bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of human variable domain antibody fragments against the U1 RNA‐associated A protein, selected from a synthetic and a patient‐derived combinatorial V gene library
Authors: de Wildt Ruud M. T. , Finnern Ricarda , Ouwehand Willem H. , Griffiths Andrew D. , van Venrooij Walther J. , Hoet René M. A.
Published: March 2007

This is the first study describing recombinant human antibody fragments directed to the U1 RNA‐associated A protein (U1A). Three anti‐U1A antibody fragments (Fab) were isolated from a semi‐synthetic human Fab library and one anti‐U1A single‐chain variable fragment (scFv) was isolated from a library which was derived from the IgG‐positive splenic lymphocytes of an autoimmune patient. Competition studies with autoantibodies against the U1 small nuclear ribonucleoprotein (snRNP) particle from patients with systemic lupus erythematosus (SLE) and SLE‐overlap syndromes revealed that U1A binding of these antibody fragments can be inhibited by about 40% of the patient sera. All antibody fragments recognized the native U1 snRNP in immunoprecipitation assays. Two of three Fab clones as well as the scFv clone derived from the repertoire of an autoimmune patient use the same heavy chain germ‐line gene DP‐65. Epitope mapping revealed that these three clones appear to recognize an identical epitope domain present on the C‐terminal RNP motif of the U1A protein. The DP‐65 heavy chain gene is used in less than 1% of the B cells in healthy individuals, while three out of four anti‐U1A antibody fragments use this gene. This points to a restricted VH gene usage in the case of U1A, suggesting that the DP‐65 heavy chain has a natural shape complementarity to the U1A protein.
The splenic marginal zone is absent in alymphoplastic aly mutant mice
Authors: Koike Ryuji , Nishimura Toshihide , Yasumizu Ryoji , Tanaka Hisako , Hataba Yoshiaki , Watanabe Toshiki , Miyawaki Shigeki , Miyasaka Masayuki
Published: March 2007

aly is a unique spontaneous autosomal recessive mutation in mice that causes a deficiency in the systemic lymph nodes (LN) and Peyer's patches (PP). aly also induces abnormal histological findings in the spleen and a deficiency in humoral and cell‐mediated immune functions, although lymphocytes of the aly/aly mouse show virtually normal immune responses in vitro. We studied the structure of the spleen of aly mice in detail by immunohistochemistry and electron microscopy. We found that the spleen of the aly/aly mouse was deficient in the expression of specific antigens for marginal metallophils (MM), marginal zone macrophages (MZM) and fibroblastic reticular cells (FRC), which are components of the marginal zone (MZ) of the spleen. Morphological analysis indicated that the aly/aly spleen is deficient in the structure of MZ, which may, in part, account for the severe immunodeficiency in the aly/aly mouse. We then performed reciprocal bone marrow transplantation experiments (BMT) between normal and aly mice and found a clear correlation between the formation of LN and the development of the splenic MZ in these mice; i.e. successful development of LN was invariably associated with the appearance of the MZ structure, whereas the failure of LN development was always associated with the absence of the development of MZ. These BMT results suggest that a common factor may regulate the generation of both LN and MZ of the spleen.
Activated B cells express functional Fas ligand
Authors: Hahne Michael , Renno Toufic , Schroeter Michael , Irmler Martin , French Lars , Bornand Thierry , MacDonald H. Robson , Tschopp Jürg
Published: March 2007

Fas ligand (FasL, Apo‐1L) is a member of the tumor necrosis factor protein family and binding to its receptor (Fas, Apo‐1, CD95) triggers cell death through apoptosis. Ligand expression is restricted to cells with known cytolytic activity and found on hematopoietic cells of the T cell and natural killer lineage. Here we provide evidence that B lymphocytes can express FasL. Flow cytometric analysis revealed that FasL is expressed on the surface of B cells upon stimulation with either lipopolysaccharide or phorbol 12‐myristute 13‐acetate/ionomycin. FasL expression on activated B cells was confirmed by Western blot and reverse transcriptase polymerase chain reaction analysis. FasL on B cells is functional since lipopolysaccharide‐activated B lymphocytes derived from wild type, but not from g/d mutant mice, were able to kill Fas‐sensitive target cells. Our data suggest that the Fas system may contribute to the control of B cell homeostasis.
CD31/PECAM‐1‐driven chemokine‐independent transmigration of human T lymphocytes
Authors: Zocchi Maria Raffaella , Ferrero Elisabetta , Leone Biagio E. , Rovere Patrizia , Bianchi Elisabetta , Toninelli Elisabetta , Pardi Ruggero
Published: March 2007

We assessed the relative contribution of CD31/PECAM‐1 (platelet‐endothelial cell adhesion molecule‐1) to T lymphocyte transmigration by the use of transfected murine fibroblasts stably expressing either the human CD31/PECAM‐1 or the intercellular adhesion molecule‐1 (CD54/ICAM‐1). Unlike CD54/ICAM‐1, CD31/PECAM‐1 supported migration of activated T cells in the absence of chemokines: most of the migrating lymphocytes were CD31+ and displayed a phenotype corresponding to the naive subpopulation (LFA‐1dull and CD45RA+). Migration of activated T lymphocytes through CD54/ICAM‐1+ transfected monolayers could be induced by creating a chemotactic gradient with the chemokine monocyte chemotactic protein‐1, and the migrating cells mainly displayed a memory phenotype (LFA‐1brightCD45RO+) under these conditions. Furthermore, we found that in transfected cells CD54/ICAM‐1 is uniformly distributed along the apical surface of the cells, while CD31/PECAM‐1 is concentrated at the intercellular junctions, suggesting the existence of a haptotactic gradient (i.e. a gradient of substrate‐ or cell‐bound molecules) responsible for T cell migration. This was also confirmed by the finding that monolayers of murine fibroblasts transfected with a CD31/PECAM‐1 mutant lacking the cytoplasmic domain (CD31/PECAM‐1‐Δcyto), which has a reduced tendency to localize at cell‐cell contact areas, supported efficient adhesion but were unable to induce migration of activated T cells unless a chemotactic gradient was created. We propose that in lymphocytes, homophilic CD31/PECAM‐1 adhesion may be primarily involved in transmigration of naive T cells and that its role is complementary to that of CD54/ICAM‐1.
Unique role of thyroxine in T cell recognition of a pathogenic peptide in experimental autoimmune thyroiditis
Authors: Dawe Kim I. , Hutchings Patricia R. , Geysen Mario , Champion Brian R. , Cooke Anne , Roitt Ivan M.
Published: March 2007

We have previously demonstrated the importance of iodination and the requirement of the thyroxine residues in thyroglobulin (Tg) for the stimulation of two clonotypically distinct murine T cell hybridomas reactive against human and mouse Tg. We are now able to show that these T cell hybridomas only recognize an 11‐residue peptide containing a thyroxine structure that has iodine at two positions on each ring. This iodination state is critical for recognition by these hybridomas as a peptide containing de‐iodinated thyroxine is nonstimulatory. Furthermore we have demonstrated that a peptide lacking the thyroxine residue or containing de‐iodinated thyroxine cannot block the recognition of the thyroxine‐containing peptide. We suggest that in our system the thyroxine residue is involved in binding to major histocompatibility complex (MHC) class II molecules. We have also been able to show that the thyroxine residue is available for contact by the T cell receptor (TCR) as recognition of the peptide/H‐2Ak complex is blockable by an antibody directed against thyroxine. Using substituted peptides, we have been able partially to define the residues within the peptide that are critical for recognition of the 11‐residue peptide by our hybridomas. From our data, we suggest that the thyroxine residue may bind the MHC and TCR, while the residues identified in the peptide backbone as important for the stimulation of the hybridomas may bind only the TCR.
Cytotoxic T lymphocytes to Plasmodium falciparum epitopes in an area of intense and perennial transmission in Tanzania
Authors: Lalvani Ajit , Hurt Nicole , Aidoo Michael , Kibatala Patience , Tanner Marcel , Hill Adrian V. S.
Published: March 2007

Studies in The Gambia have provided indirect evidence that cytotoxic T lymphocytes (CTL) play a protective role against malaria in humans and recently, using allele‐specific HLA class I peptide motifs, several peptide epitopes for CTL in four pre‐erythrocytic Plasmodium falciparum antigens have been identified in naturally exposed Gambians. However, CTL levels were low, suggesting that boosting these low levels by immunization might provide substantial protection. In the Kilombero valley of Tanzania, malaria transmission is holoendemic and 300 times more intense than in The Gambia. We report here that several of the epitopes identified in The Gambia are also recognized in naturally exposed, partially immune Tanzanian adults and that levels of CTL are similar to or slightly higher than in Gambian subjects, despite the much higher inoculation rate. We report a new HLA‐A2.1‐restricted epitope from the thrombospondin‐related anonymous protein (TRAP) and we demonstrate that peptide epitopes in TRAP are naturally processed for recognition by CTL from naturally exposed humans. The common allele of a variable HLA‐B7‐restricted epitope in the circumsporozoite protein behaved as an altered peptide ligand (APL) with respect to CTL cognate for a rarer allelic variant of this epitope, suggesting that APL antagonism may occur in natural CTL responses to P. falciparum. The moderate levels of CTL observed, even in this area of intense malaria transmission, points to the need to assess candidate vaccines aimed at increasing CTL levels.
CTX, a novel molecule specifically expressed on the surface of cortical thymocytes in Xenopus
Authors: Chrétien Isabelle , Robert Jacques , Marcuz Anne , Garcia‐Sanz Jose A. , Courtet Michèle , Du Pasquier Louis
Published: March 2007

CTX, a novel developmentally regulated type‐I transmembrane protein is expressed specifically by a large fraction of cortical thymocytes in the amphibian Xenopus. This apparently monomeric 55‐kDa glycoprotein is composed of two immunoglobulin domains, one variable (V) and one constant (C2 type), followed by a transmembrane and a 64‐amino acid cytoplasmic domain. The first immunoglobulin domain is a V‐J segment that is generated without gene rearrangement. In the genome, the V and C2 domains are both encoded by two half‐domain exons. Two CTX loci are found per haploid genome, and they exhibit sequence differences with a high replacing/silent ratio in the CDR1‐like region of the V domain, suggesting that these differences were selected. The cytoplasmic domain contains a motif that is highly conserved evolutionarily in several types of proteins, including adenylyl cyclases. Based on its unique tissue distribution, the variability of its V region and the motif of its cytoplasmic domain, CTX is a candidate for a new type of specific signaling molecule involved in thymocyte selection.
B cell early response gene expression coupled to B cell receptor, CD40 and interleukin‐4 receptor co‐stimulation: evidence for a role of the egr‐2/krox20 transcription factor in B cell proliferation
Authors: Newton Joshua S. , Li Jin , Ning Zhi‐Qiang , Schoendorf Diana E. , Norton John D. , Murphy John J.
Published: March 2007

B lymphocytes are activated following antigen stimulation of the B cell receptor but require co‐stimulation with accessory molecules provided by interleukin (IL)‐4/CD40 ligand for cell cycle progression and proliferation. By analyzing a panel of 11 early response genes induced by cross‐linking of surface immunoglobulin, we show that CD40 signaling alone induces only 2 genes, c‐myc together with an anonymous gene, 3L3, and that these are distinct from the set of genes induced in response to IL‐4. Co‐stimulation with the proliferative combination of anti‐μ, IL‐4 + CD40 signaling led to a fourfold enhancement of egr‐2/krox20 expression over that seen with anti‐μ alone. Egr‐2 expression/activity was selectively inhibited by the immunosuppressive drug cyclosporin A, and antisense oligonucleotide blockade of Egr‐2 activity elicited a dose‐dependent inhibition of B cell proliferation. Taken together, these observations show that the early gene regulatory programs coupled to different surface receptors on B cells are largely distinct from each other, but that certain genes, exemplified by egr‐2, may represent a point of convergence in the integration of different signaling pathways into the B cell proliferative response.
Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular adhesion protein‐1 (VAP‐1)
Authors: Arvilommi Anna‐Maija , Salmi Marko , Kalimo Kirsti , Jalkanen Sirpa
Published: March 2007

The binding of leukocytes to vascular endothelium and their migration into tissues is mediated by adhesion molecules on the endothelial cells and leukocytes. Vascular adhesion protein‐1 (VAP‐1) is a 170–180/90‐kDa endothelial molecule expressed most prominently in high endothelial venules in peripheral lymph node (PLN) type lymphatic tissues. VAP‐1 mediates lymphocyte binding to PLN, tonsil and synovium. The expression of VAP‐1 is induced in inflammatory diseases such as arthritis and gut inflammation. We examined the expression, structure and function of VAP‐1 in normal and inflamed skin and compared it to those of other adhesion molecules implicated in skin homing. In psoriasis, lichen ruber planus, pemphigoid and allergic lesions, VAP‐1 was markedly up‐regulated. The expression of VAP‐1 was also increased in biopsies of healthy skin of the patients. The VAP‐1 molecule induced in skin is decorated with abundant sialic acids. VAP‐1 in inflamed skin is functional, since inhibition with anti‐VAP‐1 monoclonal antibodies caused a 60% reduction in lymphocyte adhesion to vascular endothelium. Antibodies against E‐selectin, which has been regarded as the major vascular addressin directing cutaneous lymphocyte traffic, and, surprisingly, against peripheral lymph node addressin (PNAd), caused inhibitions of 30% and 60%, respectively, in the frozen section adhesion assay. These findings suggest important roles also for VAP‐1 and PNAd in lymphocyte homing into inflamed skin.
Proteasome inhibitors block VCAM‐1 and ICAM‐1 gene expression in endothelial cells without affecting nuclear translocation of nuclear factor‐ϰB
Authors: Cobb Ronald R. , Felts Katherine A. , Parry Graham C. N. , Mackman Nigel
Published: March 2007

Endothelial cells play a major role in recruiting leukocytes to sites of inflammation. This is accomplished, at least in part, by up‐regulation of cell surface adhesion molecules, including VCAM‐1 and ICAM‐1, in response to cytokines. In this report, we investigated the role of the proteasome complex in mediating the interleukin (IL)‐1β induction of VCAM‐1 and ICAM‐1 gene expression in human endothelial cells. We present evidence that a proteasome inhibitor, n‐acetyl‐leucinyl‐leucinyl‐norleucinal (norLEU), as well as specific protease inhibitors, n‐tosyl‐Lys‐chloromethylketone and n‐tosyl‐Phe‐chloromethylketone, blocked IL‐1β induction of VCAM‐1 and ICAM‐1 promoter‐driven reporter gene expression in stably transfected endothelial cells. These inhibitors also blocked cytokine induced cell surface expression of VCAM‐1 and ICAM‐1 by human umbilical vein endothelial cells. As expected, the protease inhibitors blocked the activation of nuclear factor (NF)‐ϰB in response to IL‐1β stimulation. In contrast, norLEU did not prevent IL‐1β‐induced nuclear translocation of NF‐ϰB. The effects of norLEU were specific because it did not inhibit the IL‐1β induction of plasminogen activator inhibitor type 1 gene expression. This study demonstrates that inhibition of the proteolytic activity of the proteasome blocks IL‐1β induction of VCAM‐1 and ICAM‐1 gene expression in human endothelial cells.
A new Ig‐superfamily member, molluscan defence molecule (MDM) from Lymnaea stagnalis, is down‐regulated during parasitosis
Authors: Hoek Robert M. , Smit August B. , Frings Heleen , Vink Jacqueline M. , de Jong‐Brink Marijke , Geraerts Wijnand P. M.
Published: March 2007

To survive the attacks of the internal defence system (IDS) of their host, parasites have developed various strategies to manipulate the IDS. We present evidence that the avian schistosome parasite Trichobilharzia ocellata affects gene expression in the granular cells, a cell type of the IDS of the intermediate host, the mollusc Lymnaea stagnalis. From a differential screening, a clone was isolated encoding a protein named molluscan defence molecule (MDM), which encompasses five C2‐like immunoglobulin (Ig) domains. The protein shares a domain organization and high amino acid sequence identity with hemolin, an Ig‐family member of the insect IDS. Interestingly, both MDM and hemolin have highest sequence identity with neural cell adhesion molecules, but lack the typical fibronectin repeats and motifs for membrane anchors. We find that the expression of the MDM gene is gradually down‐regulated during the course of parasitosis to ∼21% compared to the non‐parasitized level, 8 weeks post‐infection. Based on our findings, we suggest that MDM is involved in the proper functioning of the Lymnaea IDS, and that down‐regulation of MDM is part of the parasite‐induced disabling of non‐self recognition.
Non‐obese diabetic mice hemizygous at the T cell receptor α locus are susceptible to diabetes and sialitis
Authors: Elliott James I. , Altmann Daniel M.
Published: March 2007

To test the hypothesis that T cells carrying two T cell receptor (TCR)α chains play a role in autoimmunity, we backcrossed the non‐obese diabetic (NOD) strain with one carrying a TCRα gene disrupted by homologous recombination. Mice carrying one copy of the disrupted gene are incapable of generating T cells carrying two cell surface TCRα chains. Our early results suggested that either dual TCRα T cells play a role in insulin‐dependent diabetes mellitus (IDDM) induction in NOD mice or that a locus co‐segregating with the disrupted TCRα locus protected mice from diabetes induction. From the analysis both of mice in which the region co‐segregating with the disrupted TCRα locus is minimized and of the F1 offspring of NOD mice with the 129 strain (TCRα hemizygous mice), the apparent protective effect of the absence of dual TCRα T cells is lost; thus, such cells do not appear to play a critical role in autoimmune disease in NOD mice.
Activity of neutral endopeptidase and aminopeptidase N in mouse thymic stromal cells which bind double‐positive thymocytes
Authors: Small Myra , Kaiser Meirav , Tse William , Heimfeld Shelly , Blumberg Shmaryahu
Published: March 2007

The activity of two peptidases was determined in immortalized lines of thymic stromal cells. A line of total stromal cells (T‐TG‐St) was grown from transgenic mouse expressing temperature‐sensitive SV40T antigen under the control of the regulatory elements of the mouse major histocompatibility complex class I gene. From these cells we isolated a subset (DP‐TG‐St) that binds thymocytes which are mainly CD4+8+. We also assayed a clone of fetal thymic epithelial cells (BA/10) that binds CD4+8+ thymocytes. Both lines of double‐positive cell‐binding stroma exhibited strong activity of two peptidases, neutral endopepti dase (NEP; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). In contrast, the activity of both enzymes was very low in the total thymic stromal line. Use of specific inhibitors confirmed that these two enzymes were responsible for the activity observed but also suggested the presence of additional unidentified aminopeptidase(s) in the same stromal cells. The high activity of the two peptidases on stromal cells that bind thymocytes at the double‐positive stage raises the possibility that they might contribute to the microenvironment of the developing thymocytes.
Dissection of lymphocyte function‐associated antigen 1‐dependent adhesion and signal transduction in human natural killer cells shown by the use of cholera or pertussis toxin
Authors: Poggi Alessandro , Spada Franca , Costa Paola , Tomasello Elena , Revello Valentino , Pella Nicoletta , Zocchi Maria Raffaella , Moretta Lorenzo
Published: March 2007

The effect of the guanosine triphosphate‐binding protein (G‐protein) inhibitors cholera toxin (Ctx) and pertussis toxin (Ptx) has been analyzed on lymphocyte function‐associated antigen 1 (LFA‐1)‐dependent adhesion and signal transduction in human natural killer (NK) cells. Ctx, but not Ptx, inhibited the LFA‐1‐dependent adhesion of NK cells to tumor target cells which constitutively express the intercellular cell adhesion molecule‐1 (ICAM‐1) and to NIH/3T3 mouse fibroblasts stably transfected with human ICAM‐1. This effect was detectable only by the use of the entire Ctx but not of the Ctx B subunit. In addition, Ctx could inhibit both NK cell binding and spreading to purified ICAM‐1 protein. NK cell treatment with Ctx modified neither the surface expression of LFA‐1 nor its Mg2+ binding site. These findings, together with the absence of any detectable effect of Ctx on the constitutive phosphorylation of LFA‐1α, suggests that this toxin modifies the avidity of LFA‐1 for ICAM‐1 by acting on LFA‐1‐cytoskeletal protein association. Unlike Ctx, Ptx did not affect NK cell adhesion. The effects of Ctx and Ptx are unlikely to depend on intracellular levels of cyclic adenosine 3′,5′‐monophosphate (cAMP), since a strong increase of cAMP was induced by both toxins. Moreover, this was confirmed by the observation that the LFA‐1‐dependent adhesion was not inhibited by the adenylate cyclase activator forskolin (FSK), the phosphodiesterase inhibitor isobutyl‐1‐methylxantine (IBMX), or both, which increase intracellular cAMP levels. Unlike the differential effect on cell adhesion, both the intracellular calcium [Ca2+]i increase and phosphoinositide breakdown mediated via LFA‐1 were consistently inhibited in a dose‐dependent manner by both Ctx and Ptx. Also in this case, the inhibitory effect did not depend on an increase of intracellular cAMP as indicated by NK cell treatment with FSK, IBMX, or both. Further evidence of the involvement of G‐proteins in LFA‐1‐mediated signal transduction was the inhibitory effect of the GDP analog guanosine‐5′‐O‐2‐thiodiphosphate (GDPßS) on LFA‐1‐mediated calcium mobilization. Taken together, our data provide evidence that the LFA‐1‐mediated NK cell adhesion and signal transduction are partially independent phenomena which may be regulated by different G‐proteins.
A Vβ4‐specific superantigen encoded by a new exogenous mouse mammary tumor virus
Authors: Maillard Ivan , Erny Katrina , Acha‐Orbea Hans , Diggelmann Heidi
Published: March 2007

The superantigen (SAg) expressed by mouse mammary tumor virus (MMTV) has been shown to play an essential role in the course of the viral life cycle. In the present study, we describe a Vβ4‐specific SAg encoded by a new exogenous MMTV carried by the SIM mouse strain. This is the first report of a viral or bacterial SAg reacting with mouse Vβ4+ T cells. Injection of MMTV(SIM) into adult BALB/c mice leads to a rapid and strong stimulation of Vβ4+ CD4+ T cells, followed by a slow deletion of these cells. Neonatal exposure to the virus also leads to a progressive deletion of Vβ4+ T cells. In contrast to other strong MMTV SAg, this new SAg requires the presence of major histocompatibility complex class II I‐E molecules to be presented efficiently to T cells. Sequence analysis revealed a new predicted amino acid sequence in the C‐terminal polymorphic region of this SAg. Furthermore, sequence comparisons to the most closely related SAg with different Vβ specificities hint at the specific residues involved in the interaction with the T cell receptor.
Mycoplasma fermentans‐derived lipid inhibits class II major histocompatibility complex expression without mediation by interleukin‐6, interleukin‐10, tumor necrosis factor, transforming growth factor‐β, type I interferon, prostaglandins or nitric oxide
Authors: Frisch Matthias , Gradehandt Gernot , Mühlradt Peter F.
Published: March 2007

Mycoplasma cause several diseases in man and animals. Some strains can chronically infect humans, leading to fever or inflammatory syndromes such as arthritis, particularly in immunosuppressed patients. A set of pathogenicity factors shared by many mollicutes may be membrane components that activate macrophages to secrete cytokines and other inflammatory mediators. Mycoplasma‐derived high molecular weight material (MDHM) is a macrophage‐activating amphiphilic lipid which was purified from Mycoplasma fermentans. We studied the influence of MDHM on the expression of major histocompatibility complex (MHC) class II molecules by mouse resident peritoneal macrophages with an ELISA. Highly purified MDHM at 4 ng/ml and 0.8 μg/ml crude heat‐killed M. fermentans (concentrations chosen to give maximal responses) suppressed interferon (IFN)‐γ‐dependent class II MHC induction when added simultaneously with IFN‐γ. MDHM was not toxic and did not result in loss of adherent cells. Kinetic data showed that MDHM first up‐regulated, then down‐regulated the expression of preformed class II MHC molecules, while expression of Mac‐1 and F4/80 antigens remained constant. MDHM‐dependent suppression of class II MHC molecule expression resulted in impaired antigen presentation to the helper T cell line D10.G4.1. We further attempted to identify hypothetical products of MDHM‐stimulated macrophages as secondary mediators of class II MHC suppression such as were described for lipopolysaccharide (LPS)‐stimulated macrophages. Type I IFN, prostaglandins and nitric oxide, all reported to cause down‐regulation of class II MHC, could be excluded in this context. Of the cytokines tumor necrosis factor, interleukin (IL)‐6, IL‐10 and transforming growth factor‐β, only IL‐10 inhibited class II MHC expression, although less effectively than MDHM. The involvement of IL‐10 was ruled out, as no evidence for its MDHM‐dependent formation could be found. Our data suggest that MDHM interferes with class II MHC expression by up‐regulating its turnover, and at the same time, inhibits the formation of new class II MHC molecules.
An in vivo model of allergic inflammation: pulmonary human cell infiltrate in allergen‐challenged allergic Hu‐SCID mice
Authors: Duez Catherine , Tsicopoulos Anne , Janin Anne , Tillie‐Leblond Isabelle , Thyphronitis Georges , Marquillies Philippe , Hamid Qutayba , Wallaert Benoit , Tonnel André‐Bernard , Pestel Joël
Published: March 2007

CB.17 severe combined immunodeficient (SCID) mice were used to establish a model of allergic pulmonary inflammation. SCID mice were intraperitoneally reconstituted with 107 peripheral blood mononuclear cells (PBMC) from patients sensitive to Dermatophagoides pteronyssinus (Dpt) and 2 weeks later were exposed to Dpt aerosols. After Dpt nebulization, SCID mice engrafted with PBMC from Dpt‐sensitive patients developed a specific human IgE response as well as an intense pulmonary infiltrate of human cells. In contrast, SCID mice reconstituted with PBMC from patients allergic to grass pollen or from nonallergic donors failed to produce IgE or to exhibit a similar inflammatory response. The level of the IgE production was dependent on the IgE level of the allergic donor. In the lungs of the same allergic SCID mice, 2 months after Dpt inhalation, the cell infiltrate contained CD45+, CD45RO+, CD20+ and HLA‐DR+ human cells. They were located in perivascular and peribronchial areas and disseminated in the mouse lung parenchyma. Moreover, mRNA IL‐5+ cells and eosinophils were found in peribronchial infiltrates. The observations indicate that humanized allergic SCID mice may develop, after nebulization with the relevant allergen, immune reactions similar to those observed in man and suggest that SCID mice may represent a useful model to analyze the regulatory mechanisms of IgE‐associated allergic diseases.
Human basophils release interleukin‐4 after stimulation with Schistosoma mansoni egg antigen
Authors: Falcone Franco H. , Dahinden Clemens A. , Gibbs Bernhard F. , Noll Tobias , Amon Ulrich , Hebestreit Holger , Abrahamsen Ole , Klaucke Jens , Schlaak Max , Haas Helmut
Published: March 2007

The elevated interleukin (IL)‐4 and IgE production in Schistosoma mansoni infection seems to be induced essentially by the egg stage of the parasite. The underlying mechanism, however, is not known. Since basophils from human peripheral blood can produce IL‐4, we asked, whether soluble S. mansoni egg antigens (SEA) would trigger basophils to release IL‐4. Basophils from healthy human donors (n = 32) without prior history of schistosomiasis were incubated with SEA in the presence of IL‐3. In all donors, IL‐4 was produced at different concentrations. The IL‐4 production was dependent on the dose of SEA, was correlated with the purity of the basophil preparation, and the IL‐4 concentration in the culture supernatant was maximal 5 h after stimulation with SEA. In addition to its IL‐4‐stimulatory effect, SEA triggered basophils to degranulate, thereby releasing histamine and sulfidoleukotrienes. Stripping of receptor‐bound IgE from basophils inhibited both SEA‐ and anti‐IgE‐induced, but not ionomycin‐induced IL‐4 production. Moreover, resensitization of stripped basophils with stripping supernatants or human serum restored SEA‐induced IL‐4 production. This suggests that IgE is involved in the mechanism of IL‐4 induction by SEA. Since IL‐4 is induced in basophils from nonexposed donors, basophils may play a role as an early source of IL‐4 in S. mansoni infection.
Structure and chromosomal location of the mouse interleukin‐12 p35 and p40 subunit genes
Authors: Tone Yukiko , Thompson Sara A. J. , Babik Jennifer M. , Nolan Kathleen F. , Tone Masahide , Raven Catherine , Waldmann Herman
Published: March 2007

Interleukin‐12 (IL‐12) is a heterodimeric cytokine composed of p35 and p40 subunits and is required for induction of T helper 1 (Th1) responses. Knowledge of how the IL‐12 gene is regulated will permit an understanding of susceptibility and resistance to pathogenic microbes and to autoiummune diseases. In this report, we provide the gene structures, nucleotide sequences and chromosomal assignment for the p35 and p40 subunits of mouse‐IL‐12. The p35 and p40 subunit genes are distributed over 8 kb and 14 kb, and map to chromosomes 3 and 11, respectively. The p35 subunit gene consists of eight exons, including a 5′‐noncoding exon that was defined by sequence comparison of genomic DNA with the 5′ends of novel cDNA molecules. Transcription of p35 mRNA can start from the first exon but can also initiate further downstream. Potential transcription regulatory elements, AP1, AP2, AP3, NF‐kB and GATA recognition sequences, are located within 523 bp upstream of the p35 gene; however, no TATA box was identified. The p40 subunit gene consists of eight exons. A TATA box is located 30 bp upstream from the transcription start site, and AP1, AP3, GATA, and Pu.1 recognition sequences are located within 690 bp upstream of the p40 gene. An AGTTTCTACTTT sequence, which acts as an interferon‐γ response element in the promoter of the major histocompatibility complex class I gene, was also found upstream of the p40 gene.
Interleukin‐15 up‐regulates interleukin‐2 receptor α chain but down‐regulates its own high‐affinity binding sites on human T and B cells
Authors: Kumaki Satoru , Armitage Richard , Ahdieh Minoo , Park Linda , Cosman David
Published: March 2007

The cytokines interleukin (IL)‐2 and IL‐15 share many biological activities as a consequence of their utilization of the β and γ chains of the IL‐2 receptor. However, each cytokine binds to a specific receptor α chain; IL‐2 with low affinity and IL‐15 with high affinity. Here, we demonstrate that IL‐15, like IL‐2, up‐regulates expression of IL‐2Rα on human T and B cells, but rapidly down‐regulates IL‐15 high‐affinity binding sites, which represent IL‐15Rα. This leads to a decreased responsiveness to IL‐15 as measured by induction of Jak3 tyrosine phosphorylation. These results suggest a mechanism by which IL‐15, a product of activated macrophages, may cooperate with IL‐2 at the initiation of an immune response and enhance subsequent IL‐2 responsiveness during T cell expansion.
Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture
Authors: McHugh Simon , Deighton John , Rifkin Ian , Ewan Pamela
Published: March 2007

The importance of cytokine production in some disease processes is now widely recognized. To investigate temporal relationships between cytokines, we stimulated peripheral blood mononuclear cells (PBMC) in vitro using the T cell mitogen phytohemagglutinin (PHA) and various antigens chosen to induce predominantly Th1 (streptokinase: streptodornase or purified protein derivative) or Th2 (Dermatophagoides pteronyssinus, bee or wasp venom: allergens in sensitive subjects) responses. Cytokine production was measured by sensitive bioassays or enzyme‐linked immunosorbent assays. Of the 30 subjects studied, 10 were normal and 20 individuals were allergic to either D. pteronyssinus (n = 10) or bee venom (n = 10) (examined before specific allergen immunotherapy). We examined the temporal profiles of a panel of cytokines produced in prmary culture. In PHA‐driven cultures, cytokines were found to be sequentially produced in the order interleukin (IL)‐2, IL‐4, IL‐5, IL‐3, interferon (IFN)‐γ, IL‐10, IL‐6, IL‐12 and tumor necrosis factor (TNF)‐α. The response to allergen in allergic patients was predominantly Th2 in nature, with the production of IL‐4, IL‐5, IL‐6 and IL‐10, but little or no IFN‐γ. IL‐2, IL‐3, TNF‐α and IL‐12 were also produced in low amounts. The response of both atopic and normal subjects to recall bacterial antigens was predominantly Th1, with high levels of IFN‐γ, IL‐2 and TNF‐α. The relevance of the order, amount and speed of production, characteristic kinetics (production, consumption, homeostatic regulation) and the cell source of the cytokines are discussed.
Expression of human NKRP1A by CD34+ immature thymocytes: NKRP1A‐mediated regulation of proliferation and cytolytic activity
Authors: Poggi Alessandro , Costa Paola , Morelli Luigia , Canton Claudia , Pella Nicoletta , Spada Franca , Biassoni Roberto , Nanni Luca , Revello Valentino , Tomasello Elena , Mingari Maria Cristina , Moretta Alessandro , Moretta Lorenzo
Published: March 2007

In this study, we show that NKRP1A is expressed and functions on a subset of immature human thymocytes. We took advantage of the monoclonal antibody (mAb) 191B8 that was obtained by immunizing mice with cultured human thymocytes characterized by an immature surface phenotype [CD2− CD3− CD4− CD8− stem cell factor receptor (SCFR)+] and expressing cytoplasmic CD3ϵ chain. The 191B8 antibody homogeneously reacted with the immunizing population but not with most unfractionated thymocytes. It stained a minor population of resting immature thymocytes co‐expressing CD34, SCFR, or both. Following culture of the CD34+ or CD34− fractions of CD2− CD3− CD4− CD8− purified immature thymocytes with recombinant interleukin‐2 (rIL‐2), the 191B8‐defined antigen was expressed on virtually all cells even when 191B8+ cells were removed from the starting population. On the other hand, no 191B8+ cells were detected in fresh or cultured thymocytes expressing a more mature phenotype. Biochemical analysis of 191B8 mAb‐reactive molecules revealed, under non‐reducing conditions, two bands displaying apparent molecular masses of 80 and 44 kDa and a single band of 44 kDa under reducing conditions. Digestion with proteases indicated that the 80‐kDa form represented a homodimeric form of two 44‐kDa molecules, while deglycosylation with N‐glycanase suggested the existence of four N‐glycosylation sites. Transfection of COS7 or NIH3T3 cells with hNKRP1A cDNA showed that the 191B8 mAb recognized NKRP1A as shown by both immunofluorescence analysis and immu‐noprecipitation experiments. Functional studies showed that the 191B8/NKRP1A molecule mediated strong inhibition of the cytolytic activity of culturd CD2− CD3− immature thymocytes against a panel of tumor target cells. More importantly, 191B8 mAb induced proliferation of CD2− CD3− fresh thymocytes which was not increased by rIL‐2. Thus, we propose that NKRP1A molecules, which are expressed in highly immature thymocytes, may play a regulatory role in their growth and function.
Cyclic AMP‐dependent protein kinase (cAK) in human B cells: co‐localization of type I cAK (RIα2C2) with the antigen receptor during anti‐immunoglobulin‐induced B cell activation
Authors: Levy Finn Olav , Rasmussen Anne‐Marie , Taskén Kjetil , Skålhegg Bjørn S. , Huitfeldt Henrik S. , Funderud Steinar , Smeland Erlend B. , Hansson Vidar
Published: March 2007

Cyclic AMP (cAMP) inhibits antigen‐stimulated B cell proliferation through activation of cAMP‐dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RIα, RIIα, Cα and Cβ, barely detectable levels of RIβ mRNA, and no detectable RIIβ or Cγ mRNA. At the protein level, using Western blotting and subunit‐specific antibodies against the different R subunits, we find RIα and RIIα, but no RIβ or RIIβ. The presence of catalytic subunits was demonstrated using a nonselective anti‐C antiserum. By photoaffinity labeling of R subunits with 8‐azido‐[32P]cAMP, followed by immunoprecipitation with subunit‐specific antibodies, we were also able to demonstrate low levels of RIβ. Immunofluorescence staining of RIα and RIIα demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RIα, whereas the RIIα subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti‐Ig‐mediated capping of B cells, RIα subunits redistribute to the cap, co‐localizing with the antigen‐receptors, whereas the intracellular localization of RIIα subunits remains unchanged.
Interleukin‐15 induces adhesion receptor redistribution in T lymphocytes
Authors: Nieto Marta , del Pozo Miguel Angel , Sánchez‐Madrid Francisco
Published: March 2007

Chemotactic factors such as cytokines and chemokines direct the migration of leukocytes into inflammatory sites. Chemokines play a role regulating both the expression and adhesive properties of leukocyte integrins. We have recently described an additional function of chemokines in the induction of cell polarization and adhesion receptor redistribution during the initial step of leukocyte locomotion. We herein report that interleukin (IL)‐15, a newly described cytokine with chemotactic properties, is able to induce uropod formation on T lymphoblasts to which intercellular adhesion molecule (ICAM)‐3, a leukocyte‐restricted counter‐receptor for the lymphocyte function‐associated antigen (LFA)‐1 integrin, is redistributed. Other adhesion molecules, such as ICAM‐1, ICAM‐2, CD43 and CD44, also redistributed to the uropod, although in a lower proportion of the cells. The induction of uropod formation by IL‐15 was observed on T lymphoblasts adhering to the integrin ligands fibronectin, vascular cell adhesion molecule (VCAM)‐1 and ICAM‐1, but not to bovine serum albumin or poly‐L‐lysine. The effect of IL‐15 was dose dependent and specifically inhibited by a monoclonal antibody (mAb) against this cytokine. Blocking experiments with anti‐IL‐2 receptor β chain mAb showed an inhibitory effect on IL‐15‐mediated redistribution of ICAM‐3, whereas no effect was observed in the presence of anti‐IL‐2 receptor α chain mAb. The uropod induced by IL‐15 is enriched in many different adhesion receptors and, being well exposed to the external milieu, is likely to modulate the adhesive properties of lymphocytes.
Hydrogen peroxide secreted by tumor‐derived macrophages down‐modulates signal‐transducing zeta molecules and inhibits tumor‐specific T cell‐and natural killer cell‐mediated cytotoxicity
Authors: Kono Koji , Salazar‐Onfray Flavio , Petersson Max , Hansson Johan , Masucci Giuseppe , Wasserman Ken , Nakazawa Tsutomu , Anderson Paul , Kiessling Rolf
Published: March 2007

Although alterations in CD3‐associated signal‐transducing molecules in tumor‐infiltrating T cells of patients with advanced cancer have been previously described, the mechanism behind these changes is not known. We demonstrate that macrophages isolated from metastatic lymph nodes of patients with malignant melanoma down‐regulate levels of CD3ζ in autologous peripheral blood T cells. Lipopolysaccharide (LPS)‐ or phorbol 12‐myristate 13‐acetate (PMA)‐stimulated monocytes derived from peripheral blood of healthy donors also induced decreased expression of CD3 and CD16‐associated ζ chains similar to that observed in T cells and natural killer (NK) cells of patients with advanced cancer. Co‐culture with activated monocytes impaired Ca2+ mobilization in peripheral blood derived‐T cells when stimulated with monoclonal antibodies to CD3 and also strongly inhibited melanoma‐specific cytotoxic T lymphocyte (CTL) activity and NK activity. The presence of catalase, a scavenger of H2O2, during co‐culture almost totally abrogated the inhibitory effect of activated monocytes on melanoma‐specific CTL lines and on NK cells. Pre‐treatment of CTL or NK cells with nontoxic concentrations (1 × 10−5 M) of H2O2 also severely reduced their cytotoxic activity which could be prevented by catalase. The decrease in CD3ζ and in CD16ζ expression, induced by macrophages isolated from metastatic lymph nodes or by LPS‐stimulated monocytes, was also prevented by catalase when maintained throughout the co‐culture period. The possibility that monocyte/macrophage‐derived reactive oxygen metabolites contribute directly to alterations in signal transducing molecules of T cells and NK cells and to the mechanism of immunosuppression in individuals with cancer should be considered.
Characterization of CD4−CD8− T cell receptor αβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis
Authors: Matsumoto Yoh , Abe Satoshi , Tsuchida Masanori , Hirahara Hirouki , Abo Toru , Shin Taekyun , Tanuma Naoyuki , Kojima Takashi , Ishihara Yoshihiro
Published: March 2007

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion‐forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR αβ+ T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4+ or CD8+. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4−CD8− T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4−CD8− T cells in the SAS are precursors of lesion‐forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double‐negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.
Ligation of TAPA‐1 (CD81) or major histocompatibility complex class II in co‐cultures of human B and T lymphocytes enhances interleukin‐4 synthesis by antigen‐specific CD4+ T cells
Authors: Secrist Heather , Levy Shoshana , Dekruyff Rosemarie H. , Umetsu Dale T.
Published: March 2007

We have previously shown that CD4+ T cells from allergic individuals are predisposed to producing interleukin (IL)‐4 in response to allergens. IL‐4 production could be modulated by antigen concentration as well as by the type of antigen‐presenting cells (APC), with B lymphocytes inducing greater quantities of IL‐4 than monocytes. Using this system we examined IL‐4 synthesis after culture of CD4+ T cells with B cells, monocytes, or both, as APC in the presence of allergen and a monoclonal antibody against CD81 (TAPA‐1), a member of the TM4 superfamily of proteins that regulates activation, proliferation and trafficking of B cells. Addition of anti‐CD81 mAb during culture enhanced IL‐4 synthesis by 2‐ to 70‐fold over that using an isotype‐matched control mAb. Furthermore, anti‐CD81 mAb enhanced IL‐4 synthesis in CD4+ T cells only when CD4+ T cells were cultured with B cells but not monocytes as APC, indicating that anti‐CD81 mAb affected IL‐4 synthesis in T cells via interactions with B cells. However, pretreatment of either population separately with anti‐CD81 mAb prior to culture had no effect on subsequent IL‐4 synthesis, suggesting a requirement for temporal or cooperative interactions between T and B lymphocytes. In addition, anti‐CD81 mAb enhanced IL‐4 production but reduced CD4+ T cell antigen‐specific proliferation, demonstrating that IL‐4 production and proliferation by CD4+ T cells were inversely related. Finally, mAb to major histocompatibility complex class II but not to anti‐CD19 also enhanced IL‐4 synthesis when B lymphocytes were used as APC. In all instances, enhancement of CD4+ IL‐4 synthesis correlated with the presence of large cell aggregates in T‐B lymphocyte cocultures. These results indicate that the capacity of B cells to induce IL‐4 can be significantly enhanced by ligation of particular molecules on their surface and should aid in the design of treatments for diseases in which modulation of the cytokine profile would be beneficial.
Activation of mitogen‐activated protein kinases via CD40 is distinct from that stimulated by surface IgM on B cells
Authors: Kashiwada Masaki , Kaneko Yoriaki , Yagita Hideo , Okumura Ko , Takemori Toshitada
Published: March 2007

CD40 plays critical roles in B cell proliferation and differentiation in response to T cell‐dependent antigenic stimulation. It has been suggested that CD40‐mediated biological activities are transduced by a CD40 receptor‐associated factor, CRAF1 and probably by protein tyrosine kinase Lyn and its substrates, phospholipase Cγ (PLCγ) and phosphatidylinositol‐3 kinase (PI‐3 kinase). Here, we describe the novel finding that a mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated protein kinase (ERK) cascade is involved in CD40 signaling in mouse B cells. Analysis of ERK activities in the B cell lymphoma cell line WEHI 231, which shows an increase in DNA synthesis or arrest of the cell cycle by cross‐linking of CD40 or surface IgM (sIgM) cross‐linking, respectively, indicated that one of the ERK isoforms, ERK2, was preferentially and rapidly activated after CD40 cross‐linking. The CD40‐mediated ERK2 activation was comparable to that after sIgM stimulation, although the activity was reduced toward the basal level within several minutes after stimulation. In contrast, ERK1 and ERK2 were activated to a similar extent by sIgM cross‐linking, and the activities remained stable for at least 10 min. Furthermore, similar features of differential activation of ERK isoforms were observed in normal resting B cells in CD40 and sIgM signaling. These results suggest divergent regulatory pathways for ERK1 and ERK2 activation, and they support the notion that CD40 signaling may utilize a limited set of elements in the ERK cascade. Co‐stimulation of WEHI 231 cells with anti‐CD40 mAb rescues the cells from anti‐IgM‐mediated apoptosis, whereas this co‐stimulation resulted in activation of ERK isoforms comparable to that in sIgM stimulation, without a synergistic effect. This result indicates the dominance of ERK activation in sIgM signaling over that of CD40, and it suggests that ERK activation may not be linked to the biological effect that CD40 stimulation in this cell line.