Microvascular display of xanthine oxidase and NADPH oxidase in the spontaneously hypertensive rat.

Objective: Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation.

Investigating antibody-catalyzed ozone generation by human neutrophils.

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H(2)O(3)) and ozone (O(3)) from singlet oxygen ((1)O(2)(*)) and water. Given that neutrophils have the potential both to produce (1)O(2)(*) and to bind antibodies, we considered that these cells could be a biological source of O(3). We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O(3).

Evidence for antibody-catalyzed ozone formation in bacterial killing and inflammation.

Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H2O2 production by antibodies alone was found to be not sufficient for bacterial killing.

JFC1, a novel tandem C2 domain-containing protein associated with the leukocyte NADPH oxidase.

We have employed a yeast two-hybrid system to screen a B lymphoblast-derived cDNA library, searching for regulatory components of the NADPH oxidase. Using as bait the C-terminal half of p67(phox), which contains both Src homology 3 domains, we have cloned JFC1, a novel human 62-kDa protein. JFC1 possesses two C2 domains in tandem.

Purification and characterization of a C5a-inactivating enzyme from human peritoneal fluid.

Earlier work has suggested that familial Mediterranean fever, an inherited disorder characterized by sporadic episodes of inflammation involving the pleural and peritoneal cavities and the joints, is caused by the lack of a C5a inactivator normally found in serosal fluid. We have purified this inactivator from ascites fluid and obtained a protein of molecular weight 53 to 56 kD with a specific activity 10,000-fold greater than the crude material. On Western blot, an inhibitory antibody recognized a single antigenic species at the same molecular weight.

Cell-free activation of the respiratory burst oxidase by protein kinase C.

In intact neutrophils, phorbol ester treatment activates the respiratory burst oxidase, the enzyme responsible for O2-production by phagocytes. This effect is thought to be dependent on protein kinase C and on the phosphorylation of p47phox. In this paper, we report that protein kinase C activates the respiratory burst oxidase in a cell-free system consisting of isolated neutrophil cytosol and membrane.

Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation.

The respiratory burst oxidase is responsible for O2- production in stimulated neutrophils and B lymphocytes. Components of this oxidase include cytochrome b558, a membrane-bound flavohemoprotein; the cytosolic polypeptides p47phox and p67phox; and one or more small G proteins including Rac1, Rac2, and/or Rap1A. We found that when normal neutrophils were activated, small percentages of each of the cytosolic proteins p47phox, p67phox, and Rac2 were transferred to the membrane cytoskeleton.

The reduction of cytochrome b558 and the activity of the respiratory burst oxidase from human neutrophils.

It is known that in respiratory burst oxidase preparations engaged in O2- production, cytochrome b558, a characteristic oxidase component, is partly reduced. This result has been interpreted in terms of a mechanism in which cytochrome b558 functions as an electron-carrying component of the respiratory burst oxidase, its level of reduction reflecting a steady-state partitioning of the cytochrome between reduced and oxidized forms as it ferries electrons from NADPH to oxygen. Kinetic arguments based on this interpretation have supported the proposal that the cytochrome is reduced at a rate sufficient to account for the rate of O2- production by activated neutrophils.

O2- production by B lymphocytes lacking the respiratory burst oxidase subunit p47phox after transfection with an expression vector containing a p47phox cDNA.

The respiratory burst oxidase of phagocytes and B lymphocytes is a complicated enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2- production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or other appropriate stimuli. The activity of this enzyme is greatly decreased or absent in patients with chronic granulomatous disease, an inherited disorder characterized by a severe defect in host defense against bacteria and fungi.

The cytosolic components of the respiratory burst oxidase exist as a M(r) approximately 240,000 complex that acquires a membrane-binding site during activation of the oxidase in a cell-free system.

Sodium dodecyl sulfate (SDS) treatment of a mixture of cytosol and plasma membranes from resting neutrophils resulted in the activation of the respiratory burst oxidase, a complicated enzyme that catalyzes the production of O2- from NADPH and oxygen. Activation was accompanied by translocation to the plasma membranes of the oxidase components p47phox and p67phox, which in resting cytosol were found in a M(r) approximately 240,000 complex. This translocation, which appeared to take place without a major change in the size of the cytosolic complex, did not occur if the membranes lacked cytochrome b558, and was inhibited by the peptide PRGV-HFIFNK, a sequence found near the carboxyl terminus of cytochrome b558 that was known from earlier work to inhibit O2- production by the cell-free system (Rotrosen, D.

Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils.

Resting and phorbol-activated human neutrophils were separated by treatment with Triton X-100 into detergent-extractable and cytoskeleton fractions. Respiratory burst oxidase activity was restricted entirely to the cytoskeleton. The cytoskeleton also contained approximately 15% of the neutrophil cytochrome b558, an oxidase-associated heme protein, as well as most of the oxidase-related cytosolic polypeptide p67phox.

Characterization of soluble vs membrane-bound human placental 5'-nucleotidase.

Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM).

Human T cell activation. Synergy between CD73 (ecto-5'-nucleotidase) and signals delivered through CD3 and CD2 molecules.

Interaction of the glycosyl phosphatidylinositol-linked differentiation Ag CD73 (ecto-5'-nucleotidase) with the CD73-specific mAb 1E9 generates agonistic signals that strongly synergize with T cell activation induced by CD3 and CD2 mAb. This synergy is observed only when 1E9 is immobilized on plastic and occurs in the absence of accessory cells or exogenous lymphokines. 1E9 induces a rapid (though transient) increase in [Ca2+]i in a minor proportion (20 to 30%) of unfractionated T lymphocytes (presumably CD73+ cells).

Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (CD73).

A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (ecto-5'-NT, CD73) was produced using highly purified human placental 5'-NT as immunogen. Antibodies 1E9.28.

Antibodies to 5'-nucleotidase (CD73), a glycosyl-phosphatidylinositol-anchored protein, cause human peripheral blood T cells to proliferate.

Human peripheral blood T cells were stimulated to proliferate when cultured with submitogenic doses of PMA and goat antibodies to 5'-nucleotidase (5'-NT). The degree of proliferation, as measured by [3H]TdR incorporation on day 3, was similar to that achieved by stimulation with PHA. Anti-5'-NT antibodies had no effect on PHA-induced proliferation.

Functional characterization of ecto-5'-nucleotidase-positive and -negative human T lymphocytes.

Functional studies were performed on human peripheral blood T lymphocytes stained with goat anti-5'-nucleotidase antibodies and separated into ecto-5'-nucleotidase (ecto-5'-NT)-positive and -negative populations using the FACSTAR fluorescence-activated cell sorter. On the average, ecto-5'-NT+ T cells contained 34 +/- 13% CD4+ and 55 +/- 15% CD8+ cells, whereas ecto-5'-NT-T cells contained 65 +/- 12% CD4+ and 23 +/- 8% CD8+ cells. Staining with anti-5'-NT antibodies did not significantly alter the ability of unseparated T cells to proliferate in response to PHA or PMA, or in a MLR.

Synthesis of immunoglobulin G by pokeweed mitogen- or Epstein-Barr virus-stimulated human B cells in vitro is restricted to the ecto-5'-nucleotidase positive subset.

Ecto-5'-nucleotidase (ecto-5'-NT) is believed to be a maturation marker for human B lymphocytes because its expression increases during normal development and is reduced in many patients with B cell immunodeficiencies. To determine whether this enzyme defines functional subsets of B lymphocytes, human peripheral blood B cells, separated into ecto-5'-NT positive and negative populations by using goat anti-5'-NT antibodies and the fluorescence-activated cell sorter, were compared for their ability to secrete polyclonal immunoglobulin. Both populations synthesized equivalent quantities of IgM in response to a T cell-dependent (PWM) or T cell-independent (EBV) stimulator of polyclonal immunoglobulin biosynthesis.

Distribution of ecto-5'-nucleotidase on subsets of human T and B lymphocytes as detected by indirect immunofluorescence using goat antibodies.

Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin.

Purification of 5'-nucleotidase from human placenta after release from plasma membranes by phosphatidylinositol-specific phospholipase C.

5'-Nucleotidase was purified greater than 1000-fold from human placenta by treatment of plasma membranes with S. aureus phosphatidylinositol-specific phospholipase C and affinity chromatography on Con A Sepharose and AMP-Sepharose. The resulting enzyme had a specific activity of greater than 5000 mumol/hr/mg protein and a subunit molecular weight of 73,000.

Ecto-5'-nucleotidase expression during human B cell development. An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia.

Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in human B cells at different stages of development. Ecto-5'-NT activity of B cell preparations from fetal spleen and cord blood was 5.08 and 5.

Lymphocyte ecto-5'-nucleotidase activity in infancy: increasing activity in peripheral blood B cells precedes their ability to synthesize IgG in vitro.

Ecto-5'-nucleotidase activity was measured in peripheral blood lymphocytes isolated from serial specimens from nine healthy full-term infants and two premature infants at 0, 2, 4, and 6 mo of age. The postnatal nadir in activity was 7.1 +/- 2.

Lack of pokeweed mitogen-induced IgE formation in vitro by human peripheral blood mononuclear cells: detection of cross-reacting idiotypic determinants on polyclonal Ig by antibodies to a single IgE myeloma protein.

The ability of human peripheral blood mononuclear cells to synthesize IgE in vitro in response to pokeweed mitogen (PWM) is controversial. To determine whether the conflicting results obtained by different laboratories could be due to inherent qualitative differences in the anti-IgE antibodies used to measure low concentrations of IgE in culture supernatants, we compared the specificities of anti-IgE reagents prepared by various methods. Immunoadsorbent-purified antibodies were isolated from a goat antiserum to the lambda, IgE myeloma protein PS and a rabbit antiserum to the kappa, IgE protein Bed in three ways: 1) antibodies to IgE PS (anti-PS) were isolated from the goat antiserum by affinity chromatography with PS coupled to Sepharose 4B; these antibodies consisted of anti-epsilon chain-specific and anti-idiotypic antibodies to protein PS; 2) antibodies specific for the epsilon-chain (anti-epsilon) were purified by affinity chromatography with IgE myeloma proteins that were not used for immunization; and 3) antibodies to idiotypic determinants of proteins PS (anti-id PS) and Bed (anti-id Bed) were isolated on affinity columns with the respective myeloma proteins after absorption of the epsilon-chain-specific antibodies.