bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-linking of major histocompatibility complex class II molecules by staphylococcal enterotoxin A superantigen is a requirement for inflammatory cytokine gene expression.
Authors: Mehindate K , Thibodeau J , Dohlsten M , Kalland T , Sékaly R P , Mourad W
Published: June 2004

Staphylococcal enterotoxin A (SEA) has two distinct binding sites for major histocompatibility complex (MHC) class II molecules. The aspartic acid located at position 227 (D227) in the COOH terminus of SEA is one of the three residues involved in its interaction with the DR beta chain, whereas the phenylalanine 47 (F47) of the NH2 terminus is critical for its binding to the DR alpha chain. Upon interaction with MHC class II molecules, SEA triggers several cellular events leading to cytokine gene expression. In the present study, we have demonstrated that, contrary to wild-type SEA, stimulation of the THP1 monocytic cell line with SEA mutated at position 47 (SEAF47A) or at position 227 (SEAD227A) failed to induce interleukin 1 beta and tumor necrosis factor-alpha messenger RNA expression. Pretreatment of the cells with a 10-fold excess of either SEAF47A or SEAD227A prevented the increase in cytokine messenger RNA induced by wild-type SEA. However, cross-linking of SEAF47A or SEAD227A bound to MHC class II molecules with F(ab')2 anti-SEA mAb leads to cytokine gene expression, whereas cross-linking with F(ab) fragments had no effect. Taken together, these results indicate that cross-linking of two MHC class II molecules by one single SEA molecule is a requirement for cytokine gene expression.
CD73 is involved in lymphocyte binding to the endothelium: characterization of lymphocyte-vascular adhesion protein 2 identifies it as CD73.
Authors: Airas L , Hellman J , Salmi M , Bono P , Puurunen T , Smith D J , Jalkanen S
Published: June 2004

We have recently described a monoclonal antibody (mAb) 4G4 recognizing a 70-kD molecule constitutively expressed on human endothelial cells and on subpopulations of lymphocytes. We showed that this molecule, which we named lymphocyte-vascular adhesion protein 2 (L-VAP-2), mediates lymphocyte adhesion to cultured endothelial cells. Protein sequencing of tryptic peptides from immunoaffinity-purified L-VAP-2 revealed sequence identity between L-VAP-2 and CD73 (ecto-5'-nucleotidase, E.C.3.1.3.5), and COS cells transfected with a CD73 cDNA were positively stained with the mAb 4G4, which recognizes L-VAP-2. mAb 4G4 was also able to partially inhibit the ecto-5'-nucleotidase activity of peripheral blood lymphocytes. Moreover, cross-precipitation studies performed with mAb 4G4 and a CD73 workshop mAb 1E9 showed that these two antibodies recognize the same molecule. Since the tissue distribution and biochemical characteristics of the two molecules are also similar, we conclude that L-VAP-2 and CD73 are the same glycoprotein. Adhesion experiments showed significantly increased binding of freshly isolated lymphocytes to COS cells transfected with a CD73 cDNA, as compared to mock-transfected COS cells, and binding of lymphocytes to CD73-expressing COS cells was inhibited by the presence of mAb 4G4 in the adhesion assay. CD73 is a glycosyl phosphatidylinositol-linked molecule previously shown to have a cosignalling role in T lymphocyte proliferation. Our data suggest that it also has a function in mediating lymphocyte adhesion to the endothelium.
Strictly transporter of antigen presentation (TAP)-dependent presentation of an immunodominant cytotoxic T lymphocyte epitope in the signal sequence of a virus protein.
Authors: Hombach J , Pircher H , Tonegawa S , Zinkernagel R M
Published: June 2004

Peptides presented by major histocompatibility complex (MHC) class I molecules are derived from intracellularly synthesized proteins. Cytosolic proteins are fragmented into peptides, which are subsequently transported via the transporter of antigen presentation (TAP) into the endoplasmic reticulum (ER), where they bind to MHC class I molecules. We have investigated the requirements for MHC class I presentation of the immunodominant gp33 cytotoxic T lymphocyte epitope of the lymphocytic choriomeningitis virus. This epitope is located within the leader peptide of the virus glycoprotein. Such an epitope is expected to be presented in a TAP-independent manner, since it is released into the ER by signal peptidase. Taking advantage of TAP1-/- mice, however, we show both in vitro and in vivo that, after virus infection, the presentation of the gp33 epitope is strictly dependent on a functional TAP heterodimer. The results are discussed with respect to peptide trimming processes in the ER.
Inactivated influenza virus, when presented on dendritic cells, elicits human CD8+ cytolytic T cell responses.
Authors: Bender A , Bui L K , Feldman M A , Larsson M , Bhardwaj N
Published: June 2004

Inactivated or subunit virus preparations have been excellent vaccines for inducing antibody responses. Generation of cytolytic T cell responses, however, is thought to require replicating virus, primarily to provide sufficiently large amounts of cytoplasmic proteins for processing and presentation on major histocompatibility complex class I molecules by antigen-presenting cells. Potent human CD8+ cytolytic T cell responses to live replicating influenza A virus are generated when dendritic cells are used as the antigen-presenting cells. Here, we demonstrate that dendritic cells pulsed with poorly replicating, heat- or ultraviolet-inactivated influenza virus, induce equally strong CD8+ cytolytic T lymphocyte responses. The cytotoxic T lymphocytes are generated in the apparent absence of CD4+ helper cells or exogenous cytokines. Active viral protein synthesis is not required to charge class I molecules on dendritic cells. When pulsed with inactivated virus, < 1% of dendritic cells express nonstructural protein 1, which is only synthesized in the infectious cycle. To be optimally effective, however, the inactivated virus must retain its fusogenic activity, and presumably access the cytoplasm of dendritic cells. The data indicate, therefore, that dendritic cells require only small amounts of viral protein to charge class I molecules, most likely via traditional class I processing pathways. These results reopen the potential use of inactivated virus preparations as immunogens for cytotoxic T lymphocyte responses.
Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses.
Authors: Kusunoki T , Hailman E , Juan T S , Lichenstein H S , Wright S D
Published: June 2004

Mammals mount a rapid inflammatory response to gram-negative bacteria by recognizing lipopolysaccharide (LPS, endotoxin). LPS binds to CD14, and the resulting LPS-CD14 complex induces synthesis of cytokines and up-regulation of adhesion molecules in a variety of cell types. Gram-positive bacteria provoke a very similar inflammatory response, but the molecules that provoke innate responses to these bacteria have not been defined. Here we show that protein-free, phenol extracts of Staphylococcus aureus contain a minor component that stimulates adhesion of neutrophils and cytokine production in monocytes and in the astrocytoma cell line, U373. Responses to this component do not absolutely require CD14, but addition of soluble CD14 enhances sensitivity of U373 cells by up to 100-fold, and blocking CD14 on monocytes decreases sensitivity nearly 1,000-fold. Deletion of residues 57-64 of CD14, which are required for responses to LPS, also eliminates CD14-dependent responses to S. aureus molecules. The stimulatory component of S. aureus binds CD14 and blocks binding of radioactive LPS. Unlike LPS, the activity of S. aureus molecules was neither enhanced by LPS binding protein nor inhibited by bactericidal/permeability increasing protein. The active factor in extracts of S. aureus is also structurally and functionally distinct from the abundant species known as lipoteichoic acid (LTA). Cell-stimulating activity fractionates differently from LTA on a reverse-phase column, pure LTA fails to stimulate cells, and LTA antagonizes the action of LPS in assays of IL-6 production. These studies suggest that mammals may use CD14 in innate responses to both gram-negative and gram-positive bacteria, and that gram-positive bacteria may contain an apparently unique, CD14-binding species that initiates cellular responses.
T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen.
Authors: Burrows S R , Silins S L , Moss D J , Khanna R , Misko I S , Argaet V P
Published: June 2004

Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self-reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area that mimics a structure presented on HLA B*4402. Thus, tolerance to a background major histocompatibility antigen can effectively diversify the TCR repertoire for a foreign epitope by deflecting the response away from an immunodominant combination of TCR-binding residues.
Activation of virus replication after vaccination of HIV-1-infected individuals.
Authors: Staprans S I , Hamilton B L , Follansbee S E , Elbeik T , Barbosa P , Grant R M , Feinberg M B
Published: June 2004

Little is known about the factors that govern the level of HIV-1 replication in infected individuals. Recent studies (using potent antiviral drugs) of the kinetics of HIV-1 replication in vivo have demonstrated that steady-state levels of viremia are sustained by continuous rounds of de novo infection and the associated rapid turnover of CD4+ T lymphocytes. However, no information is available concerning the biologic variables that determine the size of the pool of T cells that are susceptible to virus infection or the amount of virus produced from infected cells. Furthermore, it is not known whether all CD4+ T lymphocytes are equally susceptible to HIV-1 infection at a given time or whether the infection is focused on cells of a particular state of activation or antigenic specificity. Although HIV-1 replication in culture is known to be greatly facilitated by T cell activation, the ability of specific antigenic stimulation to augment HIV-1 replication in vivo has not been studied. We sought to determine whether vaccination of HIV-1-infected adults leads to activation of virus replication and the targeting of vaccine antigen-responsive T cells for virus infection and destruction. Should T cell activation resulting from exposure to environmental antigens prove to be an important determinant of the steady-state levels of HIV-1 replication in vivo and lead to the preferential loss of specific populations of CD4+ T lymphocytes, it would have significant implications for our understanding of and therapeutic strategies for HIV-1 disease. To begin to address these issues, HIV-1-infected individuals and uninfected controls were studied by measurement of immune responses to influenza antigens and quantitation of virion-associated plasma HIV-1 RNA levels at baseline and at intervals after immunization with the trivalent influenza vaccine. Influenza vaccination resulted in readily demonstrable but transient increases in plasma HIV-1 RNA levels, indicative of activation of viral replication, in HIV-1-infected individuals with preserved ability to immunologically respond to vaccine antigens. Activation of HIV-1 replication by vaccination was more often seen and of greater magnitude in individuals who displayed a T cell proliferative response to vaccine antigens at baseline and in those who mounted a significant serologic response after vaccination. The fold increase in viremia, as well as the rates of increase of HIV-1 in plasma after vaccination and rates of viral decline after peak viremia, were higher in individuals with higher CD4+ T cell counts.(ABSTRACT TRUNCATED AT 400 WORDS)
Identification of a CD4+ T cell-stimulating antigen of pathogenic bacteria by expression cloning.
Authors: Sanderson S , Campbell D J , Shastri N
Published: June 2004

Identifying the immunogenic proteins that elicit pathogen-specific T cell responses is key to rational vaccine design. While several approaches have succeeded in identifying major histocompatibility complex (MHC) class I bound peptides that stimulate CD8+ T cells, these approaches have been difficult to extend to peptides presented by MHC class II molecules that stimulate CD4+ T cells. We describe here a novel strategy for identifying CD4+ T cell-stimulating antigen genes. Using Listeria monocytogenes-specific, lacZ-inducible T cells as single-cell probes, we screened a Listeria monocytogenes genomic library as recombinant Escherichia coli that were fed to macrophages. The antigen gene was isolated from the E. coli clone that, when ingested by the macrophages, allowed generation of the appropriate peptide/MHC class II complex and T cell activation. We show that the antigenic peptide is derived from a previously unknown listeria gene product with characteristics of a membrane-bound protein.
T helper type 1/T helper type 2 cytokines and T cell death: preventive effect of interleukin 12 on activation-induced and CD95 (FAS/APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons.
Authors: Estaquier J , Idziorek T , Zou W , Emilie D , Farber C M , Bourez J M , Ameisen J C
Published: June 2004

Human immunodeficiency virus (HIV) infection leads to a progressive loss of CD4+ T helper (Th) type 1 cell-mediated immunity that is associated with defective in vitro CD4+ T cell proliferation and abnormal T cell death by apoptosis in response to T cell receptor (TCR) stimulation. Quantification of interleukin (IL)-2, interferon gamma, IL-4, IL-5, and IL-10 secretion by immunoassays, and of interferon gamma, IL-4 and IL-10 messenger RNA expression by competitive reverse transcriptase polymerase chain reaction after in vitro stimulation of the TCR revealed a similar Th1 cytokine profile in T cells from HIV-infected persons and from controls. These data indicated that the loss of CD4+ Th1 cell function in HIV-infected persons is not related to a Th1 to Th2 cytokine switch as previously proposed, but to a process of activation-induced death of CD4+ Th1 cells. Despite the absence of elevated levels of Th2 cytokines, apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented in vitro by antibodies to IL-10 or IL-4, two Th2 cytokines that downregulate Th1 cell responses, or by the addition of recombinant IL-12, a cytokine that upregulates Th1 functions. TCR-induced apoptosis of T cell hybridomas and preactivated T cells has been shown to involve the CD95 (Fas/Apo-1) molecule. CD4+ and CD8+ T cells from HIV-infected persons expressed high levels of the CD95 molecule, and, in contrast to T cells from controls, were highly sensitive to antibody-mediated CD95 ligation, which induced apoptosis in a percentage of T cells similar to that induced by TCR stimulation. As TCR-induced apoptosis, CD95-mediated apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented by the addition of recombinant IL-12. Together, these findings suggest that apoptosis of CD4+ T cells from HIV-infected persons involves an abnormal sensitivity to CD95 ligation, and to TCR stimulation in the presence of normal levels of Th2 cytokines. The preventive effect of IL-12 on both mechanisms has potential implications for the design of immunotherapy strategies aimed at the upregulation of CD4+ Th1 cell functions in AIDS.
Immunopathology of interleukin (IL) 2-deficient mice: thymus dependence and suppression by thymus-dependent cells with an intact IL-2 gene.
Authors: Krämer S , Schimpl A , Hünig T
Published: June 2004

Interleukin (IL) 2-deficient mice develop a fatal immunopathology characterized by lymphoadenopathy, splenomegaly, T cell infiltration of the bone marrow, loss of B cells, anemia, and inflammation of the gut. The thymus dependence of these disease symptoms was tested by introducing the IL-2 mutation into athymic mice. With the exception of an increase in CD8+ intrahepatic alpha/beta T cells, IL-2 deficiency had no detectable effect on leukocyte composition or health of athymic mice, indicating a key role for thymus-derived T cells in the initiation of disease and demonstrating that B cell development and survival are independent of IL-2. In adoptive transfer studies, lymph node and spleen cells from euthymic IL-2-deficient mice induced disease in athymic mice with an intact IL-2 gene, suggesting that thymus-independent IL-2-expressing cells are unable to control the development of immune pathology. Both IL-2+ and IL-2-/- bone marrow cells repopulated the thymus and the peripheral T cell compartment of the recombination activator gene 2-deficient recipients, and chimeras that had received IL-2-deficient bone marrow developed immune pathology. Disease development was, however, fully or at least partially prevented when 30% of the bone marrow inoculum was derived from mice able to express IL-2. These results demonstrate that the IL-2 deficiency syndrome depends on the intrathymic differentiation of T cells carrying the IL-2 mutation, and that the abnormal activation of IL-2-deficient lymphocytes can be controlled by thymus-derived but not thymus-independent lymphocytes.
Thiols decrease human interleukin (IL) 4 production and IL-4-induced immunoglobulin synthesis.
Authors: Jeannin P , Delneste Y , Lecoanet-Henchoz S , Gauchat J F , Life P , Holmes D , Bonnefoy J Y
Published: June 2004

N-Acetyl-L-cysteine (NAC) is an antioxidant precursor of intracellular glutathione (GSH), usually given in human as a mucolytic agent. In vitro, NAC and GSH have been shown to act on T cells by increasing interleukin (IL) 2 production, synthesis and turnover of IL-2 receptors, proliferation, cytotoxic properties, and resistance to apoptosis. We report here that NAC and GSH decrease in a dose-dependent manner human IL-4 production by stimulated peripheral blood T cells and by T helper (Th) 0- and Th2-like T cell clones. This effect was associated with a decrease in IL-4 messenger RNA transcription. In contrast, NAC and GSH had no effect on interferon gamma and increased IL-2 production and T cell proliferation. A functional consequence was the capacity of NAC and GSH to selectively decrease in a dose-dependent manner IL-4-induced immunoglobulin (Ig) E and IgG4 production by human peripheral blood mononuclear cells. Interestingly, NAC and GSH also acted directly on purified tonsillar B cells by decreasing the mature epsilon messenger RNA, hence decreasing IgE production. In contrast, IgA and IgM production were not affected. At the same time, B cell proliferation was increased in a dose-dependent manner. Not all antioxidants tested but only SH-bearing molecules mimicked these properties. Finally, when given orally to mice, NAC decreased both IgE and IgG1 antibody responses to ovalbumin. These results demonstrate that NAC, GSH, and other thiols may control the production of both the Th2-derived cytokine IL-4 and IL-4-induced Ig in vitro and in vivo.
Complement-mediated regulation of tissue factor activity in endothelium.
Authors: Saadi S , Holzknecht R A , Patte C P , Stern D M , Platt J L
Published: June 2004

Inflammation and immunity may be associated with endothelial cell (EC) injury and thrombus formation. We explored the mechanisms through which a humoral immune response directed against the endothelium might promote coagulation. Using the interaction of anti-EC antibodies and complement (C) with cultured EC as a model, we studied the expression and function of tissue factor, a cofactor for factor VIIa-mediated conversion of factor X to Xa. Exposure of EC to anti-EC antibodies and C in sublytic amounts stimulated the synthesis of tissue factor over a period of 16-42 h. Cell surface expression of tissue factor activity required activation of C and assembly of the membrane attack complex, because expression was inhibited by soluble CR1 and was not detected in the absence of C8. Elaboration of tissue factor messenger RNA was observed over a period of 8-30 h and required protein synthesis. Expression of tissue factor was not a direct consequence of the action of C on the EC but was a secondary response that required as an intermediate step the release of interleukin 1 alpha, an early product of the EC response to C activation. These findings suggest that, after the assembly of membrane attack complex on EC, the production of tissue factor and initiation of coagulation in a blood vessel depend on the production of interleukin 1 alpha and on its availability to stimulate affected EC.
Regulation of the balance of cytokine production and the signal transducer and activator of transcription (STAT) transcription factor activity by cytokines and inflammatory synovial fluids.
Authors: Wang F , Sengupta T K , Zhong Z , Ivashkiv L B
Published: June 2004

The balance between type 1 and 2 T helper cell cytokine production plays an important role in several animal models of autoimmunity, and skewed patterns of cytokine expression have been described in human inflammatory diseases. Many cytokines activate signal transducer and activation of transcription (STAT) transcription factors, which, in turn, activate transcription of inflammatory effector genes. We used mononuclear cell priming cultures and inflammatory synovial fluids (SFs) derived from arthritis patients to examine the regulation of cytokine production and STAT activity by an inflammatory synovial microenvironment. Exposure to SFs during priming resulted in an 81% inhibition of interferon (IFN)-gamma, but not interleukin (IL) 4, production by effector cells generated in priming cultures. SF suppression was mediated by IL-4 and IL-10 and inhibition of IL-12 expression, and it was reversed in a dominant fashion by exogenous IL-12. SFs blocked the sustained activity of transcription factor Stat1, but not Stat3, during the priming period, and Stat1 activity was differentially regulated by cytokines in parallel with their positive or negative regulation of IFN-gamma production. Active Stat3, but not Stat1, was detected in cells from inflamed joints. These results suggest a role for altered balance of Stat1 and Stat3 transcriptional activity in the regulation of T cell differentiation and in the pathogenesis of inflammatory synovitis.
Superantigen binding to a T cell receptor beta chain of known three-dimensional structure.
Authors: Malchiodi E L , Eisenstein E , Fields B A , Ohlendorf D H , Schlievert P M , Karjalainen K , Mariuzza R A
Published: June 2004

The three-dimensional structure of an unglycosylated T cell antigen receptor (TCR) beta chain has recently been determined to 1.7 A resolution. To investigate whether this soluble beta chain (murine V beta 8.2J beta 2.1C beta 1) retains superantigen (SAG)-binding activity, we measured its affinity for various bacterial SAGs in the absence of MHC class II molecules. Dissociation constants (KDs) were determined using two independent techniques: surface plasmon resonance detection and sedimentation equilibrium. Specific binding was demonstrated to staphylococcal enterotoxins (SEs) B, C1, C2, and C3 and to streptococcal pyrogenic exotoxin A (SPEA), consistent with the known proliferative effects of these SAGs on T cells expressing V beta 8.2. In contrast, SEA, which does not stimulate V beta 8.2-bearing cells, does not bind the recombinant beta chain. Binding of the beta chain to SAGs was characterized by extremely fast dissociation rates (> 0.1 s-1), similar to those reported for certain leukocyte adhesion molecules. Whereas the beta chain bound SEC1, 2, and 3 with KDs of 0.9-2.5 microM, the corresponding value for SEB was approximately 140 microM. The much weaker binding to SEB than to SEC1, 2, or 3 was surprising, especially since SEB was found to actually be 3- to 10-fold more effective, on a molar basis, than the other toxins in stimulating the parental T cell hybridoma. We interpret these results in terms of the ability of SEC to activate T cells independently of MHC, in contrast to SEB. We have also measured SE binding to the glycosylated form of the beta chain and found that carbohydrate apparently does not contribute to recognition, even though the N-linked glycosylation sites at V beta 8.2 residues Asn24 and Asn74 are at or near the putative SAG-binding site. This result, along with the structural basis for the V beta specificity of SEs, are discussed in relation to the crystal structure of the unglycosylated beta chain.
Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals.
Authors: Yellin M J , Brett J , Baum D , Matsushima A , Szabolcs M , Stern D , Chess L
Published: June 2004

CD40 is expressed on a variety of cells, including B cells, monocytes, dendritic cells, and fibroblasts. CD40 interacts with CD40L, a 30-33-kD activation-induced CD4+ T cell surface molecule. CD40L-CD40 interactions are known to play key roles in B cell activation and differentiation in vitro and in vivo. We now report that normal human endothelial cells also express CD40 in situ, and CD40L-CD40 interactions induce endothelial cell activation in vitro. Frozen sections from normal spleen, thyroid, skin, muscle, kidney, lung, or umbilical cord were studied for CD40 expression by immunohistochemistry. Endothelial cells from all tissues studied express CD40 in situ. Moreover, human umbilical vein endothelial cells (HUVEC) express CD40 in vitro, and recombinant interferon gamma induces HUVEC CD40 upregulation. CD40 expression on HUVEC is functionally significant because CD40L+ Jurkat T cells or CD40L+ 293 kidney cell transfectants, but not control cells, upregulate HUVEC CD54 (intercellular adhesion molecule-1), CD62E (E-selectin), and CD106 (vascular cell adhesion molecule-1) expression in vitro. Moreover, the kinetics of CD40L-, interleukin 1-, or tumor necrosis factor alpha-induced CD54, CD62E, and CD106 upregulation on HUVEC are similar. Finally, CD40L-CD40 interactions do not induce CD80, CD86, or major histocompatibility complex class II expression on HUVEC in vitro. These results demonstrate that CD40L-CD40 interactions induce endothelial cell activation in vitro. Moreover, they suggest a mechanism by which activated CD4+ T cells may augment inflammatory responses in vivo by upregulating the expression of endothelial cell surface adhesion molecules.
The cleavage preference of the proteasome governs the yield of antigenic peptides.
Authors: Eggers M , Boes-Fabian B , Ruppert T , Kloetzel P M , Koszinowski U H
Published: June 2004

Proteasomes degrade endogenous proteins in the cytosol. The potential contribution of the proteasome to the effect of flanking sequences on the presentation of an antigenic epitope presented by the major histocompatibility complex class I allele Ld was studied. Peptides generated in cells from minigenes coding for peptides of 17- and 19-amino acid length were compared with the in vitro 20S proteasome degradation products of the respective synthetic peptides. The quality of generated peptides was independent of ubiquitination. In vivo and in vitro processing products were indistinguishable with respect to peptide size and abundance. Altering the neighboring sequence substantially improved the yield of the final antigenic nonapeptide by 20S proteasome cleavage. These results suggest that, in addition to the presence of major histocompatibility complex class I allelic motifs, the cleavage preference of the proteasome can define the antigenic potential of a protein.
Altered hepatic transport of immunoglobulin A in mice lacking the J chain.
Authors: Hendrickson B A , Conner D A , Ladd D J , Kendall D , Casanova J E , Corthesy B , Max E E , Neutra M R , Seidman C E , Seidman J G
Published: June 2004

We have created J chain knockout mice to define the physiologic role of the J chain in immunoglobulin synthesis and transport. The J chain is covalently associated with pentameric immunoglobulin (Ig) M and dimeric IgA and is also expressed in most IgG-secreting cells. J chain-deficient mice have normal serum IgM and IgG levels but markedly elevated serum IgA. Although polymeric IgA was present in the mutant mice, a larger proportion of their serum IgA was monomeric than was found in wild-type mouse serum. Bile and fecal IgA levels were decreased in J chain-deficient mice compared with wild-type mice, suggesting inefficient transport of J chain-deficient IgA by hepatic polymeric immunoglobulin receptors (pIgR). The pIgR-mediated transport of serum-derived IgA from wild-type and mutant mice was assessed in Madin-Darby canine kidney (MDCK) cells transfected with the pIgR. These studies revealed selective transport by pIgR-expressing MDCK cells of wild-type IgA but not J chain-deficient IgA. We conclude that although the J chain is not required for IgA dimerization, it does affect the efficiency of polymerization or have a role in maintaining IgA dimer stability. Furthermore, the J chain is essential for efficient hepatic pIgR transport of IgA.
Ontogenic development and tissue distribution of V gamma 1-expressing gamma/delta T lymphocytes in normal mice.
Authors: Pereira P , Gerber D , Huang S Y , Tonegawa S
Published: June 2004

A hamster monoclonal antibody (mAb) recognizing an epitope in the V gamma 1-J gamma 4-C gamma 4 chain of the gamma/delta T cell receptor has been generated. Using this mAb, we have quantitated the occurrence of V gamma 1-bearing gamma/delta T cells in the developing thymus and in the lymphoid organs and several epithelia of adult mice. The V gamma 1-expressing cells constitute a minor gamma/delta T cell subpopulation during fetal and early postnatal life, but they constitute a major population of gamma/delta T cells in the thymus and in the peripheral lymphoid organs in adult mice. In addition, we found that V gamma 1-bearing cells comprise a large proportion (15-60%) of the gamma/delta T cells present in the intestinal epithelium (i-IEL) in all strains of mice tested. V gamma 1+ i-IEL are present in athymic (nude) mice and in antigen-free mice, demonstrating that they can develop extrathymically and that their presence in the intestinal epithelium is independent of the antigenic load of the gut. Our results show that V gamma 1-bearing lymphocytes account for the largest population of gamma/delta T cells in the mouse. This population includes a thymus-dependent component that homes to the secondary lymphoid organs and a thymus-independent component that constitutes a major fraction of the gamma/delta i-IELs.
Abnormal glucocorticoid receptor-activator protein 1 interaction in steroid-resistant asthma.
Authors: Adcock I M , Lane S J , Brown C R , Lee T H , Barnes P J
Published: June 2004

Glucocorticosteroids are a very effective treatment for asthma and other chronic inflammatory diseases. However, a small proportion of patients is resistant to the therapeutic effects of glucocorticoids. Pharmacokinetic and ligand binding studies suggest that the molecular abnormality in steroid resistance lies distal to nuclear translocation. We have previously reported that there is a decreased ability of glucocorticoid receptors (GR) to bind to the DNA-binding site in peripheral blood mononuclear cells (PBMC) after dexamethasone treatment. This reduced DNA binding was due to a decrease in the number of receptors available rather than an alteration in affinity for DNA. To study this reduced DNA binding, we examined the ability of the nuclear translocated transcription factors activator protein 1 (AP-1), nuclear factor kappa B (NF-kappa B) and cyclic AMP response element-binding protein (CREB) to bind to their DNA-binding sites and to interact with GR in PBMC from patients with steroid-sensitive and steroid-resistant asthma. There was a significant reduction in the interaction between GR and AP-1 in these steroid-resistant patients, although interaction with other transcription factors activated in inflammation (NF-kappa B and CREB) was unaffected. An increase in the basal levels of AP-1 DNA binding was also detected in the nuclei from steroid-resistant asthmatic patients. There were no differences in the amount of messenger RNA detected for the components of AP-1, c-Fos and c-Jun, nor in the sequences of these messenger RNAs. These results suggest either that the ability of the GR to bind to glucocorticoid response elements and AP-1 is altered in steroid-resistant patients or that increased levels of AP-1 prevent GR DNA binding, and that this may be the molecular basis of resistance to the antiinflammatory effect of steroids in these cells.
On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety.
Authors: Bhakdi S , Dorweiler B , Kirchmann R , Torzewski J , Weise E , Tranum-Jensen J , Walev I , Wieland E
Published: June 2004

Combined treatment with trypsin, cholesterol esterase, and neuraminidase transforms LDL, but not HDL or VLDL, to particles with properties akin to those of lipid extracted from atherosclerotic lesions. Single or double enzyme modifications, or treatment with phospholipase C, or simple vortexing are ineffective. Triple enzyme treatment disrupts the ordered and uniform structure of LDL particles, and gives rise to the formation of inhomogeneous lipid droplets 10-200 nm in diameter with a pronounced net negative charge, but lacking significant amounts of oxidized lipid. Enzymatically modified LDL (E-LDL), but not oxidatively modified LDL (ox-LDL), is endowed with potent complement-activating capacity. As previously found for lipid isolated from atherosclerotic lesions, complement activation occurs to completion via the alternative pathway and is independent of antibody. E-LDL is rapidly taken up by human macrophages to an extent exceeding the uptake of acetylated LDL (ac-LDL) or oxidatively modified LDL. After 16 h, cholesteryl oleate ester formation induced by E-LDL (50 micrograms/ml cholesterol) was in the range of 6-10 nmol/mg protein compared with 3-6 nmol/mg induced by an equivalent amount of acetylated LDL. At this concentration, E-LDL was essentially devoid of direct cytotoxic effects. Competition experiments indicated that uptake of E-LDL was mediated in part by ox-LDL receptor(s). Thus, approximately 90% of 125I-ox-LDL degradation was inhibited by a 2-fold excess of unlabeled E-LDL. Uptake of 125I-LDL was not inhibited by E-LDL. We hypothesize that extracellular enzymatic modification may represent an important step linking subendothelial deposition of LDL to the initiation of atherosclerosis.
Bcl-XL displays restricted distribution during T cell development and inhibits multiple forms of apoptosis but not clonal deletion in transgenic mice.
Authors: Grillot D A , Merino R , Núñez G
Published: June 2004

The survival of T lymphocytes is tightly controlled during development. Here, we show that Bcl-xL, a protein homologue of Bcl-2, is highly regulated in the thymus in a pattern different than that of Bcl-2. The maximum expression was in CD4+CD8+ thymocytes, a developmental stage where Bcl-2 is downregulated. To assess the role of Bcl-xL in thymocyte apoptosis, we generated mice overexpressing an E mu-bcl-x transgene within the T cell compartment. Constitutive expression of Bcl-xL resulted in accumulation of thymocytes and mature T cells in lymphoid organs. Thymocytes overexpressing Bcl-xL exhibited increased viability in vitro and were resistant to apoptosis induced by different signals, including glucocorticoid, gamma irradiation, calcium ionophore, and CD3 cross-linking. However, Bcl-xL was unable to block clonal deletion of thymocytes reactive with self-superantigens or H-Y antigen. These studies demonstrate that Bcl-2 and Bcl-xL, two functionally related proteins, are regulated independently during T cell development. In contrast to Bcl-2, which has been implicated in the maintenance of mature T cells, Bcl-xL appears to provide a survival signal for the maintenance of more immature CD4+CD8+ thymocytes before positive selection.
Inflammatory microcrystals differentially regulate the secretion of macrophage inflammatory protein 1 and interleukin 8 by human neutrophils: a possible mechanism of neutrophil recruitment to sites of inflammation in synovitis.
Authors: Hachicha M , Naccache P H , McColl S R
Published: June 2004

Human neutrophils at inflammatory sites may be an important source of the chemotactic cytokines macrophage inflammatory protein 1 alpha (M1P-1 alpha; a C-C chemokine) and interleukin 8 (IL-8; a C-X-C chemokine). In this study, we show that the inflammatory microcrystals monosodium urate monohydrate (MSU) and calcium pyrophosphate dihydrate (CPPD), the major mediators of gout and pseudogout, differentially regulate the production of these two chemokines by human neutrophils. Both MSU and CPPD increased the secretion of IL-8 by neutrophils in a dose- and time-dependent manner, but had no effect on that of MIP-1 alpha. Since inflammatory cytokines are likely to be present in the synovium during crystal-induced inflammation, we examined the interaction between TNF-alpha and GM-CSF and the crystals. Both TNF-alpha and GM-CSF stimulated IL-8 production; however, only TNF-alpha exerted a significant effect on MIP-1 alpha secretion in neutrophils. IL-8 production induced by TNF-alpha and GM-CSF was synergistically enhanced in the presence of MSU or CPPD, whereas MIP-1 alpha secretion induced by TNF was completely inhibited in the presence of either MSU or CPPD. Interestingly, no interaction between the crystals and the inflammatory cytokines was observed with respect to synthesis of the C-X-C chemokine MGSA in neutrophils. These results suggest that the combination of TNF-alpha and GM-CSF with MSU or CPPD will lead to the production of IL-8 by neutrophils and abolish the release of MIP-1 alpha, an event that will theoretically lead to recruitment of neutrophils but not mononuclear cells. These results are in accordance with the pathological state of gout and pseudogout, where the predominant inflammatory cell is the neutrophil.
Inhibition of invariant chain (Ii)-calnexin interaction results in enhanced degradation of Ii but does not prevent the assembly of alpha beta Ii complexes.
Authors: Romagnoli P , Germain R N
Published: June 2004

Calnexin is a resident protein of the endoplasmic reticulum (ER) that associates with nascent protein chains. Among the newly synthesized integral membrane proteins known to bind to calnexin is invariant chain (Ii), and Ii release from calnexin coincides with proper assembly with major histocompatibility complex (MHC) class II heterodimers. Although calnexin association with several membrane glycoproteins depends on interactions involving N-linked glycans, we previously reported that a truncation mutant of mouse Ii (mIi1-107) lacking both N-glycosylation sites was highly effective in associating with MHC class II heterodimers and escorting these dimers through the secretory pathway. This could indicate that calnexin, despite binding to both Ii and class II, is not necessary for the proper interaction of these proteins, or that in contrast to most membrane glycoproteins, the N-linked glycans of Ii are not critical to its interaction with this chaperone. To examine this issue, we have directly explored the binding of calnexin to both Ii truncation mutants lacking the typical sites of N-glycosylation or Ii produced in cells treated with tunicamycin to prevent glycan addition. These experiments revealed that either method of eliminating N-linked carbohydrates on Ii also inhibited association with calnexin. A lumenally truncated form of Ii (mIi1-131) that still has N-linked carbohydrates showed a decreased affinity for calnexin compared with intact Ii, however, indicating that calnexin-Ii binding is not determined solely by the sugar moieties. All forms of Ii lacking N-linked sugars and showing defective association with calnexin also had enhanced rates of preendosomal degradation. Despite this effect on degradation rate, tunicamycin treatment did not inhibit the association of class II with glycan-free Ii. These data support the view that calnexin is not an absolute requirement for the proper assembly of class II-Ii nonamers, but rather acts primarily to retain Ii in the ER and to inhibit its degradation. These two properties of calnexin-Ii interaction may help ensure that sufficient intact Ii is available for efficient inactivation of the binding sites of newly synthesized class II molecules, while limiting the ability of excess free Ii to alter the transport properties of the early endocytic pathway.
Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes.
Authors: Bock T A , Orlic D , Dunbar C E , Broxmeyer H E , Bodine D M
Published: June 2004

We have generated immunodeficient scid-/scid- (SCID)-transgenic mice expressing the genes for human interleukin 3, granulocyte/macrophage-colony stimulating factor, and stem cell factor. We have compared engraftment and differentiation of human hematopoietic cells in transgenic SCID mice with two strains of nontransgenic SCID mice. Human bone marrow cells carrying the CD34 antigen or human umbilical cord blood were injected into sublethally irradiated recipients. Human DNA was detected by polymerase chain reaction in peripheral blood and bone marrow of 14 of 28 transgenic SCID mice after transplantation, but in only 2 of 15 nontransgenic SCID littermates at a 10-fold lower level. Bone marrow cultures 8 wk after transplantation of cord blood gave rise to human burst-forming unit erythroid, colony-forming unit granulocyte/macrophage, or granulocyte/erythroid/macrophage/megakaryocyte colonies. Engraftment was observed for up to 6 mo in transgenic SCID mice, twice as long as nontransgenic littermates or previous studies in which transplanted SCID mice were given daily injections of growth factors. We conclude that the level and duration of engraftment of human cells in SCID mice can be improved by expression of human cytokine transgenes and that transgenic SCID mice are an efficient model system for the study of human hematopoiesis.
gro-beta, a -C-X-C- chemokine, is an angiogenesis inhibitor that suppresses the growth of Lewis lung carcinoma in mice.
Authors: Cao Y , Chen C , Weatherbee J A , Tsang M , Folkman J
Published: June 2004

We have found that two chemokines, recombinant gro-alpha and gro-beta, specifically inhibit growth factor-stimulated proliferation of capillary endothelial cells in a dose-dependent manner, whereas gro-gamma has no inhibitory effect. In vivo, gro-beta inhibits blood vessel formation in the chicken chorioallantoic membrane assay. It is sufficiently potent to effectively suppress basic fibroblast growth factor-induced corneal neovascularization after systemic administration in mice. Further, gro-beta significantly inhibits the growth of murine Lewis lung carcinoma in syngeneic C57B16/J and immunodeficient nude mice without toxicity. In vitro, Lewis lung carcinoma cells are completely insensitive to recombinant gro-beta at high concentrations that significantly inhibit endothelial cell proliferation. This finding supports the conclusion that gro-beta inhibits Lewis lung tumor growth by suppression of tumor-induced neovascularization.
Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes.
Authors: Bendelac A
Published: June 2004

Mouse NK1+ T cells constitute a subset of alpha/beta TCR+ T cells that specialize in the rapid production of cytokines, in particular IL-4, and may promote the differentiation of Th2-type CD4 T cells. Their TCRs, like those of a homologous subset of human T cells, use an invariant TCR alpha chain and were recently shown to be specific for the beta 2-microglobulin-associated, MHC class I-like CD1 molecules, which are encoded outside the MHC. In contrast to mainstream thymocytes, which recognize their positively selecting MHC ligand on thymic epithelial cells, positive selection of NK1+ T cells requires their CD1 ligand to be expressed on bone marrow-derived cells. To investigate the nature of the bone marrow-derived cell involved, chimeric mice were constructed with tissues from normal, SCID, and MHC-deficient mice, so that CD1 could be selectively expressed by different subsets of bone marrow-derived cells in the thymus. CD1 expression was also directly assessed using an anti-CD1 mAb, and a CD1-specific T cell hybridoma. The results suggest that immature (CD4+8+ double-positive) cortical thymocytes are the source of CD1 presentation for positive selection of NK1+ T cells.