bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibrinolytic response to tumor necrosis factor in healthy subjects.
Authors: van der Poll T , Levi M , Büller H R , van Deventer S J , de Boer J P , Hack C E , ten Cate J W
Published: June 2004

Tumor necrosis factor (TNF) may be involved in the disturbance of the procoagulant-fibrinolytic balance in septicemia, leading to microvascular thrombosis. To assess the dynamics of the fibrinolytic response to TNF in humans, we performed a crossover saline-controlled study in six healthy men, investigating the effects of a bolus intravenous injection of recombinant human TNF (50 micrograms/m2) on the stimulation and inhibition of plasminogen activation as well as on plasmin activity and inhibition. TNF induced a brief fourfold increase in the overall plasma plasminogen activator (PA) activity peaking after 1 h (p less than 0.0001), which was associated with rises in the antigenic levels of urokinase-type plasminogen activator (p less than 0.0001) and tissue-type plasminogen activator (p less than 0.0001). Plasminogen activator inhibitor type I antigen remained unchanged in the first hour, but showed a rapid eightfold increase thereafter (p less than 0.0001), which coincided with the decrease in PA activity. Generation of plasmin activity in the first hour was signified by an 11-fold rise in D-dimer levels (p less than 0.0001); inhibition of plasmin was reflected by a 36-fold rise in plasmin-alpha 2 antiplasmin complexes (p less than 0.0001), as well as by a transient 16% decrease in alpha 2-antiplasmin activity (p less than 0.01). In conclusion, TNF induced an early activation of the fibrinolytic system becoming maximal in 1 h, with a rapid inhibition thereafter. Earlier observations in the same subjects showed sustained coagulation activation for 6-12 h. The observed disbalance between the procoagulant and fibrinolytic mechanisms after TNF injection confirms the in vivo relevance of the effects of TNF on vascular endothelium in vitro and may explain the tendency towards microvascular thrombosis in septicemia.
Neutrophil-mediated dissolution of infected host cells as a defense strategy against a facultative intracellular bacterium.
Authors: Conlan J W , North R J
Published: June 2004

The rate of growth of Listeria monocytogenes in the livers of mice infected intravenously with a lethal or sublethal inoculum of this facultative intracellular bacterium is greatly increased if neutrophils and other host cells are prevented from accumulating at foci of infection during the first 24 h by treatment with a monoclonal antibody (5C6) specific for the type 3 complement receptor of myelomonocytic cells. A histological examination of the livers of control mice showed that the accumulation of neutrophils at infectious foci resulted in the focal destruction of infected hepatocytes. In contrast, failure of neutrophils to accumulate at these sites in 5C6-treated mice allowed Listeria to multiply extensively in hepatocytes without destroying them. The results indicate that neutrophils play an important role in early defense against listeriosis in the liver by destroying infected hepatocytes, thereby reducing the opportunity for Listeria to multiply in permissive cells. In this way, neutrophils serve to break the chain of cell-to-cell spread of infection.
Antileishmanial defense in macrophages triggered by tumor necrosis factor expressed on CD4+ T lymphocyte plasma membrane.
Authors: Sypek J P , Wyler D J
Published: June 2004

In our studies of host defense to the intracellular protozoan Leishmania major, we uncovered a novel mechanism of antileishmanial defense that involves direct cell contact between effector CD4+ lymphocytes and Leishmania-infected macrophages. The mechanism is distinctive because it does not involve lymphokine secretion and induces no cytotoxic effects in the host cells; its expression is antigen-specific and genetically restricted. We now demonstrate that these effector CD4+ cells display tumor necrosis factor (TNF) on their surface and provide evidence that the membrane-associated TNF is involved in the activation of the antileishmanial defense. Using a Leishmania-specific cloned T-T cell hybridoma line (1B6; CD4+, T helper type 1) that activates antileishmanial defense in macrophages through cell contact and does not secrete TNF, we noted that only cells bearing surface TNF (TNF+), but not ones lacking surface TNF (TNF-), exerted these effects. Moreover, the antileishmanial effects excreted by TNF+ 1B6 cells as well as by lymph node CD4+ TNF+ lymphocytes could be blocked with anti-TNF antibody. We propose that membrane-associated TNF on CD4+ T cells may provide a mechanism of targeting activation signals to macrophages in an antigen-specific and genetically restricted manner.
Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide.
Authors: Kwon N S , Stuehr D J , Nathan C F
Published: June 2004

Macrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate-limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting L-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N omega-methyl-L-arginine, or N omega-amino-L-arginine. L-Arginine, but not D-arginine, abolished the protective effect of N omega-amino-L-arginine. The prototypic pharmacologic inhibitor of RR is hydroxyurea. Its structural resemblance to N omega-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H2O2 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3- generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR.
T cell receptor gamma and delta gene junctional sequences in SCID mice: excessive P nucleotide insertion.
Authors: Kienker L J , Kuziel W A , Tucker P W
Published: June 2004

The severe combined immunodeficiency (SCID) mutation has been postulated to affect a V(D)J recombinase activity involved in coding joint formation. Analysis of 38 joints from 34 distinct sequences of normally rearranged T cell receptor (TCR) gamma and delta genes from adult, SCID thymocytes reveals coding joints with an increased number of P nucleotides. One-third of P sequences are greater than or equal to 4 nucleotides in length and P elements of up to 15 bases are observed. This suggests that the SCID defect deregulates P nucleotide addition. Consequently, essential V(D)J recombination intermediates may seldom be generated.
Platelet-derived interleukin 1 induces human endothelial adhesion molecule expression and cytokine production.
Authors: Hawrylowicz C M , Howells G L , Feldmann M
Published: June 2004

Interleukin 1 (IL-1) plays a central role in the regulation of the body's response to infectious and inflammatory stimuli. Recent evidence has shown that human platelets express a cell associated form of this proinflammatory cytokine very rapidly following activation. Since one of the earliest events in inflammation is frequently the rapid adhesion of platelets to injured endothelium, it was of interest to determine whether platelets express IL-1 in a functionally relevant form that can alter the phenotype of human endothelial cells in vitro. Thrombin activated platelets induced significant expression of the adhesion molecule intercellular adhesion molecule 1, as well as secretion of the IL-1 inducible cytokines IL-6 and granulocyte macrophage colony stimulating factor by cultured human umbilical cord and saphenous vein endothelial cells. This was inhibited by prior treatment of the platelets with antibody specific for IL-1. These results suggest that platelet delivered IL-1 might initiate and regulate some of the earliest phases of the inflammatory response. An additional observation of interest was differential induction of endothelial leucocyte adhesion molecule 1 by activated platelets on saphenous vein but not umbilical vein but not umbilical vein endothelial cells, which suggests functional heterogeneity of the endothelial cells.
Antigen-driven bystander suppression after oral administration of antigens.
Authors: Miller A , Lider O , Weiner H L
Published: June 2004

Suppression of experimental autoimmune encephalomyelitis (EAE) in Lewis rats by the oral administration of myelin basic protein (MBP) is mediated by CD8+ T cells that can be isolated from the spleens of MBP-fed animals. These cells adoptively transfer protection to naive animals subsequently immunized with MBP and complete Freund's adjuvant (CFA) and suppress in vitro MBP proliferative responses. Using a transwell system in which the modulator spleen cells from MBP-fed animals are separated by a semipermeable membrane from responder cells, MBP, or OVA-specific T cell lines, we have found that cell contact is not required for in vitro suppression to occur. In vitro suppression is dependent, however, upon antigen-specific triggering of modulator T cells. Once antigen-specific triggering occurs, suppression across the transwell is mediated by an antigen-nonspecific soluble factor that equally suppresses an MBP line or an ovalbumin (OVA) line. This phenomenon of antigen-driven bystander suppression was also demonstrated in vivo. Specifically, Lewis rats fed OVA which were then immunized with MBP/CFA plus OVA given separately subcutaneously were protected from EAE. Animals fed OVA and then immunized with MBP/CFA without OVA given subcutaneously were not protected. The protective effect of feeding OVA could be adoptively transferred by CD8+ T cells from OVA-fed animals into MBP/CFA plus OVA-injected animals. Feeding bovine serum albumin (BSA) or keyhole limpet hemocyanin did not suppress EAE in animals immunized with MBP/CFA plus OVA. EAE was suppressed, however, if BSA was fed and animals then immunized with MBP/CFA plus BSA given subcutaneously. Antigen-driven bystander suppression appears to be an important mechanism by which antigen-driven peripheral tolerance after oral administration of antigen is mediated, and presumably occurs in the microenvironment accounting for the antigen specificity of suppression generated by oral tolerization to antigens.
Helper activity for immunoglobulin synthesis of T helper type 1 (Th1) and Th2 human T cell clones: the help of Th1 clones is limited by their cytolytic capacity.
Authors: Del Prete G F , De Carli M , Ricci M , Romagnani S
Published: June 2004

A large number of CD4+ human T helper type 1 (Th1) clones specific for purified protein derivative and of Th2 clones specific for the excretory/secretory antigen of Toxocara canis, derived from the same individuals, were analyzed for both cytotoxic capacity and helper function for immunoglobulin (Ig) synthesis. The great majority of Th1, but only a minority of Th2 clones exhibited cytolytic activity. All Th2 (noncytolytic) clones induced IgM, IgG, IgA, and IgE synthesis by autologous B cells in the presence of the specific antigen, and the degree of response was proportional to the number of Th2 cells added to B cells. Under the same experimental conditions, Th1 (cytolytic) clones provided helper function for IgM, IgG, and IgA, but not IgE, synthesis with a peak response at 1:1 T/B cell ratio. At higher T/B cell ratios, a strong decrease of Ig synthesis was observed. All Th1 clones lysed Epstein-Barr virus transformed autologous B cells pulsed with the specific antigen. The decrease of Ig production at high T/B cell ratios correlated with the lytic activity of Th1 clones against autologous antigen-presenting B cell targets. These data suggest that Th1 differ from Th2 human T cell clones not only for their profile of cytokine secretion, but also for cytolytic potential and mode of help for B cell Ig synthesis.
Rapid and specific conversion of precursor interleukin 1 beta (IL-1 beta) to an active IL-1 species by human mast cell chymase.
Authors: Mizutani H , Schechter N , Lazarus G , Black R A , Kupper T S
Published: June 2004

Secretory granules of human dermal mast cells contain a chymotrypsin-like serine proteinase called chymase. In this study, we demonstrate that the inactive cytokine, 31 kD interleukin 1 beta (IL-1 beta), can be converted rapidly to an 18 kD biologically active species by human mast cell chymase. The product formed is three amino acids longer at the amino terminus than the mature IL-1 beta produced by peripheral blood mononuclear cells and has comparable biological activity. Because chymase is a secretory granule constituent, it is likely to be released into the surrounding tissue when mast cells degranulate. It is also known that non-bone marrow derived cells resident in skin (keratinocytes, fibroblasts) produce but do not process 31 kD IL-1 beta. In this context, chymase may be a potent activator of locally produced 31 kD IL-1 beta. Mast cells lie in close apposition to blood vessels in dermis; therefore, chymase mediated conversion of 31 kD IL-1 beta might be expected to have a critical role in the initiation of the inflammatory response in skin.
Macrosialin, a macrophage-restricted membrane sialoprotein differentially glycosylated in response to inflammatory stimuli.
Authors: Rabinowitz S S , Gordon S
Published: June 2004

Rat monoclonal antibody FA/11 has been used to identify macrosialin, a sialoglycoprotein confined to murine mononuclear phagocytes and related cells. Originally identified as a macrophage-associated glycoprotein predominantly localized in intracellular membranes (Smith, M.J., and G.L.E. Koch. 1987. J. Cell Sci. 87:113), the antigen is widely expressed on tissue macrophages, including those in lymphoid areas, and is expressed at low levels on isolated dendritic cells. Immuno-adsorption experiments reported here show that macrosialin is identical to the major 87-115-kD sialoglycoprotein previously identified by lectin blotting in exudate but not resident peritoneal macrophages (Rabinowitz, S., and S. Gordon. 1989. J. Cell Sci. 93:623). Resident peritoneal macrophages express low levels of macrosialin antigen in a glycoform that does not bind 125I wheat germ agglutinin or 125I peanut agglutinin; inflammatory stimuli upregulate expression of this antigen (up to 17-fold), in an alternative glycoform that is detected by these lectins. Pulse-chase experiments reveal a 44-kD core peptide that initially bears high-mannose chains (giving Mr 66 kD) and is subsequently processed to a mature protein of Mr 87-104 kD. Each glycoform contains N-linked glycan, as well as O-linked sugar structures that show alternative processing. Poly-N-acetyllactosamine structures are detected in the exudate cell glycoform only. This new marker for mononuclear phagocytes illustrates two strategies by which macrophages remodel their membranes in response to inflammatory stimuli. Its predominantly intracellular location and restricted cell distribution suggest a possible role in membrane fusion or antigen processing.
Regulation of the immune response to peptide antigens: differential induction of immediate-type hypersensitivity and T cell proliferation due to changes in either peptide structure or major histocompatibility complex haplotype.
Authors: Soloway P , Fish S , Passmore H , Gefter M , Coffee R , Manser T
Published: June 2004

The immunodominant CD4 T cell epitope of the bacteriophage lambda cI repressor protein in several inbred mouse strains can be represented by a peptide encompassing amino acids 12-26. Here, we show that this peptide, and a variety of its sequence variants, can induce immediate-type hypersensitivity in mice. 12-26 variants that differ by as little as single amino acid residues deviate greatly in their ability to induce hypersensitivity. Further, differences in major histocompatibility complex class II alleles appear to be as influential as changes in peptide structure in determining whether hypersensitivity is developed. The ability of a given peptide-class II combination to induce hypersensitivity correlates with production of peptide-specific antibody, but not with ability or inability to induce a T cell proliferative response. Administration of anti-interleukin 4 (IL-4) mAb prevents the development of hypersensitivity, and analysis of cytokine production by T cell hybridomas derived from peptide-immunized mice suggests that whether a given peptide-class II combination can induce hypersensitivity depends on its ability to induce IL-4 production. The data demonstrate that changes in the nature of the epitope(s) recognized by the CD4 T cell population can result in qualitative differences in the response elicited in this population, ultimately leading to dramatic quantitative and qualitative variations in the effector phase of the immune response.
Chromosomal translocations joining LCK and TCRB loci in human T cell leukemia.
Authors: Tycko B , Smith S D , Sklar J
Published: June 2004

A case of T lymphoblastic leukemia (T-ALL) showing t(1;7)(p34;q34) as the sole karyotypic abnormality was investigated at the molecular level. Screening of a phage library of tumor DNA with a probe for the beta T cell receptor gene (TCRB), which maps to chromosomal band 7q34, resulted in the isolation of a clone containing DNA spanning the translocation breakpoint of the der(1) chromosome. This clone contained chromosome 1 DNA juxtaposed upstream of a D beta-J beta joint. Cloning of the corresponding germline region of chromosome 1 resulted in the isolation of a phage containing the breakpoint from the reciprocal, der(7), product, which showed chromosome 1 DNA joined downstream to a V beta segment. Comparison of germline and translocation clones demonstrated that breakage of chromosome 1 had occurred at the border of a tandem repeat of Alu sequences. To search for transcripts from DNA near the breakpoint, a chromosomal walk was initiated along chromosome 1. A probe consisting of chromosome 1 DNA from 24-30 kb upstream of the breakpoint hybridized to a transcript derived from the gene encoding the lymphocyte-specific tyrosine kinase p56lck, previously mapped to chromosomal band 1p34. The nonrandom nature of the breakpoints in this case was confirmed by the analysis of a second independent case of T-ALL containing a t(1;7) translocation, which was also found to show breakage within the LCK locus. The chromosomal breakpoint in the first case was localized 2 kb upstream of the lck upstream promoter and first nontranslated exon, while the breakpoint of the second case lay between the two alternative lck promoters, upstream of the second exon. Relative to normal thymus and activated T cells, levels of lck mRNA were greatly elevated in the first case and moderately elevated in the second. The existence of these translocations raises the possibility that alterations in the promoter region of the LCK locus may play a role in human cancer.
Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast.
Authors: Hart P D , Young M R
Published: June 2004

The weak base ammonium chloride has been previously reported to inhibit lysosomal movements and phagosome-lysosome (Ph-L) fusion in cultured mouse macrophages (M phi), thus reducing delivery, to an intraphagosomal infection, of endocytosed solutes that have concentrated in secondary lysosomes. We have now addressed the question, whether NH4Cl might affect any direct interaction (if it exists) between such infection phagosomes and earlier, nonlysosomal compartments of the endocytic pathway, i.e., solute-containing endosomes. The phagosomes studied were formed after ingestion of the mouse pathogen Mycobacterium microti and the nonpathogenic yeast Saccharomyces cerevisiae; and the endosomes were formed after nonreceptor-mediated endocytosis of electronopaque and fluorescent soluble markers. By electron microscopy, survey of the cell profiles of M phi that had been treated with 10 mM NH4Cl so that Ph-L fusion was prevented, and that displayed many ferritin-labeled endosomes, revealed numerous examples of the fusion of electronlucent endosomes, revealed numerous examples of the fusion of electronlucent vesicles with phagosomes, whether containing M. microti bacilli or S. cerevisiae yeasts. Fusion was recognized by transfer of label and by morphological evidence of fusion in progress. The fusing vesicles were classed as endosomes, not NH4Cl-lysosomes, by their appearance and provenance, and because lysosome participation was excluded by the concurrent, NH4Cl-caused block of Ph-L fusion and associated lysosomal stasis. No evidence of such phagosome-endosome (Ph-E) fusion was observed in profiles from M phi treated with chloroquine, nor in those from normal, untreated M phi. NH4Cl-treated living M phi that had ingested yeasts at 37 degrees C, followed by endocytosis of lucifer yellow at 17 degrees C (to accumulate labeled endosomes and postpone label passing to lysosomes), were then restored to 37 degrees C. Fluorescence microscopy showed that as many as half of the yeast phagosomes (previously unlabeled) rapidly became colored. We inferred that this transfer was from endosomes (by Ph-E fusion) because Ph-L passage was blocked (by the NH4Cl). We conclude that NH4Cl induces Ph-E fusion at the same time as it suppressed Ph-L fusion. We discuss the mechanisms of these concurrent effects and suggest that they are independent; and we consider the implications of NH4Cl opening a direct route for endocytosed molecules to reach an intraphagosomal infection without involving lysosomes.
Characterization of human T cells reactive with the Mycoplasma arthritidis-derived superantigen (MAM): generation of a monoclonal antibody against V beta 17, the T cell receptor gene product expressed by a large fraction of MAM-reactive human T cells.
Authors: Friedman S M , Crow M K , Tumang J R , Tumang M , Xu Y Q , Hodtsev A S , Cole B C , Posnett D N
Published: June 2004

While all known microbial superantigens are mitogenic for human peripheral blood lymphocytes (PBL), the functional response induced by Mycoplasma arthritidis-derived superantigen (MAM) is unique in that MAM stimulation of PBL consistently results in T cell-dependent B cell activation characterized by polyclonal IgM and IgG production. These immunostimulatory effects of MAM on the humoral arm of the human immune system warranted a more precise characterization of MAM-reactive human T cells. Using an uncloned MAM reactive human T cell line as immunogen, we have generated a monoclonal antibody (mAb) (termed C1) specific for the T cell receptor V beta gene expressed by the major fraction of MAM-reactive human T cells, V beta 17. In addition, a V beta 17- MAM-reactive T cell population exists, assessed by MAM, induced T cell proliferation and cytotoxic T cell activity. mAb C1 will be useful in characterizing the functional properties of V beta 17+ T cells and their potential role in autoimmune disease.
Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1.
Authors: van Seventer G A , Newman W , Shimizu Y , Nutman T B , Tanaka Y , Horgan K J , Gopal T V , Ennis E , O'Sullivan D , Grey H
Published: June 2004

Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen-specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM-1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation-independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply that adhesion per se is not sufficient for costimulation, but rather that the costimulation conferred by the VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions reflects specialized accessory functions of these integrin pathways. The new finding that VLA-4/VCAM-1 mediates costimulation adds significance to observations that VCAM-1 is expressed on a unique set of potential antigen-presenting cells in vivo.
Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression.
Authors: de Waal Malefyt R , Haanen J , Spits H , Roncarolo M G , te Velde A , Figdor C , Johnson K , Kastelein R , Yssel H , de Vries J E
Published: June 2004

Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma- or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Furthermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.
Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.
Authors: Hatzfeld J , Li M L , Brown E L , Sookdeo H , Levesque J P , O'Toole T , Gurney C , Clark S C , Hatzfeld A
Published: June 2004

We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.
Human and rat mesangial cell receptors for glucose-modified proteins: potential role in kidney tissue remodelling and diabetic nephropathy.
Authors: Skolnik E Y , Yang Z , Makita Z , Radoff S , Kirstein M , Vlassara H
Published: June 2004

Advanced glycosylation endproducts (AGEs) are derived from the nonenzymatic addition of glucose to proteins. AGEs have been found to accumulate on tissue proteins in patients with diabetes, and their accumulation is thought to play a role in the development of diabetic complications. The finding that macrophages and endothelial cells contain AGE-specific receptors led us to examine whether mesangial cells (MCs) also possess a mechanism for recognizing and processing AGEs. Membrane extracts isolated from rat and human MCs were found to bind AGE-bovine serum albumin (BSA) in a saturable fashion, with a binding affinity of 2.0 +/- 0.4 x 10(6) M-1 (500 nM). The binding was specific for the AGE adduct, since AGE-modified collagen I and ribonuclease both competitively inhibited 125I-AGE-BSA binding to MC membranes, while the unmodified proteins did not compete. Binding of AGE proteins was followed by slow internalization and degradation of the ligand. Ligand blotting of MC membrane extracts demonstrated three distinct AGE-binding membrane proteins of 50, 40, and 30 kD. Growth of MCs on various AGE-modified matrix proteins resulted in alterations in MC function, as demonstrated by enhanced production of fibronectin and decreased proliferation. These results point to the potential role that the interaction of AGE-modified proteins with MCs may play in vivo in promoting diabetic kidney disease.
Exogenous peptides compete for the presentation of endogenous antigens to major histocompatibility complex class II-restricted T cells.
Authors: Adorini L , Moreno J , Momburg F , Hämmerling G J , Guéry J C , Valli A , Fuchs S
Published: June 2004

Antigen-presenting cells (APC) transfected with a construct encoding the hen egg-white lysozyme (HEL) amino acid sequence 1-80 constitutively present HEL peptides complexed to major histocompatibility complex (MHC) class II molecules to specific T cell hybridomas, indicating that endogenous cellular antigens can be efficiently presented to class II-restricted T cells. Here we show that exogenous peptide competitors added to HEL-transfected APC can inhibit the presentation of endogenous HEL peptides to class II-restricted T cells. The inhibition is specific for the class II molecule binding the competitor peptide, and it affects to the same extent presentation of exogenous or endogenous HEL peptides. These results, demonstrating that an exogenous competitor can inhibit class II-restricted T cell activation induced by endogenous as well as exogenous antigen, suggest lack of strict compartmentalization between endogenous and exogenous pathways of antigen presentation. Since autoreactive T cells may recognize endogenous, as well as exogenous antigens, the results have implications for the treatment of autoimmune diseases by MHC blockade.
Nitric oxide derived from L-arginine impairs cytoplasmic pH regulation by vacuolar-type H+ ATPases in peritoneal macrophages.
Authors: Swallow C J , Grinstein S , Sudsbury R A , Rotstein O D
Published: June 2004

The ability of macrophages (Møs) to function within an acidic environment has been shown to depend on cytoplasmic pH (pHi) regulation by vacuolar-type H+ ATPases. Møs metabolize L-arginine via an oxidative pathway that generates nitric oxide, nitrate, and nitrite. Since each of these products could potentially inhibit vacuolar-type H+ ATPases, we investigated the effect of L-arginine metabolism on Mø pHi regulation in thioglycolate-elicited murine peritoneal Møs. H+ ATPase-mediated pHi recovery from an imposed cytoplasmic acid load was measured fluorometrically. When Møs were incubated with L-arginine (0.25-2.0 mM), their rate of pHi recovery declined progressively from 2 to 6 h of incubation. By contrast, the recovery rate of cells incubated in arginine-free medium remained stable over the same period. The impairment of pHi recovery was specific for L-arginine, and was blocked competitively by NG-monomethyl-L-arginine, demonstrating its dependence on L-arginine metabolism. In addition, the inhibition of pHi recovery was enhanced by lipopolysaccharide, an agent known to stimulate L-arginine metabolism by Møs. Scavenging the L-arginine metabolite nitric oxide with either ferrous sulphate or ferrous myoglobin prevented the inhibition of pHi recovery, implying that L-arginine-derived nitric oxide was the species responsible for the inhibition. This concept was supported by the finding of elevated nitrite levels in the supernatant of cells incubated in L-arginine. Furthermore, incubation of Møs with sodium nitroprusside mimicked the L-arginine-dependent inhibition of H+ ATPase activity. Treatment with the cyclic GMP analogue, 8-bromoguanosine 3':5'-cyclic monophosphate, similarly impaired Mø pHi recovery, suggesting that a nitric oxide-stimulated elevation of cyclic GMP may contribute to the L-arginine-dependent inhibition of pHi regulation.
Selective autoantibody production by Yaa+ B cells in autoimmune Yaa(+)-Yaa- bone marrow chimeric mice.
Authors: Merino R , Fossati L , Lacour M , Izui S
Published: June 2004

The accelerated autoimmune syndrome observed in BXSB/MpJ male mice is associated with the presence on the Y chromosome of an as yet unidentified mutant gene, designated Y chromosome-linked autoimmune acceleration (Yaa). To study the mechanisms by which the Yaa gene accelerates and/or induces the production of autoantibodies, we have developed double-congenic bone marrow chimeras containing B cells from autoimmune males carrying the Yaa gene, and from nonautoimmune male or female mice lacking it and differing by the Igh allotype. The analysis of the allotype of total immunoglobulins and anti-DNA antibodies in Yaa+ male-normal female (Yaa-) chimeric mice revealed that the selective activation of B cells from autoimmune Yaa+ male mice was responsible for the hypergammaglobulinemia and autoantibody production. This phenomenon was not due to an anti-HY interaction between female T helper cells and male B cells, because first, Yaa+ B cells were selectively stimulated to produce autoantibodies in Yaa+ male-Yaa- male chimeric mice; and second, normal male and female chimeras failed to develop an autoimmune syndrome. In addition, the fact that both B cell populations in Yaa(+)-Yaa- chimeras similarly responded to a foreign antigen, human IgG, argues against the possibility that the selective activation of Yaa+ B cells may be due to their hyper-responsiveness to T helper signals. We propose that a cognate interaction of T helper cells with Yaa+ B cells, because of possible T cell recognition of a Yaa-related molecule expressed on Yaa+ B cells, may be responsible for the acceleration and/or induction of autoantibodies in BXSB/MpJ mice.
On the nature of peptides involved in T cell alloreactivity.
Authors: Rötzschke O , Falk K , Faath S , Rammensee H G
Published: June 2004

The strong reaction of T cells against foreign major histocompatibility complex (MHC) antigens, commonly termed "alloreactivity", is not only a nuisance for clinical organ transplantation; it also remains a puzzling question for immunologists. By making use of recent technical developments, alloreactive T cells nominally directed against a mutation in a single MHC class I molecule were found to fall into several major categories. One is recognizing peptides whose occurrence is dependent on one particular MHC allele, another is recognizing peptides supported by several MHC alleles, and a third is recognizing peptides occurring independently of MHC alleles. In a fourth category, the binding to MHC of any of a broad range of peptides appears sufficient. In addition, there are T cells for which no peptide involvement could be detected at all. Even within these categories, the heterogeneity of T cells is considerable: among 16 Kb-reactive T cells analyzed, 15 different modes of reactions were found.
Molecular definition of a polymorphic antigen (LA45) of free HLA-A and -B heavy chains found on the surfaces of activated B and T cells.
Authors: Madrigal J A , Belich M P , Benjamin R J , Little A M , Hildebrand W H , Mann D L , Parham P
Published: June 2004

A monomoprhic monoclonal antibody (LA45 antibody) reactive with "a new activation-induced surface structure on human T lymphocytes" (LA45 antigen) that resembled free class I heavy chains has recently been described (Schnabl, E., H. Stockinger, O. Majdic, H. Gaugitsch, I.J.D. Lindley, D. Maurer, A. Hajek-Rosenmayr, and W. Knapp. 1990. J. Exp. Med. 171:1431). This antibody was used to clone a class I-like heavy chain (LA45 gene) from the HUT 102 tumor cell, which paradoxically did not give rise to the LA45 antigen on transfection into monkey COS cells. We show here that the LA45 gene is HLA-Aw66.2, a previously uncharacterized allele of the HLA-A locus. The previously determined LA45 sequence differs from that of HLA-Aw66.2, from HUT 102, and the CR-B B cell line derived from the same individual as HUT 102 by substitution of tryptophan for serine at position 4 in the alpha 1 domain. Transfection of HLA-Aw66.2, and of a mutant of this gene with serine 4 substituted for tryptophan, into a human B cell line (C1R) both resulted in expression of the LA45 epitope. Furthermore, we find expression of the LA45 epitope on Epstein Barr virus-transformed B cell lines as well as lectin-activated T cells, but not on long-term T cell lines or unstimulated peripheral blood T cells. The specificity of the LA45 antibody is polymorphic and the presence of the LA45 epitope is precisely correlated with the sequence arginine, asparagine (RN) at residues 62 and 63 of the helix of the alpha 1 domain. The LA45 epitope is broadly distributed, being associated with half the alleles of both HLA-A and -B loci but none of the HLA-C locus. All the results are consistent with the presence of pools of free HLA-A and -B heavy chains at the surfaces of certain cell types but not others. Such molecules are probably responsible for the HLA-associated class I alloantigens of lectin-activated T cells. We hypothesize the free heavy chains result from dissociation of beta 2-microglobulin from subpopulations of empty HLA-A,B molecules, or molecules with weakly bound peptides, that vary in size depending on cellular activation and peptide supply.
COOH terminus of membrane IgM is essential for an antigen-specific induction of some but not all early activation events in mature B cells.
Authors: Parikh V S , Nakai C , Yokota S J , Bankert R B , Tucker P W
Published: June 2004

Transfectants of mature B cell lines that bind phosphorylcholine were made in order to understand the role of the COOH terminus of the mu chain of membrane IgM (mIgM) in generation of antigen-specific signals. A chimeric receptor (I-A alpha tail) was constructed by replacing 40 amino acids from the mu COOH terminus with that of major histocompatibility complex class II I-A alpha chain. The effect of wild-type and chimeric tails were studied on representative immediate-early antigen-specific signals. The I-A alpha tail hybrid, but not the wild-type receptor, was defective in antigen-driven Ca2+ mobilization, although it could effectively endocytose ligand-receptor complexes. Signal(s) transduced through the wild-type receptor led to transient induction of selected immediate-early gene messages (Egr-1, c-fos, Jun) above basal levels. However, the signal(s) generated after crosslinking of the I-A alpha tail receptor either showed no effect (c-fos) or actually repressed basal level expression of Egr-1 and Jun. Thus, we have established that receptor-mediated endocytosis can be distinguished from other early events associated with B cell activation, based on their differential dependence upon the structural fidelity of the COOH-terminal sequence of mIgM.
Enhanced binding of peptide antigen to purified class II major histocompatibility glycoproteins at acidic pH.
Authors: Jensen P E
Published: June 2004

Helper T lymphocytes recognize peptide antigens stably associated with class II major histocompatibility complex (MHC) glycoproteins on the surface of antigen-presenting cells and serve to regulate a wide variety of immune responses. A previous study from our laboratory had demonstrated that the functional association of various peptide antigens with the antigen-presenting cell membrane was increased at pH 5 as compared to pH 7, consistent with the potential role of acidic endosomal compartments in antigen processing. The mechanism for this effect was not determined. In the present study, assays using purified class II glycoprotein were used to further define this mechanism. The potential requirement for pH-dependent interactions involving non-MHC membrane components was excluded in functional assays with purified class II reconstituted in artificial membranes containing only neutral phospholipids and cholesterol. The association of HEL(104-120) with I-Ed, and OVA(323-339) with I-Ad, was increased at pH 5, as measured by activation of specific T cell hybridomas. An enzyme immunoassay was developed to measure the binding of biotin-labeled peptides to purified class II in detergent micelles. The pH dependence of binding paralleled our previous functional results. Optimum binding of biotin-HEL(104-120) to I-Ed was observed at pH approximately 4.5, whereas maximum binding of biotin-Myo(106-118) to I-Ad occurred at pH approximately 5.5. The latter peptide also bound weakly to I-Ed, but with a pH dependence similar to that observed using HEL(104-120). Further experiments with biotin-HEL(104-120)/I-Ed indicated that both the apparent affinity and the apparent concentration of peptide-binding sites are increased as hydrogen ion concentration is increased from pH 7 to pH 5. The effect of pH in this range was largely reversible and was not associated with a change in peptide dissociation that could be measured with our assay system. Binding was not inhibited in the presence of 1.5 M NaCl, suggesting that electrostatic interactions between HEL(104-120) and I-Ed are not essential for binding. It is proposed that protonation of a critical group(s) in the class II molecule regulates its capacity to form stable complexes with peptide. However, this effect alone does not fully account for the rapid kinetics of peptide binding observed in experiments with intact antigen-presenting cells.
Reduction of disulfide bonds during antigen processing: evidence from a thiol-dependent insulin determinant.
Authors: Jensen P E
Published: June 2004

Previous studies have demonstrated that insulin, like other protein antigens, requires processing in metabolically active antigen-presenting cells (APC) before it can be recognized by class II-restricted T lymphocytes. Unlike many other proteins, insulin peptides of minimal size retain the requirement for antigen processing. We demonstrate that this requirement can be bypassed by incubation of insulin with reducing agents in the presence of aldehyde-fixed APC. Fixed APC treated in this way were able to stimulate I-Ab- and I-Ad-restricted T cell hybridomas. Data are presented that demonstrate that cloned and polyclonal T cells recognize a determinant within the NH2-terminal 14 residues of the beef insulin A chain with no requirement for B chain residues. The common feature among peptides capable of stimulating these cells in the presence of live APC is the chemical form of the cysteine thiol groups. Those forms that produce free thiols upon reduction are active, whereas those with irreversibly protected sulfhydryls are not. Functional experiments with fixed APC and competition binding experiments with purified I-Ad indicate that only A chain peptides with free thiols are able to stably associate with the peptide-binding site on class II in a form that is recognized by specific T cells. Our findings indicate that reduction of disulfide bonds is both necessary and sufficient for presentation of insulin to a major population of class II-restricted T cells. The results provide strong support for the hypothesis that protein disulfides can be reduced during physiologic antigen processing.