bluuebunny/crossref_metadata_2025 huggingface dataset:
Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity.
Authors: Bonnema J D , Karnitz L M , Schoon R A , Abraham R T , Leibson P J
Published: June 2004

Although diverse signaling events are initiated by stimulation of multichain immune recognition receptors on lymphocytes, it remains unclear as to which specific signal transduction pathways are functionally linked to granule exocytosis and cellular cytotoxicity. In the case of natural killer (NK) cells, it has been presumed that the rapid activation of protein kinase C (PKC) enables them to mediate antibody-dependent cellular cytotoxicity (ADCC) and "natural" cytotoxicity toward tumor cells. However, using cloned human NK cells, we determined here that Fc receptor stimulation triggers granule release and ADCC through a PKC-independent pathway. Specifically, pretreatment of NK cells with the selective PKC inhibitor, GF109203X (using concentrations that fully blocked phorbol myristate acetate/ionomycin-induced secretion) had no effect on FcR-initiated granule release or ADCC. In contrast, FcR ligation led to the rapid activation of phosphatidylinositol 3-kinase (PI 3-kinase), and inhibition of this enzyme with the selective inhibitor, wortmannin, blocked FcR-induced granule release and ADCC. Additional experiments showed that, whereas FcR-initiated killing was wortmannin sensitive and GF109203X insensitive, natural cytotoxic activity toward the tumor cell line K562 was wortmannin insensitive and GF109203X sensitive. Taken together, these results suggest that: (a) PI 3-kinase activation induced by FcR ligation is functionally coupled to granule exocytosis and ADCC; and (b) the signaling pathways involved in ADCC vs natural cytotoxicity are distinct.
Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.
Authors: Ho J L , Zhu B , He S , Du B , Rothman R
Published: June 2004

Interleukin 4 (IL-4) diminishes cytokine activation of human macrophage. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacyglycerol (DAG), an activator of PKC, from membrane phospholipids was investigated to define the proximal events of IL-4R signaling. IL-4 induced a statistically significant time-and dose-dependent generation of DAG. The IL-4-triggered production of DAG was not derived from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in response to IL-4. Experiments were performed using [14C-methyl]choline-labeled U937 cells and monocytes to determine whether IL-4R activated phospholipase C (PLC), PLD, or PLA2 to use membrane phosphatidylcholine (PC) to form DAG. IL-4 induced a time- and dose-dependent increase of phosphocholine (pchol) with concomitant degradation of membrane PC (p < 0.05 compared with control). The finding that the peak reduction of PC was equivalent to peak production of pchol suggested that IL-4R signaling involved the activation of a PC-specific PLC. Changes in choline (chol) or lyso-PC and glycerolphosphocholine, the respective products of PC cleavage by PLD or PLA2, were not detected in IL-4-treated cells. In contrast, exogenous PLD induced an increase in chol and concomitant loss of membrane PC. Additional investigation suggested that IL-4R signaling does not involve PLD. In cells labeled with L-lyso-3-PC 1-[1-14C]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with ethanol. Propranolol, an inhibitor of phosphatidate phosphohydrolase, and calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG production in response to FMLP but not to IL-4. In propranolol pretreated cells, PMA but not IL-4 triggered the production of PA and lowered the amount of DAG. Evidence that PLA2 is not coupled to IL-4R is the detection of arachidonate production in response to FMLP but not to IL-4. Furthermore, IL-4R is not coupled to sphingomyelinase (SMase) since IL-4, unlike exogenous SMase, did not generate ceramide but induced the hydrolysis of PC to pchol that was comparable to exogenous PLC. In summary, IL-4R signaling in monocytes and U937 cells involves PLC and not PLD, PLA2, or SMase, and it uses PC and not PIP2 to form DAG.
Sublethal gamma-radiation induces differentiation of CD4-/CD8- into CD4+/CD8+ thymocytes without T cell receptor beta rearrangement in recombinase activation gene 2-/- mice.
Authors: Zúñiga-Pflücker J C , Jiang D , Schwartzberg P L , Lenardo M J
Published: June 2004

DNA recombination of the immunoglobulin (Ig) or T cell receptor (TCR) gene loci is an essential step in the production of lymphocytes bearing antigen-specific receptors. Mice that lack the ability to rearrange their Ig and TCR gene loci are devoid of mature B and T cells. Complete rearrangement and expression of the TCR-beta chain has been suggested to allow immature thymocytes to switch from the CD4-/CD8- to the CD4+/CD8+ stage of thymic development. Thus, thymocytes from severe combined immune deficient (SCID) mice or mice deficient in recombinase activation genes (RAG), which do not undergo proper DNA rearrangement, are arrested at the early CD4-/CD8- stage of development. B cell precursors in SCID or RAG mice do not progress from the B220+/sIgM-/heat stable antigen (HSA)+/CD43+ to the B220+/sIgM-/HSA+/CD43- stage. In an attempt to reconstitute RAG-2-/- mice with bone marrow- or fetal liver-derived progenitor cells, we subjected these mice to sublethal doses of gamma-radiation. It is surprising that in the absence of donor cells, irradiated RAG-2-/- mice revealed a dramatic change in their lymphoid phenotype. 14 d after irradiation, the majority of thymocytes had advanced to the CD4+/CD8+ stage of T cell development and a small number of bone marrow precursors had progressed to the CD43-, HSAhi stage of B cell development. Analysis of the resulting CD4+/CD8+ thymocytes revealed no surface expression of the TCR/CD3 complex and no V-D-J rearrangement of the TCR-beta gene locus. Our findings provide evidence for a novel pathway that allows the transition of thymocytes from the CD4-/CD8- to the CD4+/CD8+ stage and that does not appear to require TCR-beta chain rearrangement.
A pathway for entry of reoviruses into the host through M cells of the respiratory tract.
Authors: Morin M J , Warner A , Fields B N
Published: June 2004

Many microorganisms gain access to the systemic circulation after entering the respiratory tract. The precise pathways used to cross the mucosal barriers of the lungs have not been clearly described. We have used the mammalian reoviruses in order to determine the pathway that a systemic virus uses to penetrate the mucosal barrier and enter the systemic circulation after entering the airways of the lungs. Reoviruses enter through pulmonary M cells, which overlie bronchus-associated lymphoid tissue, and subsequently spread to regional lymph nodes. Thus, the pathway through M cells represents a strategy by which viruses and probably other microorganisms can penetrate the mucosal surface of the respiratory tract and thereby enter the systemic circulation.
Transcriptional regulation of the T cell antigen receptor zeta subunit: identification of a tissue-restricted promoter.
Authors: Rellahan B L , Jensen J P , Weissman A M
Published: June 2004

The cell surface expression of T cell antigen receptors (TCR) is regulated in part by the limiting synthesis of the zeta subunit. Utilizing fragments from the 5' region of the human zeta gene, two discrete regions that promote transcription were characterized. Both of these elements are located within 125 bases of the most 3' site of transcription initiation. The more proximal (3') promoter exhibits activity in lymphoid as well as nonlymphoid cells. In contrast, the more distal (5') promoter element functions in a tissue-restricted fashion. The tissue-specific promoter is localized to a 29-base fragment. The sequence of this region is remarkable for a stretch of 11 consecutive purines that are required for activity. This element constitutes the only known tissue-specific promoter for an invariant TCR subunit. Consistent with the unique role served by the zeta subunit in assembly of the TCR, this study demonstrates that the expression of the zeta gene is regulated in a fashion distinct from other TCR components.
Cytosolic-free calcium elevation in Trypanosoma cruzi is required for cell invasion.
Authors: Moreno S N , Silva J , Vercesi A E , Docampo R
Published: June 2004

To replicate, the trypomastigote form of Trypanosoma cruzi must invade host cells. Since a role for Ca2+ in the process of cell invasion by several intracellular parasites has been postulated, changes in the intracellular Ca2+ concentration in T. cruzi trypomastigotes and in tissue culture L6E9 myoblasts during their interaction were studied at the single cell level using digital imaging fluorescence microscopy or in cell suspensions by fluorescence spectrophotometry. An increase in cytosolic Ca2+ in T. cruzi trypomastigotes was detected at the single cell level after association of the parasites with the myoblasts. Ca2+ mobilization in the host cells was also detected upon contact with trypomastigotes either at the single cell level or in cells grown in coverslips and exposed to suspensions of trypomastigotes. Pretreatment of the parasites with the Ca2+ chelators quin 2 (50 microM) or bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM) decreased the trypomastigotes' association to myoblasts by approximately 40 and 63%, respectively, thus indicating that an increase in intracellular Ca2+ concentration in the parasites is required for cell invasion in addition to Ca2+ mobilization in the host cells.
Quantitative analysis of mononuclear cells expressing human immunodeficiency virus type 1 RNA in esophageal mucosa.
Authors: Smith P D , Fox C H , Masur H , Winter H S , Alling D W
Published: June 2004

The mucosa of the gastrointestinal tract is presumably an important reservoir for human immunodeficiency virus type 1 (HIV-1), but the level of virus-expressing cells within the mucosa of infected patients is not known. To study this issue, we identified HIV-1 mRNA-expressing (positive) mononuclear cells by in situ hybridization in specimens of esophageal mucosa from eight patients with acquired immune deficiency syndrome (AIDS) and esophageal infections. Such cells were not found in four patients with AIDS and no esophageal disease. Immunocytochemical staining revealed that the mononuclear cells expressing HIV-1 mRNA were lamina propria macrophages. The prevalence of positive cells was measured by triplicate determinations in each of three experiments using an inverse sampling technique. No significant differences in prevalence were found among patients or among experiments. The overall prevalence of HIV-1 mRNA-expressing cells in the esophageal lamina propria was 0.059 +/- 0.01%. This prevalence of cells expressing HIV-1 mRNA in the mucosa of patients with mucosal infections may reflect the local abundance of stimuli such as bacterial endotoxin and certain cytokines capable of inducing viral transcription.
Protective role of major histocompatibility complex class II Ebd transgene on collagen-induced arthritis.
Authors: Gonzalez-Gay M A , Nabozny G H , Bull M J , Zanelli E , Douhan J , Griffiths M M , Glimcher L H , Luthra H S , David C S
Published: June 2004

Collagen-induced arthritis (CIA) is an animal model of autoimmune inflammatory polyarthritis that has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC), H-2, and restricted to the H-2q and H-2r haplotypes. Whereas the role of the H-2A molecule in susceptibility to CIA is well established, little is known about the role of H-2E molecule in the disease. In this study, we analyzed the effect of a transgenic E beta d molecule on CIA susceptibility in a recombinant mouse B10.RQB3, which expresses the CIA susceptible Aq genes and an Eak gene, but does not produce an E molecule since Ebq is nonfunctional. In the presence of an Ebd transgene, a viable E molecule is generated. Whereas B10.RQB3 were susceptible to CIA, B10.RQB3-E beta d+ showed a dramatic reduction in the incidence of arthritis as well as a decrease in the level of anti-mouse and anti-bovine CII antibodies in their serum. No clear cut differences in the expression of T cell receptor (TCR) V beta was observed between E beta d+ and E beta d- transgenic mice. Mechanisms underlying the protective effect of E beta d transgenic molecule on CIA may shed light on how HLA-DR molecules influence human RA.
Protection by alpha 1-acid glycoprotein against tumor necrosis factor-induced lethality.
Authors: Libert C , Brouckaert P , Fiers W
Published: June 2004

We here report that alpha 1-acid glycoprotein, a typical acute phase protein, protects mice from lethal shock induced by tumor necrosis factor (TNF) or endotoxin. The protection is observed both in normal and in galactosamine-sensitized mice. Optimal desensitization requires at least 3 mg alpha 1-acid glycoprotein administered 2 h before the lethal challenge. Under these conditions, complete inhibition of all TNF-induced metabolic changes was observed: fall in body temperature, release of liver transaminases, enhanced clotting time, and mortality. The known platelet aggregation-inhibitory activity of alpha 1-acid glycoprotein provides a possible explanation for this protective capacity.
Identification of a unique antigen peptide pRL1 on BALB/c RL male 1 leukemia recognized by cytotoxic T lymphocytes and its relation to the Akt oncogene.
Authors: Uenaka A , Ono T , Akisawa T , Wada H , Yasuda T , Nakayama E
Published: June 2004

BALB/c radiation leukemia RL male 1 is an immunogenic tumor. We established bulk and cloned cytotoxic T lymphocyte (CTL) lines from regressor (BALB/c x C57BL/6)F1 (CB6F1) spleen cells that recognized RL male 1 specifically. We then obtained antigen peptide recognized by CTL from RL male 1 by acid extraction. Analysis of the acid extract by reversed-phase high performance liquid chromatography (HPLC) on a semipreparative C18 column revealed that fractions eluted in 23 min (peak a) and 26 min (peak b) showed sensitization activity on the P815 target for specific CTL. On further purification of these fractions by HPLC and direct sequencing by Edman degradation, we identified the CTL-recognizing RL male 1 peptide pRL1a (IPGLPLSL) in peak a and its possible precursor peptide pRL1b (SIIPGLPLSL) in peak b. Sequence homology indicated that these peptides were derived from the 5' untranslated region of c-akt oncogene.
In vivo effects of monoclonal antibodies that functionally inhibit complement regulatory proteins in rats.
Authors: Matsuo S , Ichida S , Takizawa H , Okada N , Baranyi L , Iguchi A , Morgan B P , Okada H
Published: June 2004

The present work was designed to evaluate the effects of functional suppression of complement regulatory proteins in vivo. Male Wistar rats were anesthetized with Nembutal and were intravenously injected with 1 mg/kg of F(ab')2 or Fab fraction of either monoclonal antibody 5I2, which inhibits the function of rat counterpart of mouse Crry/p65, or monoclonal antibody 6D1, which inhibits the rat counterpart of CD59. Mean arterial pressure was continuously measured for 30 min. When 5I2 was injected, there was a biphasic change of mean arterial pressure, namely, the rapid increase immediately after the injection (approximately 2 min, phase 1) and the subsequent fall and slow recovery (approximately 4-30 min, phase 2). These effects were completely abrogated by pretreatment of rats with cobra venom factor. Pretreatment with carboxypeptidase inhibitor, which inhibits inactivation of anaphylatoxins C3a and C5a, induced enhanced reduction of blood pressure. Circulating leukocytes and platelets were rapidly decreased 5 min after antibody injection and became normal by 2 h. Hematocrit and erythrocyte count were continuously increased up to 2 h after injection, suggesting that there was hemoconcentration due to increased vascular permeability. Immunofluorescence study revealed binding of antibody fragments and rat C3 along the capillaries of lung, heart, and liver 5 min after injection. In contrast to 5I2, F(ab')2 fraction of 6D1, though localized to the same areas and in similar amounts, had no significant effect on the parameters measured. These data suggest that the rat counterpart of mouse Crry/p65 plays a vital role in vivo by preventing the activation of autologous complement on vascular endothelium.
Toxoplasma gondii alters eicosanoid release by human mononuclear phagocytes: role of leukotrienes in interferon gamma-induced antitoxoplasma activity.
Authors: Yong E C , Chi E Y , Henderson W R
Published: June 2004

Toxoplasma gondii tachyzoites markedly alter the profile of eicosanoids released by human mononuclear phagocytes. Freshly isolated, 2-h adherent human monocytes release both cyclooxygenase (e.g., thromboxane [TX] B2, prostaglandin [PG] E2) and 5-lipoxygenase (e.g., leukotriene [LT] B4, LTC4) products of arachidonic acid metabolism after stimulation by the calcium ionophore A23187 or ingestion of opsonized zymosan particles or heat-killed T. gondii. However, after incubation with viable T. gondii, normal and chronic granulomatous disease monocytes release only the cyclooxygenase products TXB2 and PGE2 and fail to form LTB4, LTC4, or other 5-lipoxygenase products. Monocytes maintained in culture for 5 d lose this capacity to release TXB2 and PGE2 after incubation with T. gondii. T. gondii significantly inhibit calcium ionophore A23187-induced LTB4 release by monocyte-derived macrophages; heat-killed organisms do not affect this calcium ionophore A23187-induced release of LTB4. T. gondii-induced inhibition of LTB4 release by calcium ionophore A23187-stimulated monocyte-derived macrophage is reversed by interferon (IFN)-gamma treatment of the monolayers. LTB4 induced extensive damage to the cellular membranes and cytoplasmic contents of the organisms as observed by transmission electron microscopy. Exogenous LTB4 (10(-6) M) induced intracellular killing of ingested T. gondii by non-IFN-gamma-treated monocyte-derived macrophages. IFN-gamma-induced antitoxoplasma activity in monocyte-derived macrophages was inhibited by the selective 5-lipoxygenase inhibitor zileuton but not by the cyclooxygenase inhibitor indomethacin. These findings suggest a novel role for 5-lipoxygenase arachidonic acid products in human macrophage IFN-gamma-induced antitoxoplasma activity.
CD2 is involved in maintenance and reversal of human alloantigen-specific clonal anergy.
Authors: Boussiotis V A , Freeman G J , Griffin J D , Gray G S , Gribben J G , Nadler L M
Published: June 2004

Induction and maintenance of a state of T cell unresponsiveness to specific alloantigen would have significant implications for human organ transplantation. Using human histocompatibility leukocyte antigen DR7-specific helper T cell clones, we demonstrate that blockade of the B7 family of costimulatory molecules is sufficient to induce alloantigen-specific T cell clonal anergy. Anergized cells do not respond to alloantigen and a variety of costimulatory molecules, including B7-1, B7-2, intercellular adhesion molecule-1 (ICAM-1), and lymphocyte function-associated molecule (LFA)-3. However, after culture in exogenous interleukin (IL)-2 for at least 7 d, anergized cells can respond to alloantigen in the presence of LFA-3. LFA-3 costimulation subsequently restores responsiveness to alloantigen in the presence of previously insufficient costimulatory signals. Expression of CD2R epitope is downregulated on anergic cells and is restored after 7 d of IL-2 culture. The loss of the CD2R is temporally associated with the inability of anergized cells to respond to LFA-3. These results suggest that in addition to blockade of B7 family members, inhibition of CD2 and, potentially, other costimulatory pathways that might reverse anergy will be necessary to maintain prolonged alloantigen-specific tolerance.
Characterization and biological properties of a new staphylococcal exotoxin.
Authors: Ren K , Bannan J D , Pancholi V , Cheung A L , Robbins J C , Fischetti V A , Zabriskie J B
Published: June 2004

Staphylococcus aureus strain D4508 is a toxic shock syndrome toxin 1-negative clinical isolate from a nonmenstrual case of toxic shock syndrome (TSS). In the present study, we have purified and characterized a new exotoxin from the extracellular products of this strain. This toxin was found to have a molecular mass of 25.14 kD by mass spectrometry and an isoelectric point of 5.65 by isoelectric focusing. We have also cloned and sequenced its corresponding genomic determinant. The DNA sequence encoding the mature protein was found to be 654 base pairs and is predicted to encode a polypeptide of 218 amino acids. The deduced protein contains an NH2-terminal sequence identical to that of the native protein. The calculated molecular weight (25.21 kD) of the recombinant mature protein is also consistent with that of the native molecules. When injected intravenously into rabbits, both the native and recombinant toxins induce an acute TSS-like illness characterized by high fever, hypotension, diarrhea, shock, and in some cases death, with classical histological findings of TSS. Furthermore, the activity of the toxin is specifically enhanced by low quantities of endotoxins. The toxicity can be blocked by rabbit immunoglobulin G antibody specific for the toxin. Western blotting and DNA sequencing data confirm that the protein is a unique staphylococcal exotoxin, yet shares significant sequence homology with known staphylococcal enterotoxins, especially the SEA, SED, and SEE toxins. We conclude therefore that this 25-kD protein belongs to the staphylococcal enterotoxin gene family that is capable of inducing a TSS-like illness in rabbits.
Surface expression of functional T cell receptor chains formed by interlocus recombination on human T lymphocytes.
Authors: Davodeau F , Peyrat M A , Gaschet J , Hallet M M , Triebel F , Vié H , Kabelitz D , Bonneville M
Published: June 2004

Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire.
Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles.
Authors: Croft M , Carter L , Swain S L , Dutton R W
Published: June 2004

We have generated primary effector populations from naive CD8 T cells in response to antigen and determined their patterns of cytokine secretion upon restimulation. The effect of exogenous factors on the effector generation was examined and compared with responses of antigen-specific CD4 effectors generated under comparable conditions. CD8 cells from bm1 mice were stimulated with C57BL/6 (B6) antigen presenting cells (APCs) bearing allogeneic class I and CD8 cells from female severe combined immunodeficiency (SCID) B6 mice, transgenic for a T cell receptor alpha/beta (TCR-alpha/beta) that recognizes H-Y on Db, were stimulated with APCs from male mice. In parallel, CD4 cells from bm12 mice were stimulated with alloantigen and CD4 cells from V beta 3/V alpha 11 TCR transgenics were stimulated with a peptide of pigeon cytochrome c on IEk. T cells from both transgenic mice were of naive phenotype whereas normal mice contained 10-20% memory cells. Effector CD8 populations generated were L-selectin low, CD45RB high, and CD44 high. Naive CD8 cells from SCID anti-H-Y mice made little or no cytokine immediately upon stimulation in contrast to naive CD4 which produced large amounts of interleukin 2 (IL-2). Both populations, however, generated primary effectors over 4-5 d that made substantial quantities of many cytokines upon restimulation. Both CD8 and CD4 effectors produced similar patterns of cytokines with alloantigen or specific antigen. Cytokines present during naive CD8 stimulation influenced the cytokine secretion profile of the effectors, as previously shown for CD4 cells, although secretion by CD8 effectors was generally lower than that of CD4 effectors. CD8 cells cultured with IL-2 alone made predominantly interferon gamma (IFN-gamma) and no IL-4 or IL-5, similar to CD4 cells. Priming with IFN-gamma increased IFN-gamma secretion from CD4 effectors, but had little if any effect on CD8 cells. In contrast, priming with IL-12 generated CD8 effectors, as well as CD4 effectors, producing elevated quantities of IFN-gamma, with similar levels from both the CD4 and CD8 populations. The presence of IL-4 during effector cell generation promoted synthesis of IL-4 and IL-5 from both CD8 and CD4 cells while downregulating IFN-gamma secretion. CD8 cells made only small amounts of IL-4, more than 100-fold less than CD4 cells, whereas significant levels of IL-5 were induced, only 3-10-fold lower than from CD4.(ABSTRACT TRUNCATED AT 400 WORDS)
Human natural resistance-associated macrophage protein: cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific expression.
Authors: Cellier M , Govoni G , Vidal S , Kwan T , Groulx N , Liu J , Sanchez F , Skamene E , Schurr E , Gros P
Published: June 2004

Natural resistance to infection with unrelated intracellular parasites such as Mycobacteria, Salmonella, and Leishmania is controlled in the mouse by a single gene on chromosome 1, designated Bcg, Ity, or Lsh. A candidate gene for Bcg, designated natural resistance-associated macrophage protein (Nramp), has been isolated and shown to encode a novel macrophage-specific membrane protein, which is altered in susceptible animals. We have cloned and characterized cDNA clones corresponding to the human NRAMP gene. Nucleotide and predicted amino acid sequence analyses indicate that the human NRAMP polypeptide encodes a 550-amino acid residue membrane protein with 10-12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionary conserved consensus transport motif. Identification of genomic clones corresponding to human NRAMP indicates that the gene maps to chromosome 2q35 within a group of syntenic loci conserved with proximal mouse 1. The gene is composed of at least 15 exons, with several exons encoding discrete predicted structural domains of the protein. These studies have also identified an alternatively spliced exon encoded by an Alu element present within intron 4. Although this novel exon was found expressed in vivo, it would introduce a termination codon in the downstream exon V, resulting in a severely truncated protein. Northern blot analyses indicate that NRAMP mRNA expression is tightly controlled in a tissue-specific fashion, with the highest sites of expression being peripheral blood leukocytes, lungs, and spleen. Additional RNA expression studies in cultured cells identified the macrophage as a site of expression of human NRAMP and indicated that increased expression was correlated with an advanced state of differentiation of this lineage.
Analysis of the structure of empty and peptide-loaded major histocompatibility complex molecules at the cell surface.
Authors: Catipović B , Talluri G , Oh J , Wei T , Su X M , Johansen T E , Edidin M , Schneck J P
Published: June 2004

We compared the conformation of empty and peptide-loaded class I major histocompatibility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the alpha 2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to beta 2-microglobulin. No FRET was found between Fab fragments bound to empty H-2Kb, but FRET was detected when empty H-2Kb molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2Kb molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.
Neutrophils roll on adherent neutrophils bound to cytokine-induced endothelial cells via L-selectin on the rolling cells.
Authors: Bargatze R F , Kurk S , Butcher E C , Jutila M A
Published: June 2004

Specific arrest of neutrophils in venules is central to their rapid accumulation during local inflammatory responses. Initial neutrophil rolling on endothelium is mediated by leukocyte L-selectin and the inducible vascular adhesion proteins P- and E-selectin. This rolling is a prerequisite for endothelial-dependent neutrophil arrest. Here we describe rolling of neutrophils on the surface of previously arrested neutrophils and demonstrate that this interaction involves L-selectin exclusively on rolling cells. The adherent neutrophil support of L-selectin-dependent neutrophil rolling in vivo can promote continuous and augmented leukocyte recruitment at sites of previous neutrophil accumulation.
Anti-SCID mouse reactivity shapes the human CD4+ T cell repertoire in hu-PBL-SCID chimeras.
Authors: Tary-Lehmann M , Lehmann P V , Schols D , Roncarolo M G , Saxon A
Published: June 2004

Injecting human peripheral blood mononuclear cells into severe combined immunodeficient (SCID) mice results in long-term engraftment of human lymphocytes, of which > 98% are phenotypically mature, activated T cells. Here we have characterized the human T cells that populate such hu-PBL-SCID chimeras. We report that these human T cells do not mobilize Ca2+ after CD3 stimulation, i.e., their T cell receptor (TCR)-mediated signal transduction is deficient. Chimera-derived human T cells do not secrete lymphokines or undergo blastogenesis after CD3 stimulation, but proliferate in response to interleukin 2 (IL-2), defining the chimera derived human T cells as anergic. Anergy was seen in both the CD4+ and the CD8+ subpopulations. We established human T cell lines from chimeras. These T cells retained their anergic state for 1-2 mo in culture, after which they simultaneously regained the ability to mobilize Ca2+, secrete lymphokines, and to undergo blastogenesis following stimulation via the TCR. Once regaining proliferative responsiveness to CD3 stimulation, these CD4+ T cell lines displayed anti-SCID mouse reactivity and showed no specificity for recall antigens. All CD3-responsive CD4+ T cell clones obtained from such lines were SCID mouse specific, recognizing native major histocompatibility complex class II products on the murine cells. In contrast, chimera-derived human CD8+ cell lines and clones did not display detectable anti-mouse reactivity. The data show that the human T cell system in long term hu-PBL-SCID chimeras is nonfunctional due to both anergy and the limitation of the CD4+ repertoire to xenoreactive clones. The data suggest that long-term hu-PBL-SCID chimerism represents an atypical graft-versus-host reaction in which the human effector T cells become anergic in the murine environment.
B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells.
Authors: Caux C , Vanbervliet B , Massacrier C , Azuma M , Okumura K , Lanier L L , Banchereau J
Published: June 2004

Dendritic cells comprise a system of highly efficient antigen-presenting cells involved in the initiation of T cell responses. Herein, we investigated the role of the CD28 pathway during alloreactive T cell proliferation induced by dendritic-Langerhans cells (D-Lc) generated by culturing human cord blood CD34+ progenitor cells with granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha. In addition to expressing CD80 (B7/BB1), a subset of D-Lc expressed B70/B7-2. Binding of the CTLA4-Ig fusion protein was completely inhibited by a combination of monoclonal antibodies (mAbs) against CD80 and B70/B7-2, indicating the absence of expression of a third ligand for CD28/CTLA-4. It is interesting to note that mAbs against CD86 completely prevented the binding of CTLA4-Ig in the presence of mAbs against CD80 and bound to a B70/B7-2-transfected fibroblast cell line, demonstrating that the B70/B7-2 antigen is identical to CD86. CD28 triggering was essential during D-Lc-induced alloreaction as it was inhibited by mAbs against CD28 (9 out of 11 tested). However, none of six anti-CD80 mAbs demonstrated any activity on the D-Lc-induced alloreaction, though some were previously described as inhibitory in assays using CD80-transfected cell lines. In contrast, a mAb against CD86 (IT-2) was found to suppress the D-Lc-dependent alloreaction by 70%. This inhibitory effect was enhanced to > or = 90% when a combination of anti-CD80 and anti-CD86 mAbs was used. The present results demonstrate that D-Lc express, in addition to CD80, the other ligand for CTLA-4, CD86 (B70/B7-2), which plays a primordial role during D-Lc-induced alloreaction.
Interaction between human CD2 and CD58 involves the major beta sheet surface of each of their respective adhesion domains.
Authors: Arulanandam A R , Kister A , McGregor M J , Wyss D F , Wagner G , Reinherz E L
Published: June 2004

The CD58 binding site on human CD2 was recently shown by nuclear magnetic resonance structural data in conjunction with site-directed mutagenesis to be a highly charged surface area covering approximately 770A2 on the major AGFCC'C" face of the CD2 immunoglobulin-like (Ig-like) NH2-terminal domain. Here we have identified the other binding surface of the CD2-CD58 adhesion pair by mutating charged residues shared among CD2 ligands (human CD58, sheep CD58, and human CD48) that are predicted to be solvent exposed on a molecular model of the Ig-like adhesion domain of human CD58. This site includes beta strand residues along the C strand (E25, K29, and K30), in the middle of the C' strand (E37) and in the G strand (K87). In addition, several residues on the CC' loop (K32, D33, and K34) form this site. Thus, the interaction between CD2 and CD58 involves the major beta sheet surface of each adhesion domain. Possible docking orientations for the CD2-CD58 molecular complex are offered. Strict conservation of human and sheep CD58 residues within the involved C and C' strands and CC' loop suggests that this region is particularly important for stable formation of the CD2-CD58 complex. The analysis of this complex offers molecular insight into the nature of a receptor-ligand pair involving two Ig family members.
Fv structure of monoclonal antibody II-481 against herpes simplex virus Fc gamma-binding glycoprotein gE contains immunodominant complementarity determining region epitopes that react with human immunoglobulin M rheumatoid factors.
Authors: Johansson P J , Malone C , Swietnicki W , Dunn B M , Williams R C
Published: June 2004

Human immunoglobulin M (IgM) rheumatoid factors (RFs) show primary direct enzyme-linked immunosorbent assay (ELISA) reactivity with Fab rather than Fc fragments of monoclonal antibody (mAb) II-481 directed against the Fc gamma-binding site of herpes simplex virus glycoprotein gE. This preferential anti-Fab specificity suggests that RFs react with antigen-binding portions of mAb II-481 as anti-idiotypic antibodies directed at the combining site regions of mAb reacting with the Fc gamma-binding region of gE. Analysis of this idiotype-anti-idiotype reaction employed polymerase chain reaction amplification and sequencing of the variable heavy and light (VH and VL) regions of mAb II-481. When VH and VL regions of mAb II-481 were synthesized as overlapping 7-mer peptides on polypropylene pins, a panel of 10 polyclonal and 6 monoclonal human IgM RFs reacted primarily with epitopes within the three solvent-exposed mAb II-481 complementarity determining regions (CDRs). Preincubation of single CDR heptamer peptides with IgM RFs in free solution, resulted in 63-100% inhibition of RF binding to mAb II-481 on the ELISA plate, confirming the antigenic importance of linear CDR regions for RF reactivity. Combinations of two or three CDR peptides frequently produced 94-100% inhibition of RF binding to whole mAb II-481. Control peptides, singly or in combination, showed no inhibition. Computer modeling suggested that the RF-reactive mAb II-481 Fv region and a previously demonstrated RF-reactive CH3 epitope displayed considerable three-dimensional similarities in conformation. These studies may provide insight into limited shape homologies possibly involved in an RF anti-idiotypic reaction.
Humoral immune responses in CD40 ligand-deficient mice.
Authors: Renshaw B R , Fanslow W C , Armitage R J , Campbell K A , Liggitt D , Wright B , Davison B L , Maliszewski C R
Published: June 2004

Individuals with X-linked hyper-IgM syndrome fail to express functional CD40 ligand (CD40L) and, as a consequence, are incapable of mounting protective antibody responses to opportunistic bacterial infections. To address the role of CD40L in humoral immunity, we created, through homologous recombination, mice deficient in CD40L expression. These mice exhibited no gross developmental deficiencies or health abnormalities and contained normal percentages of B and T cell subpopulations. CD40L-deficient mice did display selective deficiencies in humoral immunity; basal serum isotype levels were significantly lower than observed in normal mice, and IgE was undetectable. Furthermore, the CD40L-deficient mice failed to mount secondary antigen-specific responses to immunization with a thymus-dependent antigen, trinitrophenol-conjugated keyhole limpet hemocyanin (TNP-KLH). By contrast, the CD40L-deficient mice produced antigen-specific antibody of all isotypes except IgE in response to the thymus-independent antigen, DNP-Ficoll. These results underscore the requirement of CD40L for T cell-dependent antibody responses. Moreover, Ig class switching to isotypes other than IgE can occur in vivo in the absence of CD40L, supporting the notion that alternative B cell signaling pathways regulate responses to thymus-independent antigens.
Human CD4-major histocompatibility complex class II (DQw6) transgenic mice in an endogenous CD4/CD8-deficient background: reconstitution of phenotype and human-restricted function.
Authors: Yeung R S , Penninger J M , Kündig T M , Law Y , Yamamoto K , Kamikawaji N , Burkly L , Sasazuki T , Flavell R , Ohashi P S , Mak T W
Published: June 2004

To reconstitute the human immune system in mice, transgenic mice expressing human CD4 and human major histocompatibility complex (MHC) class II (DQw6) molecules in an endogenous CD4- and CD8-deficient background (mCD4/8-/-), after homologous recombination, have been generated. We report that expression of human CD4 molecule in mCD4/8-/- mice rescues thymocyte development and completely restores the T cell compartment in peripheral lymphoid organs. Upon vesicular stomatitis virus (VSV) challenge, the reconstituted mature T cell population effectively provide T help to B cells in immunoglobulin class switching from IgM to specific IgG-neutralizing antibodies. Human CD4+DQw6+ double transgenic mice are tolerant to DQw6 and the DQw6 molecule functions in antigen presentation, effectively generating a human MHC class II-restricted T cell response to streptococcal M6C2 peptide. These data show that both the hCD4 and DQw6 molecules are functional in mCD4/8-/- mice, fully and stably reconstituting this limb of the human immune system in mice. This animal model provides a powerful in vivo tool to dissect the human CD4-human class II MHC interaction, especially its role in human autoimmune diseases, superantigen-mediated diseases, and acquired immunodeficiency syndrome (AIDS).
Evidence for a functional interaction between the beta chain of major histocompatibility complex class II and the T cell receptor alpha chain during recognition of a bacterial superantigen.
Authors: Deckhut A M , Chien Y , Blackman M A , Woodland D L
Published: June 2004

Several studies have suggested that there is a direct interaction between the T cell receptor (TCR) and the major histocompatibility complex (MHC) molecule during T cell recognition of superantigen. To further investigate this possibility, we have analyzed T cell recognition of a bacterial superantigen, Staphylococcal enterotoxin B (SEB), presented by a series of mutant murine I-Ek molecules in which residues of either the alpha or beta chain predicted to interact with the TCR have been substituted. Individual T cell hybridomas gave distinct patterns of responsiveness to SEB presented by the I-E beta k mutants that could not be attributed to differences in the binding of SEB to the mutants. This effect appeared to be dependent on the TCR-alpha chain because some of these hybridomas expressed identical TCR transgenic beta chains. In contrast, none of the hybridomas gave distinct patterns of responsiveness to SEB presented by the I-E alpha k mutants. Taken together, these observations support the idea that there is a functional interaction between the alpha chain of the TCR and the beta chain of the MHC class II molecule. The data also support the idea that this interaction might enhance superantigen recognition in some cases.
The role of transforming growth factor beta in the generation of suppression: an interaction between CD8+ T and NK cells.
Authors: Gray J D , Hirokawa M , Horwitz D A
Published: June 2004

CD8+ T cells have suppressor effector functions, but the mechanisms involved in the generation of this activity are poorly understood. We report that natural killer (NK) cells have an important role in the acquisition of this function. CD8+ cells induce NK cells to produce transforming growth factor-beta (TGF-beta) which, in turn, stimulates CD8+ T cells to become suppressors of antibody production. Using a monocyte-dependent and -independent method to induce antibody production, we first observed that the addition of NK cells to CD8+ cells was required for optimal suppression. Next, we determined that the interaction of CD8+ T cells with NK cells resulted in a striking increase NK cell TGF-beta mRNA and its production. This cytokine appeared to be involved in the induction of T suppressor cell activity since: (a) anti-TGF-beta 1 completely abrogated the suppression of immunoglobulin G synthesis; (b) TGF-beta 1 could substitute for NK cells in inducing CD8+ T cells to develop suppressor activity; and (c) a short exposure of T cells to TGF-beta 1 in the absence of B cells was sufficient for the generation of suppressor activity by CD8+ T cells. Interferon gamma did not have this property. These studies provide strong evidence that in addition to its suppressive properties, TGF-beta is involved in the generation of CD8+ T suppressor effector cells. Because NK cell function is decreased in many autoimmune diseases, these cells may fail to interact properly with these individuals' CD8+ cells in generating suppressors of aggressive anti-self responses.
The specific recognition by macrophages of CD8+,CD45RO+ T cells undergoing apoptosis: a mechanism for T cell clearance during resolution of viral infections.
Authors: Akbar A N , Savill J , Gombert W , Bofill M , Borthwick N J , Whitelaw F , Grundy J , Janossy G , Salmon M
Published: June 2004

During viral infections, CD8+,CD45RO+ T populations expand. These primed cells express abundant levels of cytoplasmic granules that contain perforin and TIA-1. Recent work has suggested that the majority of this CD8+ population downregulates Bcl-2 protein expression and is destined to undergo apoptosis. In this study we have investigated the elimination of these apoptotic CD8+ T cells by both human monocyte-derived and murine bone marrow macrophages. We have found that these phagocytes recognize and ingest both apoptotic CD8+ and CD4+ T cells using an alpha v beta 3 (vitronectin receptor)/CD36/thrombospondin recognition system, with the same receptors being used in the recognition of apoptotic neutrophils. These data provide new evidence for a mechanism that enables the clearance of greatly increased populations of CD8+ effector cells which are found during viral infections. This enables cellular homeostasis to occur in the host upon resolution of viral diseases in vivo.
Apolipoprotein E (ApoE), a novel heparin-binding protein inhibits the development of Kaposi's sarcoma-like lesions in BALB/c nu/nu mice.
Authors: Browning P J , Roberts D D , Zabrenetzky V , Bryant J , Kaplan M , Washington R H , Panet A , Gallo R C , Vogel T
Published: June 2004

Recombinant apolipoprotein E-3 (ApoE-3), expressed in Escherichia coli, was purified and used in an in vitro and an in vivo model system for acquired immunodeficiency syndrome-associated Kaposi's sarcoma (AIDS-KS). This protein blocked cell proliferation and chemotaxis of AIDS-KS cells in response to activated lymphocyte conditioned medium (AL-CM) and oncostatin M (OSM). ApoE-3 also inhibited the formation of neoangiogenic lesions induced in BALB/c nu/nu mice by AIDS-KS cells. These findings represent a novel and potentially less toxic therapeutic approach for the treatment of AIDS-KS.
Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.
Authors: Peschon J J , Morrissey P J , Grabstein K H , Ramsdell F J , Maraskovsky E , Gliniak B C , Park L S , Ziegler S F , Williams D E , Ware C B , Meyer J D , Davison B L
Published: June 2004

Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.