bluuebunny/crossref_metadata_2025 huggingface dataset:
Human epidermal keratinocytes are induced to secrete interleukin‐6 and co‐stimulate T lymphocyte proliferation by a CD40‐dependent mechanism
Authors: Gaspari Anthony A. , Sempowski Gregory D. , Chess Patricia , Gish Julia , Phipps Richard P.
Published: March 2007

Although the expression and function of CD40 on B lymphocytes has been well studied, the significance of CD40 on non‐lymphoid cells such as keratinocytes (KC) is not as well characterized. We demonstrate in this report that CD40 is expressed by virtually all human KC, and that it functions as an important signaling molecule. Flow cytometry of undifferentiated and terminally differentiated KC indicated that both cell types expressed CD40, as determined by binding to monoclonal antibodies and a recombinant CD40 ligand fusion protein; interferon‐γ (IFN‐γ) treatment of KC increased CD40 expression. Cultured KC also expressed 1.5‐kb CD40 transcripts. Activation of KC cell surface CD40 using the monoclonal antibody G28.5 resulted in the rapid generation of a 50‐kDa tyrosine phosphorylated polypeptide, as well as a dose‐dependent increase in the secretion of interleukin‐6, a cytokine that has been linked to KC proliferation. KC also co‐stimulated a significant T lymphocyte proliferative response to the mitogen phytohemagglutinin that was CD40 dependent. These data indicate that KC constitutively express a low level of functional CD40 and regulate their expression in response to IFN‐γ. These data support the concept that KC, via their expression of CD40, have the capacity to amplify inflammation in the skin by interacting with CD40 ligand‐bearing T cells.
Detection of primary direct and indirect human anti‐porcine T cell responses using a porcine dendritic cell population
Authors: Dorling Anthony , Lombardi Giovanna , Binns Richard , Lechler Robert I.
Published: March 2007

The pathways of human anti‐pig T cell xenorecognition have been investigated. Freshly isolated porcine alveolar lavage (AL) cells induced primary proliferative responses by human peripheral and cord blood mononuclear cells which were inhibited by anti‐HLA‐DR antibody (indirect xenorecognition). Following overnight culture, the AL cells acquired the capacity to stimulate proliferation by purified human T cells which was inhibited by anti‐SLA‐DR antibody (direct xenorecognition). The marked increase in immunogenicity in the porcine AL cells was accompanied by a phenotypic change consistent with dendritic cell maturation. Limiting dilution assays indicate that the total anti‐pig T cell response, in particular that mediated by indirect xenorecognition, is stronger than comparable alloresponses.
Contribution of interleukin‐3 to antigen‐induced Th2 cytokine production
Authors: Aoki Ichiro , Tanaka Shun‐Ichi , Ishii Norihisa , Minami Mutsuhiko , Klinman Dm
Published: March 2007

Short‐term stimulation of mouse spleen cells in vitro with interleukin (IL)‐3 induces the secretion of the Th2 cytokines IL‐4 and IL‐6. Non‐B/non‐T cells were the target of this IL‐3 effect. However, during long‐term antigen‐dependent culture, T cells are the major source of IL‐4 and IL‐6. The addition of IL‐3 to such cultures also led to a significant increase in IL‐4 and IL‐6 production. This Th2 cytokine secretion was amplified by the addition of irradiated non‐B/non‐T cells at the initiation of culture, and was inhibited by anti‐IL‐4 antibodies. These findings suggest that IL‐3 induces the rapid release of IL‐4 and IL‐6 by non‐B/non‐T cells, thereby creating an immune milieu conducive to the development of antigen‐specific IL‐4 and IL‐6‐secreting Th2 cells.
Existence of a small population of IL‐2Rβhi TCRint cells in SCG and MRL‐lpr/lpr mice which produce normal Fas mRNA and Fas molecules from the lpr gene
Authors: Yamagiwa Satoshi , Kuwano Yuh , Hasegawa Katsuhiko , Sato Kazunari , Ohtsuka Kazuo , Iiai Tsuneo , Tomiyama Katsuhiro , Watanabe Hisami , Sugahara Satoshi , Seki Shuhji , Asakura Hitoshi , Abo Toru
Published: March 2007

Mice carrying the lpr gene, SCG and MRL‐lpr/lpr mice, were used to characterize the phenotype and lpr gene of abnormally proliferating T cells in these mice. A major population which expanded in these mice were T cells expressing intermediate (int) levels of T cell receptor (TCR) (and CD3) and the phenotype of interleukin‐2 receptor (IL‐2R)βlo α− (possibly abnormal TCRint cells). The levels of TCRhi cells of thymic origin (generated through the mainstream of T cell differentiation in the thymus) profoundly decreased after the onset of disease. However, a small population of normal TCRint cells (i.e. IL‐2Rβhi α−) were also found to exist in all tested organs. For example, the majority of abnormal IL‐2Rβlo TCRint cells were CD4−8− CD2−, while normal IL‐2Rβhi TCRint cells were a mixture of single‐positive cells (mainly CD8+), CD4−8− cells and CD2+ cells. Moreover, normal TCRint cells preferentially produced normal Fas mRNA and Fas molecules from the lpr gene. This phenomenon explains the leaky appearance of normal Fas mRNA and Fas molecules in mice carrying the lpr gene. It is suggested that a small population of IL‐2RβhiTCRint cells are resistant to the lpr genetic abnormality.
Selective suppression of endothelial cell activation by arachidonic acid
Authors: Stuhlmeier Karl M. , Tarn Chi , Csizmadia Vilmos , Bach Fritz H.
Published: March 2007

Endothelial cell (EC) activation plays a key role in inflammation, thrombosis and organ rejection. Normally, EC are in a quiescent state in which their function is to prevent coagulation and thrombosis, and to participate in the regulation of leukocyte migration from the bloodstream into the tissue. Upon activation with cytokines or other stimuli, EC up‐regulate a number of genes, including E‐selectin (ELAM‐1), intercellular adhesion molecule (ICAM)‐1, vascular cell adhesion molecule (VCAM)‐1, interleukin (IL)‐1, IL‐8, tissue factor (TF), plasminogen activator inhibitor‐1 (PAI‐1), MCP‐1 (monocyte chemoattractant protein‐1) and endothelial cell inducible gene (ECI‐6). Arachidonic acid (AA) is produced by several cell types, including EC, and acts on various cells. We report here that AA inhibits the up‐regulation of some, but not all genes that are induced with EC activation in a dose‐dependent manner. AA suppresses TNF‐α, IL‐1α, LPS or PMA‐induced E‐selectin expression, as well as mRNA accumulation of E‐selectin, ICAM‐1 and IL‐8 stimulated by TNF‐α. The inhibition appears to be at the level of transcription. At the same time under the same conditions AA does not, repress mRNA accumulation for PAI‐1, ECI‐6, MCP‐1 and VCAM‐1. We suggest that the induced expression of AA with EC activation may result in a negative feedback loop regulating further activation.
Mouse γδ TCR+NK1.1+ thymocytes specifically produce interleukin‐4, are major histocompatibility complex class I independent, and are developmentally related to αβ TCR+NK1.1+ thymocytes
Authors: Vicari Alain P. , Mocci Simonetta , Openshaw Peter , O'Garra Anne , Zlotnik Albert
Published: March 2007

Mouse T cells co‐expressing an αβ T cell receptor (TCR) and the NK1.1 antigen have been shown to be major interleukin (IL)‐4‐producing cells and could therefore regulate cell‐mediated immune responses. We have identified a related subset of thymocytes co‐expressing a γδ TCR and NK1.1 which also produce IL‐4. Unlike αβ+NK1.1+ thymocytes, the selection of γδ+NK1.1+ thymocytes is not dependent upon β2‐microglobulin (β2m)‐associated class I molecule expression because these cells are present in β2m‐deficient mice. This suggests that γδ+NK1.1+ T cells may regulate immune responses to a different variety of antigens. However, the development of αβ+NK1.1+ and αβ+NK1.1+ thymocytes appears to be related. Analysis of different mutant mice lacking αβ+NK1.1+ thymocytes revealed a specific increase in γδ+NK1.1+ thymocyte production when the block in αβ+NK1.1+ thymocyte differentiation occurs after β TCR rearrangement.
Functional CD40 ligand expression on T lymphocytes in the absence of T cell receptor engagement: involvement in interleukin‐2‐induced interleukin‐12 and interferon‐γ production
Authors: Armant Myriam , Armitage Richard , Boiani Norman , Delespesse Guy , Sarfati Marika
Published: March 2007

Despite the fact that the great majority of T cells at the site of an inflammatory response are not antigen specific, the mechanisms leading to activation and recruitment of these bystander T cells are poorly understood. We previously reported that soluble (s)CD23 potentiated the interleukin (IL)‐2‐induced interferon (IFN)‐γ production by T cells co‐cultured with autologous monocytes in the absence of T cell receptor (TCR) engagement. Our present data demonstrate that the IL‐2‐induced IFN‐γ secretion, in the presence but also in the absence of sCD23, is strictly IL‐12 dependent, inasmuch as anti‐IL‐12 antibody abrogated both responses. Most interestingly, anti‐CD40 ligand (CD40L) monoclonal antibody significantly inhibited IL‐2‐induced IL‐12 as well as IFN‐γ production. These results suggest that CD40L was expressed on T cells in the absence of TCR engagement. Indeed, purified unstimulated T cells readily expressed CD40L. IL‐2 and monocytes did not up‐regulate CD40L on resting T cells. It is proposed that low levels of CD40L expression on non‐antigen stimulated T cells are sufficient to signal through CD40 molecules on accessory cells and to induce IL‐12 secretion, which in turn can synergize with IL‐2 for the induction of IFN‐γ production, thus contributing to the inflammatory process.
Homocysteine modification of HLA antigens and its immunological consequences
Authors: Gao Xiao‐Ming , Wordsworth Paul , McMichael Andrew J. , Kyaw Myo M. , Seifert Martin , Rees David , Dougan Gordon
Published: March 2007

Homocysteine‐treated cells can be specifically lysed by cytotoxic T lymphocytes (CTL) identifiable in patients with ankylosing spondylitis and reactive arthritis. Sensitization of target cells involves disulfide bonding and the interaction between homocysteine and HLA antigens occurs in a pre‐Golgi compartment in the cells. Salmonella‐infected B cells are also lysed by homocysteine‐specific CTL, suggesting that intracellular invading microorganisms may provide homocysteine which would gain access to the newly synthesized intracellular HLA molecules and modify them inside the cells. Two different mechanisms for homocysteine modification of HLA antigens are proposed: homocysteine could bind directly to the unpaired cysteine residues in HLA antigens, or it could bind indirectly to HLA antigens through cysteine‐containing peptides bound to them. Thus, HLA antigens containing unpaired cysteine residues (e.g. HLA B27) could be modified by homocysteine directly or indirectly, while HLA antigens without unpaired cysteine residues (e.g. HLA A68) could only be modified indirectly. The results are discussed in relation to the potential involvement of homocysteine‐specific CTL in ankylosing spondylitis and reactive arthritis, both of which are related to bacterial infections, associated with HLA B27, and considered to be autoimmune diseases.
T cell activation by concanavalin A in the presence of cyclosporin A: immunosuppressor withdrawal induces NFATp translocation and interleukin‐2 gene transcription
Authors: Bemer Véronique , Truffa‐Bachi Paolo
Published: March 2007

Cyclosporin A (CSA), an immunosuppressive agent used in organ transplantation and to treat some autoimmune diseases, blocks the Ca2+‐dependent steps involved in T cell receptor triggering leading to interleukin (IL)‐2 production. Considering that the early steps of T cell activation are insensitive to CSA, we asked whether the initial activation achieved in presence of this immunosuppressor could affect the capacity of the T cell to respond to a mitogenic restimulation. We found that T cells activated by concanavadin A (ConA) for 48 h in the presence of CSA retain the capacity to proliferate in response to ConA once the immunosuppressor is removed. These cells are able to transcribe anew the IL‐2 gene, without the requirement of new protein synthesis, and to up‐regulate the α chain of the IL‐2 receptor. Furthermore, we present the first direct evidence that the nuclear factor AP‐1 is present in the nucleus of the T cells primed for 48 h in presence of CSA and that withdrawal of the immunosuppressor leads to the translocation of NFATp from the cytoplasm to the nucleus.
Altered expression of host‐encoded complement regulators on human cytomegalovirus‐infected cells
Authors: Spiller O. Brad , Morgan B. Paul , Tufaro Frank , Devine Dana V.
Published: March 2007

Human cytomegalovirus (HCMV)‐infected cells persist in the presence of anti‐HCMV antibody, suggesting that HCMV has evolved mechanisms to evade host immune defenses. Insofar as no virus‐encoded complement inhibitors have been identified for HCMV, we hypothesized that HCMV infection may alter the expression of host‐encoded cell surface complement inhibitors. Herein, we report that cell surface expression of two complement regulator proteins, CD55 and CD46, which are members of the regulators of complement activation (RCA) gene cluster, increased up to eightfold following infection of fibroblasts or glioblastoma cells with HCMV, but not after infection with HSV‐1 or adenovirus. However, the cell surface expression of a third complement regulator, CD59, which is not a member of the RCA gene cluster, was not altered during HCMV infection. Functional studies using purified complement components demonstrated that up‐regulation of CD55 suppressed the activity of cell‐associated C3 convertases on HCMV‐infected cells. Furthermore, increased CD55 expression protected infected cells from complement‐mediated lysis, an effect which directly correlated with the length of HCMV infection. Increased expression of host‐encoded complement regulator proteins may provide protection of HCMV‐infected cells from the host immune response in vivo, through increasing the resistance of infected cells to complement‐mediated lysis and decreasing the deposition of C3‐derived products on the cell surface.
A tyrosine‐phosphorylated 110–120‐kDa protein associates with the C‐terminal SH2 domain of phosphotyrosine phosphatase‐1D in T cell receptor‐stimulated T cells
Authors: Frearson Julie A. , Yi Taolin , Alexander Denis R.
Published: March 2007

The role of cytosolic phosphotyrosine phosphatases (PTPase) in T cell receptor (TCR)‐mediated signaling was investigated. PTPase activity was detected in a purified immunocomplex comprising aggregated TCR from the cell surface of Jurkat T cells. Since TCR aggregation results in phosphorylation of critical immunoreceptor tyrosine‐based activation motifs (ITAM) in the TCR ζ chain, a doubly tyrosine‐phosphorylated synthetic peptide containing the membrane‐proximal ζ chain ITAM (ζp ITAM) was used to characterize TCR ζ‐associated PTPases. PTPase activity was detected in stable association with ζp ITAM and the SH2 domain‐containing PTPase PTP‐1D (Syp, SH‐PTP2) was identified in this complex. TCR stimulation resulted in increased total PTPase activity and PTP‐1D protein in ζp ITAM precipitates. TCR stimulation did not result in the tyrosine phosphorylation of PTP‐1D but caused the rapid and transient tyrosine phosphorylation of a 110–120‐kDa protein which associated selectively with the C‐terminal SH2 domain of PTP‐1D. This currently unidentified phosphotyrosine protein may be involved in localizing PTP‐1D to the TCR following receptor stimulation.
Detection of C5a receptors on human eosinophils and inhibition of eosinophil effector functions by anti‐C5a receptor (CD88) antibodies
Authors: Elsner Jörn , Oppermann Martin , Kapp Alexander
Published: March 2007

Eosinophils and complement activation are reported to play a crucial role in the pathogenesis of connective tissue diseases. Depositions of antigens and antigen‐antibody complexes lead to complement activation with the generation of anaphylatoxins, particularly C5a, which is thought to be responsible for the infiltration and activation of eosinophils in the tissue. Previous studies suggested that the eosinophil C5a receptor differs structurally from the receptor expressed on neutrophils. In this study, we investigated the expression and functional properties of C5a receptors on human eosinophils using the C5a receptor monoclonal antibody S5/1 (anti‐CD88 mAb). Flow cytometric analysis demonstrated that the anti‐CD88 mAb bound homogeneously on the surface of human eosinophils from nonatopic healthy donors. In addition, no subpopulations with respect to C5a receptor expression were identified in normodense or hypodense eosinophils of patients with hypereosinophilia. Pre‐incubation of eosinophils with anti‐CD88 specifically inhibited C5a‐induced intracellular calcium concentration transients. C5a‐induced chemotactic activity of eosinophils was significantly inhibited after pre‐incubation of cells with anti‐CD88 mAb in a dose‐dependent manner. Furthermore, anti‐CD88 mAb inhibited dose‐dependently the release of reactive oxygen species by eosinophils following stimulation with C5a. Thus, the human eosinophil C5a receptor is homogeneously expressed on normal eosinophils from healthy donors as well as on hypodense and normodense eosinophil subpopulations from patients with hypereosinophilia. Based on the inhibitory effect of the S5/1 mAb on C5a‐stimulated eosinophil effector functions, we conclude that a single C5a receptor type exists on human eosinophils. In addition, the inhibitory effect of the S5/1 mAb on C5a functions may enable a new experimental approach to the treatment of diseases that have been associated with C5a‐mediated activation.
Differential regulation of monocytic tumor necrosis factor‐α and interleukin‐10 expression
Authors: Meisel Christian , Vogt Katrin , Platzer Cornelia , Randow Felix , Liebenthal Christa , Volk Hans‐Dieter
Published: March 2007

Activation of human monocytes by bacterial endotoxin (LPS) results in an initial burst of inflammatory cytokines like tumor necrosis factor (TNF)‐α which is followed by the secretion of anti‐inflammatory mediators like interleukin (IL)‐10. The signaling pathways in IL‐10 induction are unknown. Here, we show that the regulation of IL‐10 expression is more complex than that of TNF‐α. LPS‐induced TNF‐α and IL‐10 expression requires early activation of protein tyrosine kinases (PTK). Moreover, delayed addition of PTK inhibitors blocked IL‐10, but not TNF‐α, suggesting the impact of a late PTK activity. Two inducers of PTK activity are the downstream mediators of LPS activation, TNF‐α and cyclic adenosine monophosphate (cAMP). Both mediators synergistically up‐regulate IL‐10 expression. Downstream of PTK activation, they use distinct pathways. TNF‐α, but not cAMP‐induced IL‐10 gene expression was inhibited by pyrrolidine dithiocarbamate, suggesting the involvement of reactive oxygen species. Inhibition of protein kinase C (PKC) suppressed LPS‐induced TNF‐α and IL‐10 expression as well, but, unlike TNF‐α, direct activation of PKC by phorbol 12‐myristate 13‐acetate (PMA) did not induce IL‐10 expression. Furthermore, PKC is not involved in late events of IL‐10 activation, as delayed addition of PKC inhibitors did not suppress LPS‐induced IL‐10 expression and did not influence cAMP‐ or TNF‐α‐induced IL‐10. The modulation of IL‐10 expression by inflammatory mediators suggests a regulatory circuit of the inflammatory response.
Structural properties and mutation patterns of anti‐nucleosome monoclonal antibodies are similar to those of anti‐DNA antibodies
Authors: Brard Frédéric , Jovelin Fabien , Petit Samuel , Tron François , Gilbert Danièle
Published: March 2007

Four monoclonal antibodies (mAb) derived from an (NZB × NZW)F1 mouse bound to nucleosomes, total histones and to the H2A‐H2B dimers but not to individual histones or DNA. Sequencing of their heavy (H)‐ and light (L)‐chain variable region genes showed that they derived by somatic mutations from the same B cell precursor. The distribution of negatively and positively charged amino acids in the H‐chain complementarity‐determining regions was very similar to that observed not only in anti‐H2A‐H2B mAb derived from different lupus‐prone mouse strains but also in anti‐DNA mAb. Combined analysis of the mAb structures and their interactions with immobilized H2A‐H2B dimer or total histones by plasmon resonance allowed us to assign the H‐chain mutations a major role in the binding profiles of these anti‐nucleosome mAb. Interestingly, four of the five H‐chain mutations that distinguished mAb 3F6 from 2E1 generated negatively or positively charged amino acid residues, and two of them occurred at positions 56 and 76, which are frequently involved in the maturation process of anti‐DNA antibodies. A modeling study of the 3F6 variable fragment (Fv) predicted that acidic residues occupy the cleft of the Ab combining site and have the potential to participate in electrostatic interactions. Thus, the demonstration that (NZB × NZW)F1‐derived anti‐H2A‐H2B antibodies share certain structural features and mutation patterns with anti‐DNA mAb suggest that common selection and maturation processes account for the production of these lupus‐related autoantibodies.
Germinal center formation in mice lacking αβ T cells
Authors: Dianda Lee , Gulbranson‐Judge Adam , Pao William , Hayday Adrian C. , Maclennan Ian C. M. , Owen Michael J.
Published: March 2007

T cells are essential for inducing clonal B cell expansion in germinal centers during T cell‐dependent antibody responses. However, class‐switched antibodies are readily detectable in TCRα‐deficient mice that congenitally lack αβ T cells, including those such as IgG1 that are considered to be dependent on collaboration between B cells and αβ T cells. This observation suggests that a novel form of B:T collaboration may be evident in TCRα−/− mice. We report that germinal centers develop spontaneously in mice lacking T cell receptor α genes (TCRα−/−), despite the absence of αβ T cells. They are not seen in TCRβ−/− mice kept in similar conditions. Both strains of mice have γδ T cells, but it is a subset of T cells expressing TCRβ and CD4 that is dominant in the germinal centers of TCRα−/− mice. Exceptionally, germinal centers were associated with CD4+ γδ T cells. The expression of CD4 seems to be important, for few extrafollicular T cells have CD4 and CD4 is largely absent from TCRβ−/− T cells. The CD4+ TCRβ cells may help B cells produce autoantibodies that have been identified in TCRα−/− mice.
Glutamine and tetrapeptide repeat variations affect the biological activity of different mouse interleukin‐2 alleles
Authors: Matesanz Fuencisla , Alcina Antonio
Published: March 2007

Mouse interleukin‐2 (IL‐2) was thought to be encoded by a single allele. We have recently described N‐terminal differences in five IL‐2 molecules from nine mouse strains analyzed (Matesanz, F., Alcina, A. and Pellicer, A., Immunogenetics 1993. 38: 300). In this study, we isolated and sequenced the cDNA of three polymorphic IL‐2 molecules and constructed two recombinant IL‐2 molecules to cover representative structural changes and to address the functional significance of these changes using human and mouse cellular assays in vitro. Apart from punctual codon changes, major differences include an expanding CAG codon (translated into glutamine) and the presence of the tetrapeptide Pro‐Thr‐Ser‐Ser repeated 1, 2, or 3.5 times which is also present once in human IL‐2. This tetrapeptide repeat includes an O‐glycosylation site. These recombinant IL‐2 proteins were expressed at high levels in bacteria and purified by preparative SDS‐PAGE with a complete activity recovery. Differences in growth‐inducing activity on mouse primary splenocytes were observed in some of them, although no differences were observed in proliferative stimulation of CTLL cells. In human peripheral blood lymphocytes and the T cell line Kit‐225, the growth stimulation capacity was inversely dependent on the size of the glutamine stretch and the number of tetrapeptide repeats. These results suggest an evolutionary adaptation of the mouse IL‐2/IL‐2 receptor system that maintains polyglutamine extensions in the IL‐2 molecule. In summary, mouse IL‐2 polymorphism results in different bioactivities which may determine susceptibility or resistance to disease.
A soluble deletion mutant of the human complement receptor type 1, which lacks the C4b binding site, is a selective inhibitor of the alternative complement pathway
Authors: Scesney Susanne M. , Makrides Savvas C. , Gosselin Michael L. , Ford Pamella J. , Andrews Beth M. , Hayman Edward G. , Marsh Henry C.
Published: March 2007

The human complement receptor type 1 (CR1, CD35), is a single‐chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. The SCR themselves, considered in groups of seven, form long homologous repeats (LHR) which have been designated LHR‐A, ‐B, ‐C, and ‐D for the most common human allotype of CR1. A soluble deletion mutant of CR1 which lacks the first seven N‐terminal SCR (LHR‐A) as well as the transmembrane and cytoplasmic domains was produced and characterized. The resulting protein, designated sCR1[desLHR‐A], lacks the C4b binding site found in LHR‐A, but retains the two C3b binding sites found in LHR‐B and ‐C, respectively. The functional activities of sCR1[desLHR‐A] were quantitatively compared in vitro to those of soluble complement receptor type 1 (sCR1) which has been shown to retain all known functions of the native cell surface receptor. sCR1[desLHR‐A] and sCR1 competed equally for the binding of dimeric C3b to erythrocyte CR1. sCR1[desLHR‐A] and sCR1 were similar in their capacity to serve as a cofactor in the factor I‐mediated degradation of the C3b and C4b α chains. sCR1[desLHR‐A] and sCR1 were comparable in their capacity to inhibit erythrocyte lysis and anaphylatoxin production mediated by the alternative complement pathway. sCR1[desLHR‐A], however, was significantly less effective an inhibitor of erythrocyte lysis and anaphylatoxin production than sCR1 under conditions which allow classical pathway activation. These results demonstrate sCR1[desLHR‐A] to be a selective inhibitor of the alternative complement pathway in vitro.
Association of phosphatidylinositol 3 kinase to protein kinase C ζ during interleukin‐2 stimulation
Authors: Gómez Javier , Martínez‐A Carlos , García Alphonse , Rebollo Angelita
Published: March 2007

Interleukin‐2 induces a serine‐phosphorylated phosphatidylinositol 3 kinase activity in the mouse T cell line TS1αβ. Moreover, protein kinase C (PKC) ζ directly or indirectly associates with the phosphatidylinositol 3 kinase and the association appears to be necessary for the serine‐phosphorylated phosphatidylinositol 3 kinase activity, since release of ζPKC by competition of binding with peptides spanning the p110 sequence from amino acids 907 to 925 abolishes the serine‐phosphorylated phosphatidylinositol 3 kinase activity. This kinase activity is also blocked when ζPKC expression is inhibited by antisense oligonucleotide. Inhibition of phosphatidylinositol 3 kinase activity by wortmannin does not abolish ζPKC association.
Regulation of cathepsin E expression during human B cell differentiation in vitro
Authors: Sealy Louise , Mota Fernando , Rayment Neil , Tatnell Peter , Kay John , Chain Benjamin
Published: March 2007

Cathepsin E is an aspartic proteinase which has been implicated in antigen processing in the class II major histocompatibility complex pathway. In this study we show that cathepsin E, measured at both the protein and message level, is up‐regulated late in human B cell activation. The implications of this observation in terms of cathepsin E function are discussed.
A strict requirement of interleukin‐4 for interleukin‐4 induction in antigen‐stimulated human memory T cells
Authors: Breit Susanne , Steinhoff Matthias , Blaser Kurt , Heusser Christoph H. , Sebald Walter , Levine Alan D. , Röcken Martin
Published: March 2007

The role of interleukin‐4 (IL‐4) in the induction of IL‐4 in mouse T cells is well established, but conflicting results have been reported with anti‐CD3‐primed human T cells and T cell clones. Therefore, IL‐4 regulation was investigated in short‐term cultured human T cells primed in vitro with either a superantigen or a hapten, nickel sulfate (NiSO4), for 3 days and expanded with IL‐2 for another 5 days. Under these conditions, antigen‐specific IL‐4 producing T cells were generated in 35 / 40 cultures. Priming for IL‐4 production was abrogated in all cultures by anti‐IL‐4 antibody or soluble IL‐4 receptor (sIL‐4R). Primed T cells that were IL‐4− when cultured with IL‐2 only developed an IL‐4 producing phenotype when primed and expanded in the presence of exogenous IL‐4. T cells primed in the presence of either endogenous or exogenous IL‐4 produced 10–200‐fold more IL‐4 than T cells primed in the presence of anti‐IL‐4 antibody or sIL‐4R. While IL‐4 induction was absolutely dependent on IL‐4, neither endogenous nor exogenous IL‐4 influenced IFN‐γ synthesis. Most importantly, IL‐4 induced and sIL‐4R abolished priming for IL‐4 production even in NiSO4‐specific memory T cells from sensitized individuals. Thus, IL‐4 induction in antigen‐specific human memory T cell populations absolutely required IL‐4. The IL‐4 pathway of memory T cells retained a remarkable plasticity in sensitized individuals.
The relative importance of individual DR binding motif positions as defined by peptide anchor analysis of influenza hemagglutinin peptide 306–318 and human myelin basic protein peptide 152–165 binding to several DR molecules: definition of a common extended DR binding motif
Authors: Posch Phillip E. , Hastings Alice E. , Rosen‐Bronson Sandra , Richert John R. , Hurley Carolyn Katovich
Published: March 2007

Definition of peptide binding motifs for DR molecules has proven difficult as the peptides that bind to a DR molecule have shown extensive variability at putative motif positions. Recent studies suggest that specific peptide anchor residues (motif positions) and specific DR residues can differ in importance for peptide binding to a DR molecule. To assess further the relevance of individual peptide anchor residues, the binding of serial alanine‐substituted analogs of influenza virus hemagglutinin (HA) 306–318 and human myelin basic protein (MBP) 152–165 to a panel of transfected wild‐type DR molecules was examined. This analysis included DR molecules from a wide range of allelic families and, unlike most earlier studies, multiple members of single DR allelic families. The data show that different peptide residues serve as critical anchors for binding to different DR molecules. For example, MBP binding to DR(α,β1*0303) required peptide residues F154 (i), R159 (i + 5) and R162 (i + 8). In contrast, MBP binding to DR(α,β1*0102) required peptide residues I153 (i) and L156 (i + 3). More importantly, the combination of critical anchor residues in HA and MBP differed for binding to a single DR molecule [e.g. V309 (i) for HA and I153 (i) and L156 (i + 3) for MBP binding to DR(α,β1*0102)]. Although the location of the binding pocket in each DR molecule compared to the DR (α,β1*0101) crystal is expected to be similar and suggests a common extended DR binding motif, the present results suggest that the relative importance of individual peptide anchor residues and of the corresponding DR binding pockets will differ for each DR/peptide complex.
Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model
Authors: Klonisch Thomas , Delves Peter J. , Berger Peter , Panayotou George , Lapthorn Adrian J. , Isaacs Neil W. , Wick Georg , Lund Torben , Roitt Ivan M.
Published: March 2007

We systematically screened a large panel of well‐characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of 125I‐hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3–50‐fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti‐IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3‐fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo‐hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters α2/β3/5, α2/hCGβCTP (C‐terminal peptide) and β3/5/hCGβCTP showed the strongest enhancement, with binding more than 10‐fold greater than the sum of 125I‐hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups β1/β3/5, c1/2/β3/5, β1/α2, and α2/α3/5 (3–9‐fold). The greater frequency of synergy obtained with the linear epitopes of the hCGβCTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCGα and core‐hCGβ (residues 1–112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non‐overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high‐affinity bispecific antibodies targetted to a single antigen molecule.
Antigen processing and presentation of a naturally glycosylated protein elicits major histocompatibility complex class II‐restricted, carbohydrate‐specific T cells
Authors: Michaëlsson Erik , Broddefalk Johan , Engström Åke , Kihlberg Jan , Holmdahl Rikard
Published: March 2007

It is well known that T cells recognize antigen as processed peptides bound to major histocompatibility complex molecules on the surface of antigen‐presenting cells. Recently, it has been shown that T cells can specifically recognize synthetic glycopeptides. However, whether glycopeptides are selected for presentation during antigen processing of glycoproteins and eventually elicit carbohydrate‐specific T cells is still an open question. In this study, we utilized synthetic glycopeptides to analyze T cell recognition of the naturally glycosylated immunodominant peptide representing type II collagen (CII) residues 256–270. In this peptide, lysines at positions 264 and 270 may be post‐translationally modified by hydroxylation and subsequent O‐linked glycosylation with β‐galactosyl or α‐glucosyl‐(1 → 2)‐β‐galactosyl residues. T cell hybridomas established from type II collagen‐immunized mice specifically recognized CII 256–270 with either galactose or glucosyl‐galactose at position 264. The T cell hybridoma recognizing glucosyl‐galactose displayed no cross‐reactivity either to galactose or to the structurally different α‐galactosyl‐(1 → 4)‐β‐galactose. Furthermore, the T cell hybridoma recognizing galactose did not cross‐react to glucosyl‐galactose or galactosyl‐galactose, indicating that the antigen‐presenting cells (bulk spleen cells, lipopolysaccharide‐stimulated spleen cells, anti‐CD40‐stimulated spleen cells, peritoneal exudate cells or CFA‐primed lymph node cells) inefficiently processed carbohydrates when the antigen was given as a glycopeptide.
Comparison of numerous delivery systems for the induction of cytotoxic T lymphocytes by immunization
Authors: Allsopp Catherine E. M. , Plebanski Magdalena , Gilbert Sarah , Sinden Robert E. , Harris Steve , Frankel Gadi , Dougan Gordon , Hioe Catarina , Nixon Douglas , Paoletti Enzo , Layton Guy , Hill Adrian V. S.
Published: March 2007

A variety of vaccine delivery systems including peptides with various adjuvants, recombinant particles, live recombinant viruses and bacteria and plasmid DNA were tested for their ability to induce CD8+ cytotoxic T lymphocytes (CTL) against a well‐defined epitope (amino acids 252–260) from the circumsporozoite (CS) protein of Plasmodium berghei. We compared routes of immunization that would be applicable for the administration of a malaria vaccine in humans. The majority of these vaccines did not induce high CTL responses in the spleens of immunized mice. However, both a yeast‐derived Ty virus‐like particle expressing the optimal nine‐amino acid epitope SYIPSAEKI from the CS protein (CSP‐VLP) and a lipid‐tailed peptide of this same sequence induced high levels of the major histocompatibility complex (MHC) class I‐restricted CTL with one and three subcutaneous immunizations, respectively. Moreover, these CTL were able to recognize naturally processed antigen expressed by a recombinant vaccinia virus. The levels of CTL induced by CSP‐VLP could be augmented by co‐immunization with certain cytokines. Target cells pulsed with CSP‐VLP were recognized and lysed, showing that the particles were effectively processed and presented through MHC class I presentation pathway. The levels of CTL induced using CSP‐VLP and lipopeptides are comparable to those observed after immunization with multiple doses of irradiated sporozoites.
Processing of pro‐tumor necrosis factor‐α by venom metalloproteinases: A hypothesis explaining local tissue damage following snake bite
Authors: Moura‐da‐Silva Ana Maria , Laing Gavin David , Paine Mark John Ingraham , Dennison Jeremy Michael Thomas Jonathan , Politi Vincenzo , Crampton Julian Moray , Theakston Robert David Geoffrey
Published: March 2007

Venom‐induced necrosis is a common local debilitating sequela of bites by many vipers, frequently resulting in severe permanent scarring and deformity. Antivenoms are not effective under these circumstances unless administered within a few minutes of the bite; this is unlikely to occur in the rural tropics where most victims take a long time to reach medical care. We have shown that two venom zinc metalloproteinases (jararhagin from Bothrops jararaca venom and a metalloproteinase from Echis pyramidum leakeyi venom) successfully cleaved the recombinant glutathione‐S‐transferase – tumor necrosis factor‐α fusion protein (GST‐TNF‐α) substrate to form biologically active TNF‐α which was shown to be neutralized by ovine TNF‐α Fab antibodies. This resulted in a reduction of venom‐induced necrosis in mice when injected intravenously or intradermally both before and after intradermal injections of E.p.leakeyi venom. A peptidomimetic (POL 647) was also found to inhibit the Echis metalloproteinase, thus preventing the processing of the TNF precursor; this was shown using a TNF‐α‐sensitive cell culture assay and electrophoresis. These observations demonstrate the possible importance of TNF‐α in the development of the resulting necrotic lesion and leads to the hypothesis that increased levels of venom metalloproteinases following snake bite release active TNF‐α. This cytokine may contribute to the local necrosis and also induce the production of endogenous matrix metalloproteinases, which in turn generate a positive feedback mechanism resulting in continued cleavage of pro‐TNF‐α. The results indicate that inhibition or neutralization of endogenous TNF‐α appears to result in a significant reduction in venom‐induced necrosis. This could help to explain the clinical observations that treatment of local necrosis following snake bite by antivenom is only minimally successful.
The interleukin‐8 receptor B and CXC chemokines can mediate transendothelial migration of human skin homing T cells
Authors: Babi Luis F. Santamaria , Moser Bernhard , Soler Maria Teresa Perez , Moser René , Loetscher Pius , Villiger Beat , Blaser Kurt , Hauser Conrad
Published: March 2007

We studied the involvement of chemokines that bind to G protein‐coupled receptors in the migration of skin homing T cells across a bilayer vascular construct (BVC) consisting of a fibroblast matrix underneath an activated endothelial (EC) monolayer. Based on the expression of the cutaneous lymphocyte‐associated antigen (CLA), a skin homing receptor, CD45R0+ T cells freshly isolated from blood or HUT‐78 cutaneous T lymphoma cells were separated into CLA+ and CLA− subpopulations. These T cells were incubated on interleukin (IL)‐1β and tumor necrosis factor‐α‐activated EC, and the number of transmigrated cells was determined. The chemokine IL‐8 was selectively involved in the enhanced migration of CLA+ T cells across activated EC as demonstrated by blocking antibody to IL‐8 but not to GRO‐α, MCP‐1 and RANTES. Identical results were obtained with both human umbilical vein EC (HUVEC) and microvascular skin EC (HDMEC). Pertussis toxin selectively inhibited the enhanced transendothelial migration (TEM) of CLA+ T cells, suggesting that CLA‐dependent TEM depends on Gi protein‐transmitted signals. Moreover, the IL‐8 receptor B (IL‐8RB) appeared to be functionally involved in TEM, as demonstrated by receptor desensitization with the CXC chemokines IL‐8 and GRO‐α and by blocking the IL‐8RB with specific monoclonal antibodies. Although only the IL‐8RB was involved in CLA‐dependent TEM, mRNA encoding IL‐8RA and IL‐8RB was expressed by both CLA+ and CLA− T cells. This correlated with IL‐8RA and IL‐8RB surface expression on these cells. Thus, the IL‐8RB is selectively functional in TEM of T cells expressing the skin homing receptor CLA. Our results demonstrate a critical role for IL‐8 and possibly other IL‐8RB ligands in addition to the IL‐8RB in TEM and suggest the involvement of these molecules in the homing of specific T cells to inflamed skin.
Synergistic effect between CD40 and class II signals overcome the requirement for class II dimerization in superantigen‐induced cytokine gene expression
Authors: Mehindate Khalil , Al‐Daccak Reem , Damdoumi Farida , Mourad Walid
Published: March 2007

Although staphylococcal enterotoxin A (SEA), B (SEB), and toxic shock syndrome toxin 1 (TSST‐1) bind to major histocompatibility complex (MHC) class II molecules, they differ in their mode of binding. Signaling induced by these toxins via MHC class II molecules seems to be largely mediated by their mode of interaction. In the present study, we have demonstrated that contrary to SEA, stimulation of the human monocytic cell line THP‐1 with SEB or TSST‐1 failed to induce interleukin‐1β or tumor necrosis factor‐α gene expression. Treatment of THP‐1 cells with interferon‐γ increased the level of MHC class II expression but did not enhance the SEB and TSST‐1 response. However, cross‐linking of SEB or TSST‐1 bound to MHC class II molecules with specific antibodies leads to cytokine gene expression, indicating that dimerization of class II molecules is a requirement for this superantigen‐induced response. The presence of anti‐CD40 antibodies in the course of SEB or TSST‐1 stimulation overcomes this requirement, indicating that certain signal(s) induced via CD40 molecules can replace those induced by dimerization of class II molecules. Pretreatment with anti‐lymphocyte functional antigen‐1 (LFA‐1) antibodies completely inhibited SEA‐induced response as well as that induced by SEB or TSST‐1 in the presence of CD40 antibodies, supporting the involvement of LFA‐1 intercellular adhesion molecule system in these responses. The entirety of these results demonstrate clearly that dimerization of class II molecules is a prerequisite for superantigen‐induced T cell‐independent cytokine gene expression which can be replaced by signaling via CD40 in an LFA‐1‐dependent system.
Marked depletion at the late pro‐B cell stage in the bone marrow of lpr mice correlates with the development of lymphadenopathy but not autoimmunity
Authors: Dautigny Nicole , Chabre Henri , Garcia Corinne , Ezine Sophie
Published: March 2007

We have found that old lpr mice exhibit a loss of mature B cells in the bone marrow. The deleted population is HSAlo B220hi and is generated from the peripheral pool. Abnormalities in the microenvironment could explain the absence of mature B cells. Thus, old lpr bones were grafted under the skin of normal adult Ly5.1 hosts and examined 3 weeks later for the presence of Ly5.1+ B220hi cells. Our data show that the lpr medullary compartment was efficiently restored by host B cells. These results suggest that the bone marrow microenvironment of old lpr mice is able to sustain mature B cells. However, transfer of T cell‐depleted bone marrow cells from old lpr mice to Rag‐2−/− mice leads to incomplete and inefficient repopulation of the host medullary compartment. Thus, a defect at an early stage of B cell differentiation was detected: using four‐color flow cytometry, we found a profound depletion of the late pro‐B B220+ CD43+ HSA+ BP‐1+ cell population in aging lpr mice. This depletion was not observed in old autoimmune‐prone MRL‐+/+ mice which develop only autoantibodies but was present in B6‐lpr mice which develop a lymphadenopathy and an indolent autoimmune syndrome. Altogether, our results demonstrate an age‐linked defect in the progression of B cell differentiation in lpr mice independent of the presence of autoantibodies and targeted to the late pro‐B cell subset.
Differential expression of interleukin‐5 mRNA+ cells and eosinophils in Nippostrongylus brasiliensis infection in resistant and susceptible strains of mice
Authors: Zhou Yafen , Bao Shisan , Rothwell Terence L. W. , Husband Alan J.
Published: March 2007

Interleukin (IL)‐5 is produced by both T cells and eosinophils and has been implicated in lymphocyte and eosinophil differentiation and maturation. The extent to which differences in IL‐5 expression contribute to genetic variability in parasite immunity was investigated by comparing eosinophilia, IgE production, mastocytosis and IL‐5 mRNA+ cells following Nippostrongylus brasiliensis infection of resistant (BALB/c) and susceptible (C57BL/6) mice. In uninfected mice, IL‐5 mRNA+ cells detected by in situ hybridization were distributed throughout the lamina propria and crypt regions of the small intestine in both strains, but were 1.5‐fold higher in BALB/c than in C57BL/6 mice. Following N. brasiliensis infection, the numbers of IL‐5 mRNA+ cells in BALB/c mice continued to increase until day 11 post‐infection at which time they were more than 4‐fold more numerous than in uninfected control mice of the same strain. In C57BL/6 mice, IL‐5 mRNA+ cells reached peak numbers on day 7 post‐infection, only 1.5‐fold higher than uninfected controls, but the numbers began to decline thereafter. At all time points after day 5, the numbers of IL‐5 mRNA+ cells in the gut of C57BL/6 mice were significantly lower than BALB/c mice. The differences in numbers of IL‐5 mRNA+ cells induced by infection in each strain of mice correlated with changes in blood and intestinal eosinophilia, mastocytosis and IgE production and was reflected in differences in worm expulsion and egg counts. Although numbers of intestinal IgA‐containing cells increased in both strains after infection, there was no difference between strains except at day 11 when there were significantly higher numbers in BALB/c mice than in C57BL/6 mice. These results suggest that IL‐5 is an important regulatory factor determining host immunity to parasite infection and that differential regulation of IL‐5 expression explains in part the observed strain differences with respect to parasite resistance.