelled LPS indicated that active LPSs (from R. species Sin‐1 and R. galegae) and inactive LPSs (from S. enterica and B. pertussis) bind to the same site of the constitutive LPS receptor of C3H/HeJ cells. Furthermore, binding of R. species Sin‐1 LPS, and signalling induced by this LPS, were both inhibited by pre‐exposure of C3H/HeJ cells to B. pertussis lipid A. This correlation between binding and signalling suggests that in C3H/HeJ cells, the constitutive receptor, which recognizes a large panel of LPSs from different origins, appears selectively unable to be activated by some particular LPSs, such as those of Enterobacteria and Bordetella.
Distribution of, and immune response to, chicken anti‐αGal immunoglobulin Y antibodies in wild‐type and αGal knockout mice
Authors: Walsh Jr W. E. , Anderson B. E. , Ivancic D. , Zhang Z. , Piccini J. P. , Rodgers T. G. , Pao W. , Fryer J. P.
Published: March 2003

Chicken antibodies (immunoglobulin Y; IgY) to the αGal epitope (galactose α‐1,3‐galactose) bind to αGal antigens of mouse and porcine tissues and endothelial cells in vitro and block human anti‐αGal antibody binding, complement activation and antibody‐dependent cell‐mediated lysis mechanisms. The activities and toxicity of anti‐αGal IgY have not been tested in vivo. In this study, we tested the effects of multiple injections of affinity‐purified anti‐αGal IgY (AP‐IgY) in both wild‐type (WT) and α‐1,3‐galactosyltransferase knockout (Gal KO) mice. WT and Gal KO mice were injected once, twice, three, or four times intravenously (i.v.) with AP‐IgY and killed at 1 hr or 24 hr. Mice displayed no toxicity to four injections of AP‐IgY. Heart, lung, liver, kidney, spleen and pancreatic tissue were evaluated using immunohistochemical techniques for the presence of the αGal epitope using the GSI‐B4 lectin, and for bound IgY, as well as mouse IgM and IgG. The binding of AP‐IgY antibodies to the endothelium of WT mouse tissues was essentially identical to the pattern of binding of the GSI‐B4 lectin after injection of WT mice and death at 1 hr. WT mice killed 24 hr after i.v. injection of AP‐IgY showed little remaining bound IgY in their endothelia, indicating that IgY is cleared over that time period. We also evaluated the blood drawn at the time of death for the presence of anti‐αGal IgY, anti‐IgY IgM and anti‐IgY IgG by enzyme‐linked immunosorbent assay. Anti‐αGal IgY was almost undetectable in WT mouse sera at all injection and killing times. In contrast, Gal KO mouse sera showed increasing anti‐αGal IgY levels until 24 hr after the fourth injection, when anti‐αGal IgY levels were almost undetectable. Anti‐IgY IgM and IgG levels in WT and Gal KO mouse sera showed a typical increase in anti‐IgY IgM 24 hr after the second injection (3 days after the first injection) and an increase in anti‐IgY IgG 24 hr after the third injection (5 days after the first injection). These results show that IgY binds to αGal epitopes in the WT mice and is cleared sometime over a 24‐hr time period and that IgY is an expected immunogen in mice eliciting a rather typical anti‐IgY IgM and IgG response.
Inhibition of contact sensitivity in human CD4+ transgenic mice by human CD4‐specific monoclonal antibodies: CD4+ T‐cell depletion is not required
Authors: Podolin P. L. , Webb E. F. , Reddy M. , Truneh A. , Griswold D. E.
Published: March 2003

Clenoliximab and keliximab are monkey/human chimeric monoclonal antibodies (mAbs) of the immunoglobulin G4 (IgG4) and IgG1 isotypes, respectively, that recognize the same epitope on human CD4. The two mAbs possess identical idiotypes and exhibit equal affinities for CD4. Upon administration of these mAbs to mice that express a human CD4 transgene, but not mouse CD4 (HuCD4/Tg mice), clenoliximab and keliximab exhibited similar kinetics of binding to CD4, and induced the same degree of CD4 modulation from the cell surface, although only keliximab mediated CD4+ T‐cell depletion. Epicutaneous sensitization and challenge of HuCD4/Tg mice with the hapten oxazolone resulted in a contact sensitivity response characterized by tissue swelling, and the presence of interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) in the local tissue. Administration of a single 2‐mg dose of either clenoliximab or keliximab to HuCD4/Tg mice prior to sensitization significantly reduced post‐challenge tissue swelling, and levels of IFN‐γ and IL‐4, indicating that CD4+ T‐cell depletion is not required for anti‐CD4 mAb‐mediated inhibition of contact sensitivity. Administration of either mAb prior to challenge failed to inhibit the contact sensitivity response, indicating differential sensitivity of the afferent and efferent phases of the response to inhibition by CD4‐specific mAbs. Collectively, these data indicate that CD4 functions as a positive regulatory molecule in the contact sensitivity response.
CD40–CD40 ligand (CD154) engagement is required but may not be sufficient for human T helper 1 cell induction of interleukin‐2‐ or interleukin‐15‐driven, contact‐dependent, interleukin‐1β production by monocytes
Authors: Ribbens C. , Dayer J.‐M. , Chizzolini C.
Published: March 2003

To investigate whether antigen‐independent, interleukin‐2 (IL‐2) or IL‐15 activation of polarized T helper (Th) cells would result in contact‐dependent activation of monocytes, living Th1 and Th2 cell clones were co‐cultured with THP‐1 cells or fresh peripheral blood monocytes. Under these conditions IL‐1β production was induced almost exclusively by Th1 cells and was dependent on the presence and dose of IL‐2 or IL‐15, and on cell–cell contact, as demonstrated by double‐chamber cultures. Low levels of IL‐1 receptor antagonist (IL‐1Ra) were induced by Th1 and higher levels by Th2 cells. IL‐10 production was similar in Th1/monocyte and Th2/monocyte co‐cultures, thus arguing against preferential down‐regulation of IL‐1β production by anti‐inflammatory IL‐10 in Th2 co‐cultures. In addition, IL‐4 and IL‐10 neutralization did not result in enhanced IL‐1β production in Th2/monocyte co‐cultures. Preferential expression on Th1 cells of CD11b correlated with their capacity to induce IL‐1β production by THP‐1 cells in the presence of IL‐2 or IL‐15, but anti‐CD11b monoclonal antibody could not inhibit this activity. Blockade of the CD40–CD40 ligand interaction resulted in inhibition of IL‐1β‐inducing capacity while IL‐1Ra induction was unaffected, a result previously unknown. This differential effect indicates the selective relevance of CD40–CD40 ligand engagement in inflammatory monocyte responses upon activation by T cells. CD40 ligand expression levels did not differ in Th1 and Th2 cell clones, thus indicating that additional, unidentified molecule(s) preferentially expressed by Th1 cells are involved in their IL‐1β induction capacity.
A novel CD18 genomic deletion in a patient with severe leucocyte adhesion deficiency: a possible CD2/lymphocyte function‐associated antigen‐1 functional association in humans
Authors: Allende L. M. , Hernández M. , Corell A. , García‐pérez M. A. , Varela P. , Moreno A. , Caragol I. , García‐martín F. , Guillén‐perales J. , Olivé T. , Español T. , Arnaiz‐villena A.
Published: March 2003

Leucocyte adhesion deficiency (LAD) is an autosomal‐recessive genetic disease that is characterized clinically by severe bacterial infections and caused by mutations in the CD18 gene that codes for the β2 integrin subunit. A patient with a severe LAD phenotype was studied and the molecular basis of the disease was identified as a single homozygous defect in a Herpes virus saimiri (HVS)‐transformed T‐cell line. The defect identified involves a deletion of 171 bp in the cDNA that encodes part of the proteic extracellular domain. This genetic abnormality was further studied at the genomic DNA level and found to consist of a deletion of 169 bp (from − 37 of intron 4 to + 132 of exon 5), which abolishes the normal splicing and results in the total skipping of exon 5. The 171‐bp shortened ‘in‐frame’ mRNA not only resulted in the absence of CD18 expression on the cell surface but also in its absence in the cytoplasm of HVS T‐cell lines. Functionally, the LAD‐derived HVS T‐cell lines showed a severe, selective T‐cell activation impairment in the CD2 (but not in the CD3) pathway. This defect was not reversible when exogenous interleukin‐2 (IL‐2) was added, suggesting that there is also a functional interaction of the lymphocyte function‐associated antigen‐1 (LFA‐1) protein in the CD2 signal transduction pathway in human T cells, as has been previously reported in mice and in the human Papillon–Lefèvre syndrome. Thus, HVS transformation is not only a suitable model for T‐cell immunodeficiency studies and characterization, but is also a good system for investigating the immune system in pathological conditions. It may also be used in the future in cellular models for in vitro gene‐therapy trials.
Interleukin‐16 stimulates the expression and production of pro‐inflammatory cytokines by human monocytes
Authors: Mathy N. L. , Scheuer W. , Lanzendörfer M. , Honold K. , Ambrosius D. , Norley S. , Kurth R.
Published: March 2003

Interleukin‐16 (IL‐16) acts as a chemoattractant for CD4+ cells, as a modulator of T‐cell activation, and plays a key role in asthma. This report describes the cytokine‐inducing effects of IL‐16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4+ T lymphocytes did not secrete cytokines in response to rhIL‐16, CD14+ CD4+ monocytes and maturing macrophages secrete IL‐1β, IL‐6, IL‐15 and tumour necrosis factor‐α (TNF‐α) upon rhIL‐16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr post‐stimulation, with protein being secreted by 24 hr. Secretion of IL‐1β and IL‐6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL‐16. However, for IL‐15 or TNF‐α maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL‐16. Purified monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL‐16, except for monocytes where maximal secretion of IL‐15 was, interestingly, observed with only 50 ng/ml rhIL‐16. The use of higher concentrations of rhIL‐16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL‐16‐induced cytokines are likely to be involved in the immune system's response to antigen, the data suggest that IL‐16 may play a key role in initiating and/or sustaining an inflammatory response.
Most parasite‐specific CD8+ cells in Trypanosoma cruzi‐infected chronic mice are down‐regulated for T‐cell receptor‐αβ and CD8 molecules
Authors: Grisotto M. G. , D'Império Lima M. R. , Marinho C. R. F. , Tadokoro C. E. , Abrahamsohn I. A. , Alvarez J. M.
Published: March 2003

The present study shows that CD8+ T lymphocytes expressing low levels of T‐cell receptor (TCR)αβ, CD8 and CD3 accumulate in the spleen, blood, peritoneum and liver, but not in the lymph nodes of mice chronically infected with Trypanosoma cruzi. Analysis of spleen lymphocytes reveals that most CD8LOW TCRLOW T cells have an experienced phenotype (CD44HIGH CD62LLOW and CD45RA,B,CLOW). These cells have small size, lack activation markers such as CD69, CD25 and CD11b (Mac‐1), and do not spontaneously secrete cytokines, suggesting they are at the resting state. When stimulated in vitro with T. cruzi‐infected macrophages, TCRLOW CD8LOW T cells behave as parasite‐specific memory cells, readily responding with interferon‐γ (IFN‐γ) production. Indeed, among parasite‐activated CD8+ lymphocytes, IFN‐γ production was mostly due to TCRLOW CD8LOW cells. Upon in vitro stimulation with anti‐CD3/CD28 monoclonal antibodies, down‐regulated cells produce IFN‐γ and tumour necrosis factor‐α, but not interleukin IL‐10 or IL‐4. Our results indicate that despite parasite persistence, most T. cruzi‐specific experienced CD8+ cells are resting. Nevertheless, when encountering infected macrophages these cells differentiate to Tc1 effectors.
The ability of heat‐killed Mycobacterium vaccae to stimulate a cytotoxic T‐cell response to an unrelated protein is associated with a 65 kilodalton heat‐shock protein
Authors: Skinner M. A. , Prestidge R. , Yuan S. , Strabala T. J. , Tan P. L. J.
Published: March 2003

Exogenous antigens are generally presented by Class II major histocompatibility (MHC) molecules. When administered with an adjuvant, however, they are capable of inducing a CD8+ T‐cell response where antigen recognition is associated with Class I MHC. Accordingly, immunization with soluble ovalbumin (OVA) alone does not activate CD8+ cytotoxic T cells (CTL) but when given in complete Freund's adjuvant (CFA), or in formulations of a number of novel adjuvants, an OVA‐specific CD8+ CTL response can be detected. We show in this report that immunization with soluble OVA mixed with heat‐killed Mycobacterium vaccae, but not with other common pathogenic and saprophytic mycobacteria, can activate OVA‐specific CD8+ CTL. An OVA‐specific CTL response is detected when mice are immunized by either the intraperitoneal or intranasal route and their spleen cells are re‐stimulated in vitro. Adjuvant activity of heat‐killed M. vaccae is present in M. vaccae culture filtrate, in soluble protein components of whole M. vaccae and in the 65 kDa heat‐shock protein (hsp) of M. vaccae. Mycobacterium vaccae has previously been shown to have no adverse side‐effects in humans. The current results suggest that M. vaccae may be useful as an adjuvant for vaccines and other immunotherapies where CD8+ CTL responses to exogenous proteins are crucial.
Early Vλ diversification in sheep
Authors: Jeong Youngkee , Osborne Barbara A. , Goldsby Richard A.
Published: March 2003

This study examined a number of tissues during early gestation in foetal sheep to determine the earliest site of Vλ expression and time of generation of the Vλ repertoire. Tissues, including spleen, liver, gut, blood and bone marrow, were obtained from 48, 55, 60 and 63 gestational day (g.d.) ovine foetuses and cDNA libraries were prepared from them by reverse transcription–polymerase chain reaction. Clones were randomly selected from cDNA libraries and subjected to sequencing. Analysis of these sequences and comparison with a pool of germline genes led to the following conclusions. The expression of Vλ occurs earlier in spleen (48 g.d.) than in all of the other tissues examined. Also, diversity is seen earlier and at higher levels in early foetal spleen than in all of the other tissues examined. In this regard, it is notable that splenic Vλ expression is readily apparent even before such gut‐associated lymphoid tissue as the ileal Peyer's patch (IPP) has developed. Two germline Vλ genes, 5.1 and 5.3 predominate in early immunoglobulin λ light‐chain gene rearrangement. Examination of Jλ usage revealed the existence of a new Jλ gene and its utilization during the early phases of the development of the ovine antibody repertoire. This study indicates that sites other than the IPP contribute to the diversification of the Vλ repertoire in sheep. We suggest that it is likely that foetal spleen may provide a partially diversified B‐cell repertoire before the IPP becomes active as a major site for massive clonal expansion and extensive diversification of B cells.
Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells
Authors: Sullivan Yvonne B. , Landay Alan L. , Zack Jerome A. , Kitchen Scott G. , Al‐Harthi Lena
Published: March 2003

Aside from an intermediate stage in thymic T‐cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T‐cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti‐CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA‐mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA‐mediated effect. Autologous CD4+ and CD8+ T‐cell co‐cultures in the presence of PHA induced this CD4dimCD8bright T‐cell expression by 33%, demonstrating a role for CD4 cells in the PHA‐mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4− counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominately T‐cell receptor (TCR) αβ cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.
Fcγ receptor polymorphisms in populations in Ethiopia and Norway
Authors: Van Den Berg Linda , Myhr Kjell‐Morten , Kluge Beate , Vedeler Christian A.
Published: March 2003

Seventy‐seven healthy Ethiopians were genotyped for polymorphisms in the immunoglobulin G Fc receptors (FcγR) FcγRIIa, FcγRIIIa and FcγRIIIb, including the SH allele. The genotype and allele frequencies were compared with those of 96 healthy Norwegians. Ethiopians had higher frequencies of the SH‐FcγRIIIb (P = 0·001), FcγRIIIa‐158 V (P = 0·026) and FcγRIIIb‐Na2 (P = 0·046) alleles. The genotype distributions of FcγRIIa, FcγRIIIa and FcγRIIIb, however, did not differ significantly from those of the Norwegians. The data were also compared with those reported from studies on other ethnic groups. The variation of different polymorphisms both within and between ethnic groups may influence differences in the incidence rates of infectious and autoimmune diseases.
Assessing a role for enhancer‐blocking activity in gene regulation within the murine T‐cell receptor α/δ locus
Authors: Sleckman Barry P. , Carabana Juan , Zhong Xiaoping , Krangel Michael S.
Published: March 2003

Although situated close together within the T‐cell receptor (TCR) α/δ locus, TCR δ and TCR α gene segments are controlled by two developmental stage‐specific enhancers and are activated according to distinct developmental programmes. We previously used a stable transfection colony assay to identify an enhancer‐blocking element, blocking element alpha/delta‐1 (BEAD‐1), between the TCR δ and α gene segments of the human TCR α/δ locus. We hypothesized that enhancer‐blocking by BEAD‐1 might be required to prevent the TCR δ enhancer from activating TCR α gene segment transcription and rearrangement at the double negative stage of thymocyte development. Here, we used a transfection approach to define partial enhancer‐blocking activity in an analogous position of the murine TCR α/δ locus. To test the functional significance of this activity in vivo, we used gene targeting to delete the region from the endogenous locus. We found no perturbation of TCR δ and TCR α gene expression and rearrangement on targeted alleles, indicating that enhancer‐blocking activity in this region is not required to maintain the developmentally distinct activation profiles of the two genes. We suggest that appropriate regulation may be achieved as a result of intrinsic biases in enhancer–promoter interactions or a developmental stage specificity to promoter function that is distinct from any additional specificity imposed by the enhancers themselves.
Crucial Fas–Fas ligand interaction in spontaneous acceptance of hepatic allografts in mice
Authors: Uchiyama Hideaki , Kishihara Kenji , Minagawa Ryosuke , Hashimoto Koji , Sugimachi Keizo , Nomoto Kikuo
Published: March 2003

The Fas/Fas ligand (FasL) system plays important roles in the immune system, including host immunoregulation and cytotoxicity. In this study, we investigated the involvement of Fas–FasL interactions in spontaneous acceptance of hepatic allografts in murine orthotopic liver transplantation. Liver transplantation between the C57BL/6 (B6, H‐2b) donor and the MRL/Mp (MRL, H‐2k) recipient was performed in various combinations of donor and recipient mice with wild type (+/+), Fas‐mutant (lpr) or FasL‐mutant (gld) genotypes. The prolongation and spontaneous acceptance of the fully allogeneic grafts in recipients was not observed in either MRL‐lpr recipients with B6+/+ livers or MRL+/+ recipients with B6‐gld livers. Moreover, the serum alanine aminotransferase (ALT) levels and the degree of cell infiltration into hepatic allografts on day 7 after transplantation were inversely correlated with the recipient survival time (in days). The donor‐specific cytotoxic T‐lymphocyte (CTL) activities of the graft‐infiltrating cells (GICs) from MRL‐gld recipients with B6+/+ livers were much lower than those from MRL+/+ or ‐lpr recipients on days 5 and 10 after transplantation. However, the CTL activities of the GICs from MRL+/+ and ‐gld recipients predominately disappeared by day 15 after transplantation. Furthermore, the anti‐donor CTL activities induced in MRL+/+ recipients were ascribed to CD8+ cells, and were not mediated by Fas–FasL interactions. These results strongly suggest that the Fas/FasL system plays a critical role for recipient immunoregulation, enabling recipients in accepting hepatic allografts by deletion of the donor‐specific T cells, but not for CTL/target cell interaction in MRL+/+ recipients.