bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological roles of γδ T‐cell receptor intraepithelial lymphocytes in cytoproliferation and differentiation of mouse intestinal epithelial cells
Authors: MATSUMOTO  , NANNO  , WATANABE  , MIYASHITA  , AMASAKI  , SUZUKI  , UMESAKI
Published: March 2003

In this study we aimed to elucidate the physiological role of γδ intraepithelial lymphocytes (IEL) in the mouse intestine. For this purpose, we used T‐cell receptor (TCR) Vγ4/Vδ5 transgenic mice (KN 6 Tg: BALB/c background, H‐2d), and compared the immunological and physiological characteristics of the intestinal tracts of KN 6 Tg and non‐transgenic (non‐Tg) littermates. In KN 6 Tg littermates, 95% of small intestinal (SI) and large intestinal (LI) IEL expressed γδ TCR, and their TCR was replaced by Tg γδ TCR. In these mice, class II major histocompatibility complex (MHC) expression was up‐regulated in the SI epithelium, compared with the non‐Tg littermates, under specific pathogen‐free (SPF) conditions. Competitive reverse transcription–polmerase chain reaction (RT–PCR) analysis showed that the mRNAs of the I‐Eα chain on the SI epithelial cells was higher in KN 6 Tg than in non‐Tg littermates. However, in the LI, class II MHC molecules were not expressed in either KN 6 Tg or non‐Tg littermates. The epithelial cell mitotic index in the SI, but not in the LI, was higher in KN 6 Tg than in non‐Tg littermates under SPF conditions. However, differentiation markers for SI epithelial cells, such as alkaline phosphatase and disaccharidase (lactase, maltase and sucrase) activities, were similar in KN 6 Tg and non‐Tg littermates. MHC class II molecule expression on the SI epithelium was absent in germ‐free (GF) Tg mice, but was induced under SPF conditions, coinciding with the increase of interferon‐γ (IFN‐γ) mRNA in γδ TCR SI‐IEL. These findings suggest that γδ TCR IEL regulate epithelial cell regeneration and class II MHC expression, but not cell differentiation in the SI. However, these functions were not observed in the γδ TCR IEL in the LI. In addition, the activation step in the γδ TCR SI‐IEL is dependent on the presence of gut microflora.
Anti‐CD44 treatment does not prevent the extravasation of autopathogenic T cells to the thyroid in experimental autoimmune thyroiditis
Authors: Parish  , Brennan  , Cooke
Published: March 2003

The hyaluronic acid binding glycoprotein CD44 is expressed on a wide variety of cells, and by mediating interactions between cells and extracellular matrices promotes the movement of cells from the circulation into organs. Recent reports have described the effects of an antibody specific for CD44 (IM7) that has beneficial effects in two murine models of autoimmune disease. Both experimental allergic encephalomyelitis (EAE) and collagen‐induced arthritis were ameliorated by treatment with IM7, which was considered to be acting by preventing the homing of lymphocytes to the relevant inflammatory sites, namely the central nervous system and the synovium, respectively. In this study the same anti‐CD44 antibody was used to try to prevent leucocytic infiltration of the thyroid in the murine model of Hashimoto’s thyroiditis, experimental autoimmune thyroiditis (EAT). We report that, in contrast to the previous findings, this antibody had an exacerbating effect on thyroiditis induced by immunization of mice with mouse thyroglobulin (MTg) and complete Freund’s adjuvant (CFA). Thyroid infiltrates lasted longer and showed increased severity compared with untreated or control antibody‐treated mice. Antibody responses to MTg were unaffected by antibody treatment. The data suggest that simple rules cannot be drawn that predict the potential broad therapeutic use of anti‐CD44 reagents, presumably due to differences in the cellular phenotypes and the dynamics of their movement into inflammatory sites during different disease processes.
Concanavalin A stimulation enhanced secondary VλJλ rearrangement in some human plasma B cells without up‐regulation of recombination‐activating gene expression and Vλ germline transcription
Authors: Haruta  , Tachibana  , Yamada
Published: March 2003

Light chain shifting is a phenomenon that occurs in certain human antibody‐producing plasma B cells which, when stimulated with concanavalin A (Con A), shift production of the original light chain to new light chains. Here we investigated the effect of Con A stimulation on these light chain shift‐inducible cells. Analysis of transcripts and VJ‐coding joints for new light chains revealed that a leaky amount of secondary VλJλ rearrangement occurs spontaneously, without Con A stimulation, and that Con A stimulation markedly increases VJ‐coding joints and transcripts for new light chains. It was also shown that new light chain producers, which have carried out secondary rearrangement, do not further rearrange their light chain genes, even when stimulated with Con A. Recombination‐activating gene (RAG) products and Vλ germline transcription were constitutively expressed in these cell lines and their expression levels were not affected by Con A stimulation. These results suggest that Con A stimulation enhanced secondary VλJλ rearrangement, but this was not a result of the up‐regulation of RAG expression and Vλ germline transcription, which are believed to be sufficient for the process of VλJλ rearrangement.
Co‐operation between human CR1 (CD35) and CR2 (CD21) in internalization of their C3b and iC3b ligands by murine‐transfected fibroblasts
Authors: Grattone  , Villiers  , Villiers  , Drouet  , Marche
Published: March 2003

CR1 and CR2 are expressed as associated proteins on the B‐lymphocyte surface. To investigate their respective contributions to the internalization of C3 fragments, transfected murine fibroblasts expressing human CR1, CR2, or both CR1 and CR2 were produced. CR1‐ and CR1–CR2‐expressing cells bound C3b and C3b‐dimer whereas CR2‐ and CR1–CR2‐expressing cells bound iC3b and C3de. In all cases, maximum binding was achieved at low ionic strength. CR1–CR2‐positive cells internalized two‐ to threefold more C3b and 1·5‐fold more iC3b than CR1‐ and CR2‐single‐positive cells, respectively. Internalization of the anti‐CR1 antibody J3D3, or C3de was at the same level, in both double‐transfected and single‐transfected cells. Furthermore, the internalization of C3b dimer by CR1–CR2 cells was impaired in the presence of OKB7, an anti‐CR2‐blocking antibody, but it was not altered in CR1 cells. Taken together, these findings suggest that CR1 and CR2 collaborate to internalize C3b and iC3b proteins. We suggest that the induction of conformational changes of the ligands enhances their binding to both receptors.
Dissecting the human peripheral B‐cell compartment with phage display‐derived antibodies
Authors: Van Der Vuurst De Vries  , Logtenberg
Published: March 2003

Previously we have employed a large semisynthetic phage antibody display library, in combination with subtractive selection by flow cytometry to isolate phage antibodies specific for subpopulations of leucocytes. In this study, human tonsillar B cells were incubated with the phage library and IgD− CD38− memory B lymphocytes and attached phage antibodies were selected by cell sorting. In a panel of 17 monoclonal phage antibodies obtained, five displayed binding to cells of multiple haematopoietic lineages or broadly reacted with B‐lineage cells. Immunofluorescent, immunohistochemical and biochemical studies permitted the characterization of the target molecules recognized by these phage antibodies. The remaining 12 antibodies displayed restricted binding to small subpopulations of peripheral human B cells. These results show that subtractive selections with phage antibody display libraries in combination with flow cytometry yield antibodies that bind to differentially expressed molecules on closely related cell populations, and can be used as a tool in a variety of assays.
B7 costimulatory ligand regulates development of the T‐cell response to Cryptococcus neoformans
Authors: Monari  , Kozel  , Casadevall  , Pietrella  , Palazzetti  , Vecchiarelli
Published: March 2003

The contribution of B7 molecules to the induction and maintenance of the T‐cell response to the human pathogenic fungus Cryptococcus neoformans was investigated. T‐cell activation by C. neoformans was regulated by B7 molecules. This costimulatory signal was necessary for initiation and maintenance of the T‐cell response, through early and late requirements for B7–CD28 interaction. Blocking B7‐2 inhibited the normal T‐cell proliferative response. This inhibition was due, in part, to a reduced capability of T cells to produce interleukin‐2 (IL‐2). In contrast, the same T‐cell population produced more interferon‐γ. Suppression of the normal lymphoproliferation and IL‐2 secretion responses to encapsulated C. neoformans by antibodies to B7 was largely reversed by addition of the monoclonal antibody 2H1, that is reactive with the major capsular polysaccharide, glucuronoxylomannan. Overall, our data indicate that B7 molecules play a critical role in T‐cell activation by C. neoformans and suggest that appropriate manipulation could drive T helper type 1 cell development.
Activation of complement receptor 3 on human monocytes by cross‐linking of very‐late antigen‐5 is mediated via protein tyrosine kinases
Authors: Van Den Berg  , Van Furth  , Hazenbos
Published: March 2003

Bordetella pertussis interacts with very‐late antigen‐5 (VLA‐5) receptors on the human monocyte resulting in cross‐linking of these receptors followed by activation of complement receptor 3 (CR3) and firm adhesion of B. pertussis to these monocytes. In the present study we investigated whether protein tyrosine kinases are involved in the activation of CR3 on monocytes, which was assessed by the binding of C3bi‐coated erythrocytes (EC3bi). Pre‐incubation of monocytes with tyrphostin‐A47, a specific protein tyrosine kinase inhibitor, before adherence of the cells to an anti‐VLA‐5 monoclonal antibody‐coated surface, or addition of tyrphostin‐A47 within 10 min of the adherence to such surface, reduced the binding of EC3bi to monocytes significantly. Pre‐incubation of monocytes with tyrphostin‐A47 reduced the binding of B. pertussis to such monocytes as well. Inhibitors of protein kinase A and/or C had no effect on EC3bi binding to monocytes. Cross‐linking of VLA‐5 on monocytes resulted in tyrosine phosphorylation of several proteins. Together, these results indicate that protein tyrosine kinases are involved in the VLA‐5‐induced activation of CR3 on human monocytes.
Acidic pH increases the avidity of FcγR for immune complexes
Authors: López D. H. , Trevani A. S. , Salamone G. , Andonegui G. , Raiden S. , Giordano M. , Geffner J. R.
Published: March 2003

The interaction of immunoglobulin G (IgG) antibodies with FcγR constitutes a critical mechanism through which IgG antibody effector functions are mediated. In the current work we have examined whether human neutrophil FcγR exhibit pH dependence in their association with IgG. Binding assays were performed in culture medium adjusted to different pH values. It was found that the binding of either heat‐aggregated human IgG (AIgG), soluble immune complexes (sIC) or IgG‐coated erythrocytes (IgG‐E) was markedly higher at pH 6·5 than at pH 7·3. This effect was not observed when saturation of FcγR was achieved, suggesting that acidic pH increases the avidity of FcγR for IC without modifying the total binding capacity. Similar results were observed for the binding of AIgG to either monocytes, natural killer (NK) or K562 cells, suggesting that acidic pH increases the avidity of both, FcγRII and FcγRIII. Additional experiments were performed to analyse whether the binding of IgG to FcγRI also showed pH dependence. To this aim, we employed interferon‐γ‐treated human neutrophils and mouse inflammatory macrophages, previously incubated with blocking antibodies directed to FcγRII and FcγRIII. Acidic pH did not enhance the binding of AIgG nor monomeric IgG under these experimental conditions. Further studies are required to determine whether the enhancement of FcγR avidity for IC could be attributed to titration of histidine(s) residues on the Fc fragment of IgG.
Histidine‐rich glycoprotein prevents the formation of insoluble immune complexes by rheumatoid factor
Authors: Gorgani N. N. , Altin J. G. , Parish C. R.
Published: March 2003

In previous studies we have shown that histidine‐rich glycoprotein (HRG), a relatively abundant plasma protein, can bind to immunoglobulin G (IgG) and inhibit the insolubilization of IgG‐containing immune complexes (IC). It was of interest, therefore, to determine whether HRG can inhibit the formation of insoluble IC (IIC) resulting from the interaction of rheumatoid factor (RF) with human IgG‐containing IC. Light scattering techniques were used to examine the effect of HRG on the formation of IIC between RF and IC containing human IgG according to three different models. In all three models physiological concentrations of HRG could block the formation of IIC induced by RF. Optical biosensor studies of the RF–IgG interaction also revealed that HRG can mask the epitopes on IgG recognized by RF. Additional studies examined whether HRG can solubilize already formed IIC and demonstrated that HRG can, in fact, partially solubilized IIC. These data indicate that HRG can regulate the formation of IIC induced by RF at three levels: namely by inhibiting the initial recognition of IgG containing IC by RF, by inhibiting the subsequent insolubilization of IgG containing IC by RF and by solubilizing already formed IIC. Collectively, these findings suggest that HRG may be an important inhibitor of the formation of pathogenic IC in diseases such as systemic lupus erythematosus and rheumatoid arthritis.
The effect on immunoglobulin glycosylation of altering in vivo production of immunoglobulin G
Authors: Jeddi P. , Keusch J. , Lydyard P. M. , Bodman‐Smith K. B. , Chesnutt M. S. , Wofsy D. , Hirota H. , Taga T. , Delves P. J.
Published: March 2003

The effect on murine immunoglobulin G (IgG) glycosylation of altering IgG production in vivo was assessed in interleukin (IL)‐6 transgenic and CD4 knockout mice. C57BL/6 mice carrying the IL‐6 transgene showed increased levels of circulating IgG. This was associated with decreased levels of galactose on the IgG oligosaccharides. No decrease in β4‐galactosyltransferase mRNA or in enzyme activity was seen in IL‐6 transgenic mice. MRL‐lpr/lpr mice normally have elevated levels of circulating IgG, again accompanied by decreased levels of IgG galactose. Disruption of the CD4 gene in MRL‐lpr/lpr mice led to a substantial decrease in the concentration of circulating IgG, but IgG galactose levels remained low. Thus, an enforced decrease in IgG levels in the lymphoproliferative MRL‐lpr/lpr mice did not alter the percentage of agalactosyl IgG in these mice, suggesting that agalactosyl IgG production is not simply caused by excessive IgG synthesis leading to an insufficient transit time in the trans‐Golgi, but rather to a molecular defect in the interaction between galactosyltransferase and the immunoglobulin heavy chain.
Differential expression of inducible nitric oxide synthase gene by alveolar and peritoneal macrophages in lipopolysaccharide‐hyporesponsive C3H/HeJ mice
Authors: Wang M. J. , Jeng K. C. G. , Shih P. C.
Published: March 2003

In lipopolysaccharide (LPS)‐hyporesponsive C3H/HeJ mice, alveolar macrophages (AMφ) produce much more tumour necrosis factor‐α than peritoneal macrophages (PMφ) when stimulated with LPS (10 µg/ml), but the induction of inducible nitric oxide synthase (iNOS) gene expression and production of nitric oxide (NO) in AMφ are not found. In the present study, we determined the induction of iNOS gene expression, using semi‐quantitative reverse transcription–polymerase chain reaction, and the release of NO in AMφ and PMφ from C3H/HeJ and C3H/HeN mice. The results showed the induction of iNOS mRNA accumulation in a dose‐dependent manner by LPS alone or in combination with interferon‐γ in both macrophages. The effects of the stimuli on iNOS gene expression and NO production were significantly higher in AMφ than in the PMφ of C3H/HeJ mice. The response of macrophages from C3H/HeN mice was similar to those from C3H/HeJ mice, but the difference of iNOS gene expression between AMφ and PMφ in C3H/HeN mice was not as striking as in C3H/HeJ mice. The results show that the iNOS gene expression and NO production were activated differently in AMφ and PMφ and suggest that the functional properties of macrophages isolated from distinct origins are different.
Impaired delayed‐type hypersensitivity response in mutant mice secreting soluble CD4 without expression of membrane‐bound CD4
Authors: Wang C.‐R. , Hino A. , Yoshimoto T. , Nagase H. , Kato T. , Hirokawa K. , Matsuzawa A. , Nariuchi H.
Published: March 2003

Delayed‐type hypersensitivity (DTH) is an important in vivo manifestation of cell‐mediated immunity. We examined the DTH response to methylated bovine serum albumin of a novel mutant strain of mice that have soluble CD4 (sCD4) in their circulation without expression of CD4 on the cell surface. The DTH response of the mutant mice was severely impaired, although the response of CD4 knockout (KO) mice, generated by homologous recombination, was comparable to that of wild‐type mice. The response of the mutant mice was restored by the neutralization of sCD4 with anti‐CD4, and that of CD4KO mice was markedly reduced by the implantation of a diffusion chamber containing sCD4 cDNA transfectant cells. The restored DTH response of the mutant mice treated with anti‐CD4 was abolished by treatment with anti‐interferon‐γ (IFN‐γ). IFN‐γ production by CD4 mutant and CD4KO mice was consistent with their DTH response and inversely related to the presence of sCD4 in their circulation, indicating that sCD4 impairs the DTH response by blocking the production of IFN‐γ in our mutant mice. These results raise the possibility that sCD4 could impair cell‐mediated immunity. Our mutant mice would provide a useful tool with which to analyse the mechanisms of the DTH reaction.
The role of endogenous steroid hormones in the generation of T helper 2‐mediated autoimmunity in mercuric chloride‐treated Brown–Norway rats
Authors: Macphee I. A. M. , Turner D. R. , Oliveira D. B. G.
Published: March 2003

Injection of Brown–Norway rats with mercuric chloride (HgCl2) activates a T helper type 2 (Th2) autoimmune response, with production of a number of autoantibodies and vasculitis primarily affecting the gut. Glucocorticoids have been shown to suppress Th1 and to promote the development of Th2‐type responses. Conversely dehydroepiandrosterone (DHEA) promotes Th1 responses with suppression of Th2 responses. This study set out to define the role of these hormones in this animal model. Rats were adrenalectomized (Adx) with no steroid replacement (n = 11), Adx with basal steroid replacement given by a 25 mg corticosterone pellet inserted subcutaneously (n = 13), or sham‐Adx (n = 14) prior to administration of HgCl2. In both groups of Adx animals there was a delay in the production of immunoglobulin E (IgE) and serum concentrations on day 9 were marginally lower (P = 0·035, repeated measures
anova). All of the animals Adx with no steroid replacement and two Adx animals with steroid replacement died between 10 and 14 days after HgCl2 challenge. There was no difference in the severity of caecal vasculitis between the groups. A significant increase in adrenal size was noted following administration of HgCl2. Administration of subcutaneous DHEA implants (100 mg and 200 mg) had no significant effect on IgE concentrations or severity of vasculitis. These observations do not support the hypothesis that corticosterone and DHEA play a central role in setting the Th1/Th2 balance in this experimental Th2‐mediated autoimmune disease; in contrast with the Th1‐mediated autoimmune disease experimental allergic encephalomyelitis where corticosterone plays a key role in immunoregulation.
Mucosal immunogenicity of plant lectins in mice
Authors: Lavelle E. C. , Grant G. , Pusztai A. , Pfüller U. , O’Hagan D. T.
Published: March 2003

The mucosal immunogenicity of a number of plant lectins with different sugar specificities was investigated in mice. Following intranasal (i.n.) or oral administration, the systemic and mucosal antibody responses elicited were compared with those induced by a potent mucosal immunogen (cholera toxin; CT) and a poorly immunogenic protein (ovalbumin; OVA). After three oral or i.n. doses of CT, high levels of specific serum antibodies were measured and specific IgA was detected in the serum, saliva, vaginal wash, nasal wash and gut wash of mice. Immunization with OVA elicited low titres of serum IgG but specific IgA was not detected in mucosal secretions. Both oral and i.n. delivery of all five plant lectins investigated [Viscum album (mistletoe lectin 1; ML‐1), Lycospersicum esculentum (tomato lectin; LEA), Phaseolus vulgaris (PHA), Triticum vulgaris (wheat germ agglutinin (WGA), Ulex europaeus I (UEA‐1)] stimulated the production of specific serum IgG and IgA antibody after three i.n. or oral doses. Immunization with ML‐1 induced high titres of serum IgG and IgA in addition to specific IgA in mucosal secretions. The response to orally delivered ML‐1 was comparable to that induced by CT, although a 10‐fold higher dose was administered. Immunization with LEA also induced high titres of serum IgG, particularly after i.n. delivery. Low specific IgA titres were also detected to LEA in mucosal secretions. Responses to PHA, WGA and UEA‐1 were measured at a relatively low level in the serum, and little or no specific mucosal IgA was detected.
Studies on delayed systemic effects of ultraviolet B radiation on the induction of contact hypersensitivity, 3. Dendritic cells from secondary lymphoid organs are deficient in interleukin‐12 production and capacity to promote activation and differentiation of T helper type 1 cells
Authors: Kitazawa T. , Streilein J. W.
Published: March 2003

Ultraviolet‐B radiation (UVR) of mouse skin promotes both local and systemic immune aberrations that are thought to be important in the pathogenesis of cutaneous malignancies. Acute, low‐dose UVR regimens inhibit the induction of contact hypersensitivity (CH) in genetically susceptible mice by TNF‐α‐dependent mechanisms. In addition, these regimens also promote the development of tolerance when hapten is applied to the UVR‐exposed site at the completion of the radiation treatment protocol. A third immune abnormality is also observed in mice exposed to acute, low‐dose UVR. This abnormality, which develops within 48–72 hr of the completion of the UVR regimen, has been described among antigen‐presenting cells within secondary lymphoid organs, including lymph nodes that do not drain the site of irradiation. Dendritic cells (DCs) from lymph nodes and spleens of mice exposed to UVR lack the capacity to induce CH if they are derivatized with hapten and injected intracutaneously into naive mice. The DC defect is related to the production of and systemic dissemination of interleukin‐10 (IL‐10) by keratinocytes within the epidermis of the UVR‐exposed skin. We have now examined the nature of the functional aberration that exists among DCs within the secondary lymphoid organs of UVR‐exposed mice by examining the capacity of DCs to express co‐stimulatory molecules, and their ability to activate ovalbumin (OVA) ‐specific DO11.10 T‐cell receptor transgenic T cells in vitro. Our results indicate that DCs from UVR‐exposed mice produced insufficient amounts of IL‐12. When pulsed with OVA, these cells were capable of inducing proliferation among DO11.10 T cells in vitro, but the responding cells produced neither IFN‐γ nor IL‐10 and IL‐4. A similar antigen‐presenting cell defect was generated in mice treated with a subcutaneous injection of IL‐10. We conclude that acute, low‐dose UVR creates an IL‐10‐dependent functional deficit in DCs in secondary lymphoid organs, and that this defect robs UVR‐exposed mice of the capacity to develop CH when hapten is painted epicutaneously.
Antibody cross‐linking of human CD9 and the high‐affinity immunoglobulin E receptor stimulates secretion from transfected rat basophilic leukaemia cells
Authors: Higginbottom A. , Wilkinson I. , McCullough B. , Lanza F. , Azorsa D. O. , Partridge L. J. , Monk P. N.
Published: March 2003

Previous studies have shown that antibody cross‐linking of the tetraspanin protein CD9 stimulates the degranulation of platelets and eosinophils, although the mechanism of activation is unclear. In this work we transfected human CD9 into the rat basophilic leukaemia (RBL‐2H3) cell line and studied the stimulation of secretion from these cells in response to a panel of anti‐CD9 antibodies. Intact immunoglobulin G1 (IgG1) antibodies activated transfected cells whereas F(ab′)2 fragments of antibody and an intact IgG2a did not. Stimulation of secretion was inhibited by co‐incubation with monomer murine immunoglobulin E (IgE) but not with an IgG1 isotype control, indicating that the response involves the endogenous high‐affinity IgE receptor (FcεRI). The anti‐CD9 antibody activation curve was biphasic, and supraoptimal antibody concentrations stimulated little or no degranulation, indicating that multivalent binding of human CD9 molecules is necessary for the formation of an active complex with rat FcεRI. Immunoprecipitation of FcεRI under mild detergent conditions co‐precipitated CD9, suggesting the presence of pre‐existing complexes of CD9 and FcεRI that could be activated by antibody cross‐linking. These data are further evidence that tetraspanins are involved in FcεRI signalling and may reflect the participation of tetraspanins in the formation of complexes with other membrane proteins that use components of Fc receptors for signal transduction.
Pentoxifylline treatment of mice with chronic pulmonary tuberculosis accelerates the development of destructive pathology
Authors: Turner J. , Frank A. A. , Brooks J. V. , Marietta P. M. , Orme I. M.
Published: March 2003

It is well established in animal models that production of the cytokine tumour necrosis factor‐α (TNF‐α) is essential to the proper expression of acquired specific resistance following infection with Mycobacterium tuberculosis. This gives rise to an apparent state of chronic disease which over the next 100–200 days is characterized by slowly worsening pathological changes in the lung. To determine whether continued TNF‐α production was harmful during this phase mice were treated with a TNF‐α inhibitor, pentoxifylline. It was observed that although this therapy did not alter the numbers of bacteria recovered from the lungs of the infected mice, tissue damage within the lung was accelerated. These data thus demonstrate that production of TNF‐α, already known to be important during the early expression of resistance to tuberculosis, remains important and beneficial during the chronic stage of the disease.
Kinetics of GATA‐3 gene expression in early polarizing and committed human T cells
Authors: Lantelme E. , Mantovani S. , Palermo B. , Campanelli R. , Sallusto F. , Giachino C.
Published: March 2003

Different transcription factors have been shown to control the transition of naive T cells into T helper 1 (Th1)/Th2 subsets. The T‐cell‐specific transcription factor GATA‐3 is known to be selectively expressed in murine developing Th2 cells and to exert a positive action on Th2‐specific cytokine production. Investigating GATA‐3 gene regulation in human T cells we have found that naive T cells highly express GATA‐3, and during early T2 or T1 polarization, respectively, they either maintain or quickly down‐regulate expression. In developing T2 cells, as well as in committed Th2 cell lines and clones, we found a positive correlation among GATA‐3, interleukin (IL)‐5 and IL‐4 gene expression kinetics, supporting the positive action of GATA‐3 on Th2‐specific cytokine production. A possible relationship between GATA‐3 gene expression and the down‐regulation of the IL‐12 receptor (β2‐chain; IL‐12Rβ2) gene was evident only in the early phases of T2 polarization (within 24 hr), and not demonstrated at later times. During T‐cell commitment the presence of IL‐4 in the culture was essential to maintain or enhance GATA‐3 transcription, while IL‐12 was not necessary for full repression of GATA‐3. Finally, we showed selective GATA‐3 up‐regulation in human Th2 cell lines and clones and the maintainance of a low basal level of GATA‐3 expression in Th1 cells upon activation.
Detection of anaphylatoxin receptors on CD83+ dendritic cells derived from human skin
Authors: Kirchhoff Konstanze , Weinmann Oliver , Zwirner Jörg , Begemann Gabriele , Götze Otto , Kapp Alexander , Werfel Thomas
Published: March 2003

Dendritic cells (DC) are recruited to sites of inflammation for the initiation of immune responses. As the anaphylatoxins C5a and C3a are important mediators of inflammation, we investigated the expression of their receptors (C3aR and C5aR) on human DC. DC were isolated from human skin or generated from purified blood monocytes and were identified by their expression of CD1a or CD83. Freshly isolated or cultured dermal CD1a+ and CD83+ DC bound anti‐C5aR and anti‐C3aR monoclonal antibodies (mAbs), as detected by flow cytometry. C5a induced calcium fluxes in dermal CD1a+ and CD83+ DC, which could be inhibited by C17/5, an anti‐C5a mAb. C3a did not induce calcium fluxes in these cells. Anaphylatoxin receptor expression was down‐regulated on dermal DC by adding tumour necrosis factor‐α (TNF‐α) to the culture medium. On CD1a+ CD83− cells generated from isolated blood monocytes by culture with 6·25 ng/ml of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and 125 U/ml of interleukin‐4 (IL‐4), expression of both C5aR and C3aR was observed. In these cells, both C5a and C3a induced calcium fluxes. After addition of TNF‐α to the culture medium, the majority of the CD1a+ cells expressed CD83+. These cells – expressing a phenotype of ‘mature DC’– down‐regulated the expression of the anaphylatoxin receptors and lost their reactivity to the respective ligands. Our results demonstrate the expression of the anaphylatoxin receptors C5aR and C3aR on human skin‐derived DC and blood‐derived cells expressing the DC‐associated membrane molecule, CD1a. Furthermore, the expression of anaphylatoxin receptors on CD83+ dermal DC is indicative of an intermediate stage of maturation of these cells, which was not observed on in vitro‐differentiated CD83+ cells.
Immunostimulation by the synthetic lipopeptide P3CSK4: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages
Authors: Müller Markus R. , Pfannes Silke D. C. , Ayoub Mohamed , Hoffmann Petra , Bessler Wolfgang G. , Mittenbühler Klaus
Published: March 2003

Synthetic lipopeptides based on bacterial lipoprotein are efficient activators for monocytes/macrophages inducing the release of interleukin (IL)‐1, IL‐6, tumour necrosis factor‐α (TNF‐α), reactive oxygen/nitrogen intermediates, and the translocation of nuclear factor κB (NFκB). In this report we investigate the signal transduction pathways involved in leucocyte activation by the synthetic lipopeptide N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2R,S)‐propyl]‐(R)‐cysteinyl‐seryl‐(lysyl)3‐lysine (P3CSK4). We show that P3CSK4 activates mitogen‐activated protein (MAP)‐kinases ERK1/2 and MAP kinase (MAPK)‐kinases MEK1/2 in bone‐marrow‐derived macrophages (BMDM) and in the macrophage cell line RAW 264·7. Additionally, we could detect differences between the P3CSK4 and lipopolysaccharide (LPS)‐induced phosphorylation of MAP kinases: Different levels in phosphorylation were found both in kinetics and dose–response using RAW 264·7 cells or BMDM from BALB/c and LPS responder mice (C57BL/10ScSn) or LPS non‐responder mice (C57BL/10ScCr). The lipopeptide activated the MAPK‐signalling cascade in both LPS responder and non‐responder macrophages, whereas LPS induced the MAPK signalling pathway only in macrophages derived from LPS responder mice. An approximately 70% decrease of lipopeptide induced NFκB translocation and an about 50% reduction of nitric oxide (NO) release was observed in the presence of anti‐CD14. These data correspond to the reduction of phosphorylation of ERK1/2 after stimulation with P3CSK4 in the presence of anti‐CD14 antibodies. Inhibition of MEK1/2 by PD98059 completely reduced the lipopeptide‐induced phosphorylation of ERK1/2 indicating that MEK1/2 are solely responsible for the phosphorylation of the downstream‐located MAP kinases ERK1/2.
Intensive generation of NK1.1– extrathymic T cells in the liver by injection of bone marrow cells isolated from mice with a mutation of polymorphic major histocompatibility complex antigens
Authors: Halder R. C. , Kawamura T. , Bannai M. , Watanabe H. , Kawamura H. , Mannoor M. K. , Morshed S. R. M. , Abo T.
Published: March 2003

Whether intermediate TCR (TCRint) cells and natural killer T (NKT or NK1.1+TCRint) cells are extrathymically generated remains controversial. This arises from the fact that there are few of these T cells in athymic nude mice and neonatally thymectomized mice. However, when athymic mice were provided with appropriate microenvironments or stimulation, many TCRint cells (mainly NK1.1−) were found to arise in the liver. NKT cells are known to be positively selected by monomorphic major histocompatibility complex (MHC) ‐like antigens (e.g. CD1d). This is true even if they are CD4+. In other words, a MHC class I‐like antigen is restricted to CD4 antigen. This rule is somewhat different from that seen in conventional T cells (i.e. the restriction of class II with CD4 and that of class I and CD8). In the case of NK1.1−TCRint cells, they were selected by polymorphic MHC antigens, but their MHC restriction to CD4 or CD8 antigen was incomplete. This was revealed by experiments of bone marrow transfer with class I (bm 1) or II (bm 12) disparity. Depending on the disparity, a unique cytokine profile in sera was detected. These results suggest that the development of T lineage lymphocytes and MHC restriction to CD4 and CD8 might have occurred in parallell as a phylogenic event, and that NK1.1− extrathymic T cells (i.e. NK1.1−TCRint) are at an intermediate position between NKT cells and conventional T cells in phylogeny.
Immunoglobulin VH‐gene usage of autoantibodies in mercuric chloride‐induced membranous glomerulopathy in the rat
Authors: Dammers Peter M. , Bun Judy C. A. M. , Bellon Blanche , Kroese Frans G. M. , Aten Jan , Bos Nicolaas A.
Published: March 2003

Brown‐Norway (BN) and Dorus Zadel Black (DZB) rats develop a T‐cell‐dependent membranous glomerulopathy (MGP) with high proteinuria and antiglomerular basement membrane (GBM) autoreactive antibodies (Abs), upon exposure to mercuric chloride (HgCl2). Laminin is an important autoantigenic target of the anti‐GBM Abs, absorbing ≈ 30% of the anti‐GBM reactivity. Although many anti‐GBM Abs have undergone isotype switching, it is currently unclear whether affinity maturation occurs during the HgCl2‐induced autoimmune response. To address this question we analysed the rearranged immunoglobulin heavy chain variable‐region genes (VHDJH regions) of 15 mAbs that were previously obtained from HgCl2‐treated rats. Seven of these mAbs exhibit reactivity towards laminin. Our study showed that the VH‐gene usage of antilaminin mAbs is largely restricted to the PC7183 VH‐gene family (six out of seven). In addition, we demonstrated that at least three out of six laminin reactive and five out of six non‐laminin‐binding mAbs are encoded by germline VH genes (a total of eight out of 12 mAbs). Of the eight mAbs that are encoded by germline VH genes, seven are of a non‐immunoglobulin M (IgM) isotype, indicating that isotype switching has occurred in these mAbs in the absence of somatic mutations. The mutations observed in the VH genes of the four remaining mAbs do not provide strong evidence for antigenic selection. The data support the notion that B cells in this model of MGP are not subjected to affinity maturation and probably result from polyclonal B‐cell activation.
Specific immunoglobulin E responses in ZAP‐70‐deficient patients are mediated by Syk‐dependent T‐cell receptor signalling
Authors: Toyabe Shin‐ichi , Watanabe Akihiro , Harada Waka , Karasawa Tamaki , Uchiyama Makoto
Published: March 2003

ZAP‐70 deficiency is a rare primary immunodeficiency characterized by the absence of peripheral CD8+ T cells and defects in T‐cell receptor (TCR) signalling. T cells in ZAP‐70‐deficient patients are assumed to have no helper functions for B‐cell immunoglobulin synthesis, whereas the patients rarely have antigen‐specific antibodies. We experienced a ZAP‐70‐deficient patient, who had immunoglobulin E (IgE) antibodies specific to food allergens, and we investigated the mechanisms of switching to IgE in the patient. Peripheral blood mononuclear cells from the patient did not proliferate upon stimulation with the antigens but produced distinct levels of interleukin‐4 (IL‐4). Cell sorting analysis indicated that the cells that produced IL‐4 in response to the antigens were enriched in CD4+ T cells. Purified CD4+ T cells from the patient produced IL‐4 and expressed CD40L upon stimulation with anti‐CD3. Moreover, CD4+ T cells pretreated with anti‐CD3 induced mature ε transcript on naive B cells. Since the results indicated that there remained sufficient T‐cell receptor (TCR)‐signalling in the patient's T cells to exert antigen‐specific IgE switching on B cells, we next investigated the expression of the ZAP‐70‐homologous kinase Syk. Syk was present in high levels in patient's CD4+ T cells and was tyrosine‐phosphorylated after TCR stimulation. Inhibition of Syk by piceatannol resulted in decreased production of IL‐4 and expression of CD40L on patient's CD4+ T cells. Moreover, Syk was expressed on all human T‐cell leukaemia virus (HTLV‐1)‐transformed T‐cell lines derived from peripheral blood of the patient, whereas it was low or undetectable in control lines. It was therefore concluded that specific IgE responses in the patient were most likely to be mediated by Syk‐dependent TCR‐signalling.
Tumour necrosis factor‐α potentiates CR3‐induced respiratory burst by activating p38 MAP kinase in human neutrophils
Authors: Forsberg Maria , Löfgren Ragnhild , Zheng Limin , Stendahl Olle
Published: March 2003

CR3 and FcγRs are the main receptors involved in the phagocytic process leading to engulfment and killing of microbes by production of reactive oxygen intermediates (ROI) and degranulation. Various inflammatory mediators, such as tumour necrosis factor‐α (TNF‐α) and lipopolysaccharide (LPS), are known to prime neutrophils leading to increased bactericidal responses, but the underlying mechanism of priming has only been partially elucidated. The purpose of this study was to investigate how TNF‐α primes neutrophils for subsequent stimuli via either CR3 or FcγR. The receptors were specifically activated with pansorbins (protein‐A‐positive Staphylococcus aureus) coated with anti‐CR3, anti‐FcγRIIa, or anti‐FcγRIIIb monoclonal antibody. Activation of neutrophils with these particles resulted in ROI production as measured by chemiluminescence. Anti‐CR3 pansorbins induced the most prominent ROI production in neutrophils. TNF‐α potentiated the CR3‐mediated respiratory burst but had little effect on that mediated by FcγRs. The priming effect of TNF‐α on CR3‐mediated ROI production is associated with an increased activation of p38 MAPK as well as tyrosine phosphorylation of p72syk. Pretreatment of neutrophils with the inhibitors for p38 MAPK and p72syk markedly suppressed the respiratory burst induced by CR3. Furthermore, TNF‐α induced about a three‐fold increase in the expression of CR3 in neutrophils, an effect which is blocked by the p38 MAPK inhibitor. Taken together, these results showed that TNF‐α potentiates the CR3‐mediated respiratory burst in neutrophils not only by triggering a p38 MAPK‐dependent up‐regulation of CD11b/CD18 but also by modulating the signalling pathways.
Topical tacrolimus and cyclosporin A differentially inhibit early and late effector phases of cutaneous delayed‐type and immunoglobulin E hypersensitivity
Authors: Geba Gregory P. , Ptak Wlodzimierz , Askenase Philip W.
Published: March 2003

Systemic and topical administration routes of tacrolimus and cyclosporin A (CsA) were compared in effects on early and late phases of elicited T‐cell‐mediated contact sensitivity (CS), and effects on early and late phases of cutaneous immunoglobulin E (IgE) antibody‐mediated hypersensitivity responses in mice. Thus, both CS and IgE responses in the skin have an early mast‐cell‐dependent phase, and also a late inflammatory phase. We measured the effects of both immunosuppressants on both phases of the respective T cell versus IgE responses. Systemic administration of both agents completely suppressed CS and IgE late‐phase responses, but failed to affect either early phase. In contrast, when topical CsA was used, low doses abolished the early phase of IgE responses, but even high doses did not inhibit the early phase of CS. Conversely, topical tacrolimus inhibited the early phase of CS more potently than the early phase of cutaneous IgE hypersensitivity responses. Thus, topical treatment was needed to inhibit the early phases and the two agents acted differentially, suggesting differing susceptibility of the early phases, that are probably due to different signalling mechanisms. These studies underscore the potential value of topical administration of these powerful immunosuppressive agents in the treatment of allergic diseases that exhibit features of early‐phase mast‐cell‐dependent inflammation, and late inflammation due to mast cells or to T cells, such as atopic dermatitis or asthma, since the early phase is predominantly susceptible to topical application, while the last phase of both IgE and T‐cell inflammation responds to systemic treatment with both agents.
Granule‐dependent killing of Toxoplasma gondii by CD8+ T cells
Authors: Nakano Yoko , Hisaeda Hajime , Sakai Tohru , Zhang Manxin , Maekawa Yoichi , Zhang Tianqian , Nishitani Masaaki , Ishikawa Hiroyuki , Himeno Kunisuke
Published: March 2003

Immunization of mice with live bradyzoites of a low‐virulent Beverley strain of Toxoplasma gondii has been shown to increase CD8+ T‐cell mediated immunity against a highly virulent RH strain. We found that preimmunization with an RH homogenate further enhanced this immunity. Using this model, we investigated the mechanism of CD8+ T‐cell mediated protection against T. gondii infection. Splenic cells from mice immunized with RH homogenate and live bradyzoites stimulated apoptosis of RH‐infected J774A.1 macrophages in vitro, and at the same time, the immunization significantly suppressed the proliferation of parasites within macrophages, as assessed by measuring 3H‐uracil uptake by the parasites. Splenic cells from the immunized mice produced larger amounts of interferon‐γ (IFN‐γ) than did naive splenic cells; however, the production of nitric oxide (NO) by RH‐infected macrophages was not enhanced. The elimination of CD8+ T cells from splenic cells significantly reduced their inhibitory action on parasite proliferation as well as their cytotoxic activity against RH‐infected macrophages, but it did not affect the production of IFN‐γ. Treatment of CD8+ T‐enriched splenic cells from the immunized mice with concanamycin A, but not an anti‐Fas ligand monoclonal antibody, significantly reduced their anti‐proliferative and killing capabilities, suggesting that the CD8+ T cells induced by immunization with RH antigen and live bradyzoites of the Beverley strain may exert protection against T. gondii infection at least in part through granule‐dependent cytotoxic activities.
T‐cell activation, proliferation and apoptosis in primary Listeria monocytogenes infection
Authors: Mannering Stuart I. , Zhong Jie , Cheers Christina
Published: March 2003

Listeria monocytogenes infection of mice leads to a rapid expansion of activated T cells, followed by a decline in specific cells once the bacteria are eliminated. In order to define the relationship between T‐cell proliferation and activation, and to investigate the role of apoptosis in limiting the expansion, the expression of activation markers, uptake of 5‐bromo‐2′‐deoxyuridine (BrdU) in vivo and the incidence of apoptosis was investigated. Increased numbers of T cells expressing the activated phenotype CD25+, CD44hi and CD62Llo were detected 4 days after infection. Expression of CD25 (IL‐2Rα chain) on CD4+ and CD8+ T cells peaked at this time and returned to normal by day 7. In contrast, CD44hi and CD62Llo persisted, with the maximum proportion occurring at 7 days after infection. This was accompanied by a burst of in vivo proliferation of CD4+ and CD8+ T cells occurring between day 5 and 7. Apoptosis, which is presumably needed to control this expansion of T cells, also peaked at 7 days after infection. Apoptosis occurred preferentially amongst T cells which had proliferated. Most but not all proliferating T cells had down‐regulated their CD62L marker. While most apoptotic T cells were CD62Llo, again not all had down‐regulated this marker. Hence, CD25 expression peaked early, but expression of other activation markers, in vivo proliferation and apoptosis coincided after Listeria infection. T cells that had proliferated were over‐represented in the apoptotic population.
Characterizing a soluble survival signal for activated lymphocytes from CD14+ cells
Authors: Tang Xiaolei , Yocum David E. , DeJonghe David , Nordensson Kathy
Published: March 2003

T‐cell activation requires at least two signals: antigen and a costimulatory signal. As antigen‐presenting cells play an important role in this area, the role of CD14+ cells in T‐cell activation, proliferation and activation‐induced cell death (AICD) was investigated. Using phytohaemagglutinin (PHA) activation, it was found that CD14+ cell depletion resulted in significantly greater AICD, decreased lymphocyte growth and up‐regulated interleukin‐2 (IL‐2) secretion. However, T‐cell activation was delayed according to the expression of CD69 and CD25. Dynabeads conjugated with anti‐CD14 monoclonal antibody (mAb) bound CD14+ cells and induced secretion of IL‐1β, tumour necrosis factor‐α (TNF‐α), transforming growth factor‐β (TGF‐β) and IL‐6, but not IL‐2, IL‐12 or IL‐15. Supernatants were collected from Dynabeads‐activated CD14+ cell cultures and designated as ‘CD14 cocktails’. Addition of CD14 cocktails to CD14+ cell‐depleted mononuclear cell cultures reversed the increased AICD, decreased lymphocyte growth and increased IL‐2 secretion. Depletion of IL‐1β and TNF‐α in the CD14 cocktails by panning followed by blocking with the corresponding mAbs had no effect on the active AICD protection. TGF‐β was determined not to be the active factor owing to the presence of >1·0 ng of TGF‐β in the media for culturing both CD14+ and CD14− peripheral blood mononuclear cells (PBMC). The CD14 cocktails did not contain IL‐12 and IL‐15. Depletion of IL‐6 with panning followed by blocking residual IL‐6 with anti‐IL‐6 mAb significantly reduced the protective effect of the CD14 cocktails. Human recombinant IL‐6 also partially reversed the effects of CD14+ cell depletion on AICD, lymphocyte growth and IL‐2 secretion. The data suggest that IL‐6 is one of the active factors in the survival signal from CD14+ cells.