Showing papers in "Journal of Experimental Medicine in 1995"

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.

Abstract: A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.

Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products.

Abstract: We have previously demonstrated that human peripheral blood low density mononuclear cells cultured in granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 develop into dendritic cells (DCs) that are extremely efficient in presenting soluble antigens to T cells. To identify the mechanisms responsible for efficient antigen capture, we studied the endocytic capacity of DCs using fluorescein isothiocyanate-dextran, horseradish peroxidase, and lucifer yellow. We found that DCs use two distinct mechanisms for antigen capture. The first is a high level of fluid phase uptake via macropinocytosis. In contrast to what has been found with other cell types, macropinocytosis in DCs is constitutive and allows continuous internalization of large volumes of fluid. The second mechanism of capture is mediated via the mannose receptor (MR), which is expressed at high levels on DCs. At low ligand concentrations, the MR can deliver a large number of ligands to the cell in successive rounds. Thus, while macropinocytosis endows DCs with a high capacity, nonsaturable mechanism for capture of any soluble antigen, the MR gives an extra capacity for antigen capture with some degree of selectivity for non-self molecules. In addition to their high endocytic capacity, DCs from GM-CSF + IL-4-dependent cultures are characterized by the presence of a large intracellular compartment that contains high levels of class II molecules, cathepsin D, and lysosomal-associated membrane protein-1, and is rapidly accessible to endocytic markers. We investigated whether the capacity of DCs to capture and process antigen could be modulated by exogenous stimuli. We found that DCs respond to tumor necrosis factor alpha, CD40 ligand, IL-1, and lipopolysaccharide with a coordinate series of changes that include downregulation of macropinocytosis and Fc receptors, disappearance of the class II compartment, and upregulation of adhesion and costimulatory molecules. These changes occur within 1-2 d and are irreversible, since neither pinocytosis nor the class II compartment are recovered when the maturation-inducing stimulus is removed. The specificity of the MR and the capacity to respond to inflammatory stimuli maximize the capacity of DCs to present infectious non-self antigens to T cells.

CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation.

Abstract: The importance of the B7/CD28/CTLA-4 molecules has been established in studies of antigen-presenting cell-derived B7 and its interaction with the T cell costimulatory molecule CD28. CTLA-4, a T cell surface glycoprotein that is related to CD28, can also interact with B7-1 and B7-2. However, less is known about the function of CTLA-4, which is expressed at highest levels after activation. We have generated an antibody to CTLA-4 to investigate the consequences of engagement of this molecule in a carefully defined system using highly purified T cells. We show here that the presence of low levels of B7-2 on freshly explanted T cells can partially inhibit T cell proliferation, and this inhibition is mediated by interactions with CTLA-4. Cross-linking of CTLA-4 together with the TCR and CD28 strongly inhibits proliferation and IL-2 secretion by T cells. Finally, results show that CD28 and CTLA-4 deliver opposing signals that appear to be integrated by the T cell in determining the response to activation. These data strongly suggest that the outcome of T cell antigen receptor stimulation is regulated by CD28 costimulatory signals, as well as inhibitory signals derived from CTLA-4.

Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.

Abstract: Programmed cell death (PCD) is a physiological process commonly defined by alterations in nuclear morphology (apoptosis) and/or characteristic stepwise degradation of chromosomal DNA occurring before cytolysis. However, determined characteristics of PCD such as loss in mitochondrial reductase activity or cytolysis can be induced in enucleated cells, indicating cytoplasmic PCD control. Here we report a sequential disregulation of mitochondrial function that precedes cell shrinkage and nuclear fragmentation. A first cyclosporin A-inhibitable step of ongoing PCD is characterized by a reduction of mitochondrial transmembrane potential, as determined by specific fluorochromes (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine++ + iodide; 3,3'dihexyloxacarbocyanine iodide). Cytofluorometrically purified cells with reduced mitochondrial transmembrane potential are initially incapable of oxidizing hydroethidine (HE) into ethidium. Upon short-term in vitro culture, such cells acquire the capacity of HE oxidation, thus revealing a second step of PCD marked by mitochondrial generation of reactive oxygen species (ROS). This step can be selectively inhibited by rotenone and ruthenium red yet is not affected by cyclosporin A. Finally, cells reduce their volume, a step that is delayed by radical scavengers, indicating the implication of ROS in the apoptotic process. This sequence of alterations accompanying early PCD is found in very different models of apoptosis induction: glucocorticoid-induced death of lymphocytes, activation-induced PCD of T cell hybridomas, and tumor necrosis factor-induced death of U937 cells. Transfection with the antiapoptotic protooncogene Bcl-2 simultaneously inhibits mitochondrial alterations and apoptotic cell death triggered by steroids or ceramide. In vivo injection of fluorochromes such as 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide; 3,3'dihexyloxacarbocyanine iodide; or HE allows for the detection of cells that are programmed for death but still lack nuclear DNA fragmentation. In particular, assessment of mitochondrial ROS generation provides an accurate picture of PCD-mediated lymphocyte depletion. In conclusion, alterations of mitochondrial function constitute an important feature of early PCD.

Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine.

Abstract: Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.

Antibodies to interleukin 12 abrogate established experimental colitis in mice.

Abstract: In this study, we describe a novel murine model of chronic intestinal inflammation induced by the hapten reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS). Rectal application of low doses of TNBS in BALB/c and SJL/J mice resulted in a chronic transmural colitis with severe diarrhea, weight loss, and rectal prolapse, an illness that mimics some characteristics of Crohn's disease in humans. The colon of TNBS-treated mice on day 7 was marked by infiltration of CD4+ T cells; furthermore, in situ polymerase chain reaction studies revealed high levels of interferon (IFN)-gamma mRNA in diseased colons. Isolated lamina propria (LP) CD4+ T cells from TNBS-treated mice stimulated with anti-CD3 and anti-CD28 antibodies exhibited a Th1 pattern of cytokine secretion: a 20-50-fold increase in IL-2 and IFN-gamma levels and a 5-fold decrease in IL-4 levels as compared with those of stimulated LP CD4+ T cells from control BALB/c mice. Administration of monoclonal anti-IL-12 antibodies to the TNBS-treated mice both early (at 5 d) and late (at 20 d) after induction of colitis led to a striking improvement in both the clinical and histopathological aspects of the disease and frequently abrogated the established colitis completely. Furthermore, LP CD4+ T cells isolated from anti-IL-12-treated mice failed to secrete IFN-gamma upon in vitro stimulation. In summary, the data demonstrate the pivotal role of IL-12 and IFN-gamma in a TNBS-induced murine model of chronic intestinal inflammation. Furthermore, they suggest the potential utility of anti-IL-12 antibodies in patients with Crohn's disease.

Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo.

Abstract: In a number of experimental systems in which lymphocyte depletion was induced by apoptosis-inducing manipulations, no apoptotic morphology and ladder-type DNA fragmentation were detected among freshly isolated peripheral lymphocytes ex vivo. Here we report that one alteration that can be detected among splenocytes stimulated with lymphocyte-depleting doses of dexamethasone (DEX) in vivo is a reduced uptake of 3,3'dihexyloxacarbocyanine iodide (DiOC6[3]), a fluorochrome which incorporates into cells dependent upon their mitochondrial transmembrane potential (delta psi m). In contrast, ex vivo isolated splenocytes still lacked established signs of programmed cell death (PCD):DNA degradation into high or low molecular weight fragments, ultrastructural changes of chromatin arrangement and endoplasmatic reticulum, loss in viability, or accumulation of intracellular peroxides. Moreover, no changes in cell membrane potential could be detected. A reduced delta psi m has been observed in response to different agents inducing lymphoid cell depletion in vivo (superantigen and glucocorticoids [GC]), in mature T and B lymphocytes, as well as their precursors. DEX treatment in vivo, followed by cytofluorometric purification of viable delta psi mlow splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither delta psi mlow, nor delta psi mhigh cells, exhibit detectable DNA fragmentation. However, after short-term culture (37 degrees C, 1 h) delta psi mlow cells show endonucleolysis, followed by cytolysis several hours later. Incubation of delta psi mlow cells in the presence of excess amount of the GC receptor antagonist RU38486 (which displaces DEX from the GC receptor), cytokines that inhibit DEX-induced cell death, or cycloheximide fails to prevent cytolysis. The antioxidant, N-acetylcysteine, as well as linomide, an agent that effectively inhibits DEX or superantigen-induced lymphocyte depletion in vivo, also stabilize the DiOC6(3) uptake. In contrast, the endonuclease inhibitor, aurintricarboxylic acid acts at later stages of apoptosis and only retards the transition from the viable delta psi mlow to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in delta psi m constitutes an obligate irreversible step of ongoing lymphocyte death, preceding other alterations of cellular physiology, and thus allowing for the ex vivo assessment of PCD.

Fas ligand mediates activation-induced cell death in human T lymphocytes.

Abstract: A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.

Metalloproteinase-mediated release of human Fas ligand.

Abstract: Fas ligand (FasL) is a type II integral membrane protein homologous with tumor necrosis factor (TNF). Recent studies indicate that TNF is processed to yield the soluble cytokine by metalloproteinases at the cell surface of activated macrophages and T cells. In the present study, we investigated whether FasL is also released by metalloproteinases. Treatment with hydroxamic acid inhibitors of matrix metalloproteinases specifically led to accumulation of membrane-type FasL (p40) on the surface of human FasL cDNA transfectants and activated human T cells, as estimated by surface immunofluorescence and immunoprecipitation with newly established anti-human FasL monoclonal antibodies. This surface accumulation of mFasL was associated with the decrease of soluble FasL (p27) in the supernatant as estimated by quantitative ELISA and immunoprecipitation with anti-human FasL monoclonal antibodies. These results indicate that human FasL is efficiently released from the cell surface by metalloproteinases like TNF.

The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-alpha beta-transgenic model.

Abstract: The dose of foreign antigen can influence whether a cell-mediated or humoral class of immune response is elicited, and this may be largely accounted for by the development of CD4+ T helper cells (Th) producing distinct sets of cytokines. The ability of antigen dose to direct the development of a Th1 or Th2 phenotype from naive CD4+ T cells, however, has not been demonstrated. In this report, we show that the antigen dose used in primary cultures could directly affect Th phenotype development from naive DO11.10 TCR-alpha beta-transgenic CD4+ T cells when dendritic cells or activated B cells were used as the antigen-presenting cells. Consistent with our previous findings, midrange peptide doses (0.3-0.6 microM) directed the development of Th0/Th1-like cells, which produced moderate amounts of interferon gamma (IFN-gamma). As the peptide dose was increased, development of Th1-like cells producing increased amounts of IFN-gamma was initially observed. At very high (> 10 microM) and very low (< 0.05 microM) doses of antigenic peptide, however, a dramatic switch to development of Th2-like cells that produced increasing amounts of interleukin 4 (IL-4) and diminishing levels of IFN-gamma was observed. This was true even when highly purified naive, high buoyant density CD4+ LECAM-1hi T cells were used, ruling out a possible contribution from contaminating "memory" phenotype CD4+ T cells. Neutralizing anti-IL-4 antibodies completely inhibited the development of this Th2-like phenotype at both high and low antigen doses, demonstrating a requirement for endogenous IL-4. Our findings suggest that the antigen dose may affect the levels of endogenous cytokines such as IL-4 in primary cultures, resulting in the development of distinct Th cell phenotypes.

Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage.

Abstract: Apoptosis occurs in the normal liver and in various forms of liver disease. The CD95 (APO-1/Fas) (CD95) receptor mediates apoptosis, and liver cells in animal models are acutely sensitive to apoptosis initiated by this receptor. We have used primary human hepatocytes as a model system to investigate CD95-mediated apoptotic liver damage. Treatment of fresh human hepatocytes with low concentrations of agonistic antibodies against CD95 resulted in apoptosis of > 95% of the cultured liver cells within 4 and 7.5 h. Immunohistology of a panel of explanted liver tissues revealed that hepatocytes in normal livers (n = 5) and in alcoholic cirrhosis (n = 13) expressed low constitutive levels of CD95. CD95 receptor expression was highly elevated in hepatocytes in hepatitis B virus-related cirrhosis (n = 9) and in acute liver failure (n = 8). By in situ hybridization CD95 ligand messenger RNA expression was absent in normal liver but detected at high levels in livers with ongoing liver damage. In cases of hepatitis B virus-related cirrhosis and acute hepatic failure, ligand expression was found primarily in areas with lymphocytic infiltration. In contrast, in patients with alcoholic liver damage, high CD95 ligand messenger RNA expression was found in hepatocytes. These findings suggest that liver destruction in hepatitis B may primarily involve killing of hepatocytes by T lymphocytes using the CD95 receptor-ligand system. In alcoholic liver damage, death of hepatocytes might occur by fratricide and paracrine or autocrine mechanisms mediated by the hepatocytes themselves.

Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes.

Abstract: The appearance of phosphatidylserine (PS) on the cell surface during apoptosis in thymocytes and cytotoxic T lymphocyte cell lines provokes PS-dependent recognition by activated macrophages. Flow cytometric analysis of transbilayer lipid movements in T lymphocytes undergoing apoptosis reveals that downregulation of the adenosine triphosphate-dependent amino-phospholipid translocase and activation of a nonspecific lipid scramblase are responsible for PS reaching the surface from its intracellular location. Both mechanisms are expressed at the same time, and precede DNA degradation, zeiosis, and cell lysis in the apoptotic pathway.

Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited.

Abstract: We have used the cryosection immunogold technique to study the composition of the Mycobacterium tuberculosis phagosome. We have used quantitative immunogold staining to determine the distribution of several known markers of the endosomal-lysosomal pathway in human monocytes after ingestion of either M. tuberculosis, Legionella pneumophila, or polystyrene beads. Compared with the other phagocytic particles studied, the M. tuberculosis phagosome exhibits delayed clearance of major histocompatibility complex (MHC) class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transferrin receptor, and relatively weak staining for the lysosomal membrane glycoproteins, CD63, LAMP-1, and LAMP-2 and the lysosomal acid protease, cathepsin D. In contrast to M. tuberculosis, the L. pneumophila phagosome rapidly clears MHC class I molecules and excludes all endosomal-lysosomal markers studied. In contrast to both live M. tuberculosis and L. pneumophila phagosomes, phagosomes containing either polystyrene beads or heat-killed M. tuberculosis stain intensely for lysosomal membrane glycoproteins and cathepsin D. These findings suggest that (a) M. tuberculosis retards the maturation of its phagosome along the endosomal-lysosomal pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics; and (b) the intraphagosomal pathway, i.e., the pathway followed by several intracellular parasites that inhibit phagosome-lysosome fusion, is heterogeneous.

Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production.

Abstract: During human immunodeficiency virus infection and allergic diseases, characterized by a dominant T helper (Th) 2 response, overproduction of prostaglandin E2 (PGE2) is observed. In this paper we studied the effect of PGE2 on interleukin (IL)-12 synthesis, because this cytokine has been described to be essential in induction of Th1 responses. IL-12 synthesis was induced in monocytes that were stimulated with Neisseria meningitidis-derived lipopolysaccharide in whole blood cultures. PGE2 almost completely inhibited lipopolysaccharide induced IL-12 production, whereas IL-6 production was only partially inhibited by PGE2. In contrast, the production of IL-10 was approximately twofold enhanced at these conditions. The effects of PGE2 were due to its cAMP-inducing capacity, since they could be mimicked by other cAMP inducers. Recombinant human IL-10 also inhibited IL-12 and IL-6 production. However, the inhibitory effect of PGE2 on IL-12 production was independent of IL-10 since neutralizing anti-IL-10 antibodies were unable to reverse this inhibition. These results suggest that the capacity of an antigen to induce PGE2 synthesis may play a crucial role in the development of either a Th1 or Th2 response.

Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo.

Abstract: Human interferon-inducible protein 10 (IP-10), a member of the alpha chemokine family, inhibits bone marrow colony formation, has antitumor activity in vivo, is chemoattractant for human monocytes and T cells, and promotes T cell adhesion to endothelial cells. Here we report that IP-10 is a potent inhibitor of angiogenesis in vivo. IP-10 profoundly inhibited basic fibroblast growth factor-induced neovascularization of Matrigel (prepared by H. K. Kleinman) injected subcutaneously into athymic mice. In addition, IP-10, in a dose-dependent fashion, suppressed endothelial cell differentiation into tubular capillary structures in vitro. IP-10 had no effect on endothelial cell growth, attachment, and migration as assayed in vitro. These results document an important biological property of IP-10 and raise the possibility that IP-10 may participate in the regulation of angiogenesis during inflammation and tumorigenesis.

Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+ T cells.

Abstract: Naive CD4+ T cells can differentiate into cells predominantly involved in humoral immunity, known as T helper type 2 cells (Th2), or cells involved in cell-mediated immunity, known as Th1 cells. In this report, we show that priming of CD4+ T cells bearing a transgene-encoded T cell receptor can lead to differentiation into Th1-like cells producing abundant interferon gamma when the cells are exposed to high antigen doses, while low doses of the same peptide induce cells with the same T cell receptor to differentiate into Th2-like cells producing abundant interleukin 4. Thus antigen dose is one factor that can control the differentiation fate of a naive CD4+ T cell.

Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics.

Abstract: To gain more insight into the role of HIV-1-specific cytotoxic T lymphocytes (CTL) in the pathogenesis of AIDS, we investigated temporal relations between HIV-1 Gag-specific precursor CTL (CTLp), HIV-1 viral load, CD4+ T cell counts, and T cell function. Six HIV-1-infected subjects, who were asymptomatic for more than 8 yr with CD4+ counts > 500 cells/mm3, were compared with six subjects who progressed to AIDS within 5 yr after HIV-1 seroconversion. In the long-term asymptomatics, persistent HIV-1 Gag-specific CTL responses and very low numbers of HIV-1-infected CD4+ T cells coincided with normal and stable CD4+ counts and preserved CD3 mAb-induced T cell reactivity for more than 8 yr. In five out of six rapid progressors Gag-specific CTLp were also detected. However, early in infection the number of circulating HIV-1-infected CD4+ T cells increased despite strong and mounting Gag-specific CTL responses. During subsequent clinical progression to AIDS, loss of Gag-specific CTLp coincided with precipitating CD4+ counts and severe deterioration of T cell function. The possible relationships of HIV-1 Gag-specific CTLp to disease progression are discussed.

Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.

Abstract: Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.

Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse.

Abstract: Insulin-dependent diabetes mellitus (IDDM) is thought to be an immunologically mediated disease resulting in the complete destruction of the insulin-producing islets of Langerhans. It has become increasingly clear that autoreactive T cells play a major role in the development and progression of this disease. In this study, we examined the role of the CD28/B7 costimulation pathway in the development and progression of autoimmune diabetes in the nonobese diabetic (NOD) mouse model. Female NOD mice treated at the onset of insulitis (2-4 wk of age) with CTLA4Ig immunoglobulin (Ig) (a soluble CD28 antagonist) or a monoclonal antibody (mAb) specific for B7-2 (a CD28 ligand) did not develop diabetes. However, neither of these treatments altered the disease process when administered late, at > 10 wk of age. Histological examination of islets from the various treatment groups showed that while CTLA4Ig and anti-B7-2 mAb treatment blocked the development of diabetes, these reagents had little effect on the development or severity of insulitis. Together these results suggest that blockade of costimulatory signals by CTLA4Ig or anti-B7-2 acts early in disease development, after insulitis but before the onset of frank diabetes. NOD mice were also treated with mAbs to another CD28 ligand, B7-1. In contrast to the previous results, the anti-B7-1 treatment significantly accelerated the development of disease in female mice and, most interestingly, induced diabetes in normally resistant male mice. A combination of anti-B7-1 and anti-B7-2 mAbs also resulted in an accelerated onset of diabetes, similar to that observed with anti-B7-1 mAb treatment alone, suggesting that anti-B7-1 mAb's effect was dominant. Furthermore, treatment with anti-B7-1 mAbs resulted in a more rapid and severe infiltrate. Finally, T cells isolated from the pancreas of these anti-B7-1-treated animals exhibited a more activated phenotype than T cells isolated from any of the other treatment groups. These studies demonstrate that costimulatory signals play an important role in the autoimmune process, and that different members of the B7 family have distinct regulatory functions during the development of autoimmune diabetes.

Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo.

Abstract: Experiments were designed to study the effect of systemically administered IL-5 on local eosinophil accumulation induced by the intradermal injection of the chemokine eotaxin in the guinea pig. Intravenous interleukin-5 (IL-5) stimulated a rapid and dramatic increase in the numbers of accumulating eosinophils induced by i.d.-injected eotaxin and, for comparison, leukotriene B4. The numbers of locally accumulating eosinophils correlated directly with a rapid increase in circulating eosinophils: circulating eosinophil numbers were 13-fold higher 1 h after intravenous IL-5 (18.3 pmol/kg). This increase in circulating cells corresponded with a reduction in the number of displaceable eosinophils recovered after flushing out the femur bone marrow cavity. Intradermal IL-5, at the doses tested, did not induce significant eosinophil accumulation. We propose that these experiments simulate important early features of the tissue response to local allergen exposure in a sensitized individual, with eosinophil chemoattractant chemokines having an important local role in eosinophil recruitment from blood microvessels, and IL-5 facilitating this process by acting remotely as a hormone to stimulate the release into the circulation of a rapidly mobilizable pool of bone marrow eosinophils. This action of IL-5 would be complementary to the other established activities of IL-5 that operate over a longer time course.

A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.

Abstract: Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN-gamma and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid- but not LPS-induced CAT activity by more than 70%. Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-gamma or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRE. Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-gamma activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression. These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.

Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice.

Abstract: Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen-induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.

A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma.

Abstract: Human melanoma MZ2-MEL expresses several distinct antigens that are recognized by autologous cytolytic T lymphocytes (CTL). Some of these antigens are encoded by genes MAGE-1, MAGE-3, and BAGE, which are expressed in a large fraction of tumors of various histological types but are silent in normal adult tissues with the exception of testis. We report here the identification of the gene coding for MZ2-F, another antigen recognized by autologous CTL on MZ2-MEL cells. This gene, which was named GAGE-1, is not related to any presently known gene. It belongs to a family of genes that are expressed in a variety of tumors but not in normal tissues, except for the testis. Antigenic peptide YRPRPRRY, which is encoded by GAGE-1, is recognized by anti-MZ2-F CTL on class I molecule HLA-Cw6. The two genes of the GAGE family that code for this peptide, namely GAGE-1 and GAGE-2, are expressed in a significant proportion of melanomas (24%), sarcomas (25%), non-small cell lung cancers (19%), head and neck tumors (19%), and bladder tumors (12%). About 50% of melanoma patients carry on their tumor at least one of the presently defined antigens encoded by the MAGE, BAGE, and GAGE genes.

Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines.

Abstract: Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA-A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. E75-pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA-A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens.

Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12.

Abstract: Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that can be transferred to naive mice via CD4+ T cells isolated from appropriately immunized mice. We have evaluated the effects of recombinant murine interleukin 12 (rmIL-12), a potent inducer of interferon gamma (IFN-gamma) and promoter of Th1 T cell development, on the course of adoptively transferred EAE. The transfer of lymph node cells (LNC) isolated from proteolipid protein (PLP)-primed animals and stimulated in vitro with PLP to naive mice resulted in a progressive paralytic disease culminating in complete hind limb paralysis in the majority of the recipients. When mice were injected with LNC that had been stimulated in vitro with PLP in the presence of rmIL-12, the subsequent course of disease was more severe and prolonged. The addition of rmIL-12 during the in vitro stimulation with PLP resulted in a 10-fold increase in IFN-gamma and a 2-fold increase in tumor necrosis factor (TNF) alpha in the supernatants, relative to LNC stimulated with PLP alone. However, neutralization of IFN-gamma or TNF-alpha in vitro with specific antibodies did not abrogate the ability of rmIL-12 to exacerbate the subsequent disease. Similarly, mice treated with rmIL-12 in vivo after the transfer of antigen-stimulated LNC developed a more severe and prolonged course of disease compared with vehicle-treated control animals. In contrast, treatment of mice with an antibody to murine IL-12 after cell transfer completely prevented paralysis, with only 40% of the mice developing mild disease. These results demonstrate that in vitro stimulation of antigen primed LNC with PLP and rmIL-12 enhances their subsequent encephalitogenicity. Furthermore, inhibition of endogenous IL-12 in vivo after LNC transfer prevented paralysis, suggesting that endogenous IL-12 plays a pivotal role in the pathogenesis of this model of autoimmune disease.

Sequential contribution of L- and P-selectin to leukocyte rolling in vivo.

Abstract: Leukocyte recruitment into inflammatory sites is initiated by a reversible transient adhesive contact with the endothelium called leukocyte rolling, which is thought to be mediated by the selectin family of adhesion molecules. Selectin-mediated rolling precedes inflammatory cell emigration, which is significantly impaired in both P- and L-selectin gene-deficient mice. We report here that approximately 13% of all leukocytes passing venules of the cremaster muscle of wild-type mice roll along the endothelium at < 20 min after surgical dissection. Rolling leukocyte flux fraction reaches a maximum of 28% at 40-60 min and returns to 13% at 80-120 min. In P-selectin-deficient mice, rolling is absent initially and reaches 5% at 80-120 min. Rolling flux fraction in L-selectin-deficient mice is similar to wild type initially and declines to 5% at 80-120 min. In both wild-type and L-selectin-deficient mice, initial leukocyte rolling (0-60 min) is completely blocked by the P-selectin monoclonal antibody (mAb) RB40.34, but unaffected by L-selectin mAb MEL-14. Conversely, rolling at later time points (60-120 min) is inhibited by mAb MEL-14 but not by mAb RB40.34. After treatment with tumor necrosis factor (TNF)-alpha for 2 h, approximately 24% of all passing leukocytes roll in cremaster venules of wild-type and P-selectin gene-deficient mice. Rolling in TNF-alpha-treated mice is unaffected by P-selectin mAb or E-selectin mAb 10E9.6. By contrast, rolling in TNF-alpha-treated P-selectin-deficient mice is completely blocked by L-selectin mAb. These data show that P-selectin is important during the initial induction of leukocyte rolling after tissue trauma. At later time points and in TNF-alpha-treated preparations, rolling is largely L-selectin dependent. Under the conditions tested, we are unable to find evidence for involvement of E-selectin in leukocyte rolling in mice.

Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations.

Abstract: CD4+ T helper (Th) cells can be classified into different types based on their cytokine profile. Cells with these polarized patterns of cytokine production have been termed Th1 and Th2, and can be distinguished functionally by the production of IFN-gamma and IL-4, respectively. These phenotypes are crucial in determining the type of immune response that develops after antigen priming. There are no surface markers that define them, and cytokine immunoassay or mRNA analysis both have limitations for characterization of single cells. Using immunofluorescent detection of intracellular IFN-gamma and IL-4, we have studied the emergence of Th1 and Th2 cells in response to antigen exposure and the patterns of cytokine synthesis in established T cell clones. IFN-gamma production by Th1 clones was detectable in almost all cells by 4 h, and it continued in most cells for > 24 h. IL-4 production in Th2 cells peaked at 4 h, but declined rapidly. In Th0 cells containing both cytokines, fewer cells produced IFN-gamma, which did not appear until IL-4 synthesis declined. Cocultivation of clones showed no such cross-regulation. Antigen stimulation of transgenic T cells expressing an ovalbumin-specific T cell receptor generated Th2 cells, probably as a result of endogenous IL-4 production. Addition of IL-12 and/or anti-IL-4 caused Th1 cells to develop, while some Th0 cells were seen when IL-12 alone was added. These results show that stimulation in the presence of polarizing stimuli results in cells producing either IFN-gamma or IL-4, but that coproduction can occur in rare cells under defined conditions.

The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.

Abstract: In mice, natural resistance or susceptibility to infection with intracellular parasites is determined by a locus or group of loci on chromosome 1, designated Bcg, Lsh, and Ity, which controls early microbial replication in reticuloendothelial organs. We have identified by positional cloning a candidate gene for Bcg, Nramp1, which codes for a novel macrophage-specific membrane transport protein. We have created a mouse mutant bearing a null allele at Nramp1, and we have analyzed the effect of such a mutation on natural resistance to infection. Targeted disruption of Nramp1 has pleiotropic effects on natural resistance to infection with intracellular parasites, as it eliminated resistance to Mycobacterium bovis, Leishmania donovani, and lethal Salmonella typhimurium infection, establishing that Nramp1, Bcg, Lsh, and Ity are the same locus. Comparing the profiles of parasite replication in control and Nramp1-/- mice indicated that the Nramp1Asp169 allele of BcgS inbred strains is a null allele, pointing to a critical role of this residue in the mechanism of action of the protein. Despite their inability to control parasite growth in the early nonimmune phase of the infection, Nramp1-/- mutants can overcome the infection in the late immune phase, suggesting that Nramp1 plays a key role only in the early part of the macrophage-parasite interaction and may function by a cytocidal or cytostatic mechanism distinct from those expressed by activated macrophages.

CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2.

Abstract: Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig-treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig-treated animals was associated with increased staining for the Th2-related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig-treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig-treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo.

Sustained signaling leading to t cell activation results from prolonged t cell receptor occupancy. role of t cell actin cytoskeleton

Abstract: Using antigen-specific T cell clones and peptide-pulsed antigen-presenting cells (APCs) we investigated the mechanisms that lead to sustained signaling, known to be required for activation of effector function. Four lines of evidence indicate that the T cell actin cytoskeleton plays a crucial role in T cell activation by antigen-pulsed APCs, but is not required when T cell receptor (TCR) is cross-linked by soluble antibodies. First, addition of antibodies to the major histocompatibility complex molecules recognized by the TCR aborts the ongoing intracellular calcium concentration ([Ca2+]i) increase in performed T-APC conjugates, indicating that the sustained signaling requires the continuous occupancy of TCR. Second, time-lapse image recording shows that T lymphocytes conjugated to peptide-pulsed APCs undergo a sustained [Ca2+]i increase, which is accompanied by the formation of a large and changing area of contact between the two opposing membranes. Third, drugs that disrupt the actin cytoskeleton, Cytochalasin D and and C2 Clostridium botulinum toxin induce a rapid block of [Ca2+]i rise, coincident with a block of the cyclic changes in T cell shape. Finally, the addition of Cytochalasin D or of anti-MHC antibodies to preformed conjugates inhibits interferon gamma production in an 1-antigen dose- and time-dependent fashion. These results identify T cell actin cytoskeleton as a major motor for sustaining signal transduction and possibly for driving TCR cross-linking and offer an explanation for how T cells equipped with low affinity TCR can be triggered by a small number of complexes on APCs.