Algirdas J. Jesaitis

Compromised Host Defense on Pseudomonas aeruginosa Biofilms: Characterization of Neutrophil and Biofilm Interactions

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H2O2 accumulation was transient, reaching a maximal measured value of 1 μM. Following contact, stimulated degranulation was 20–40% (myeloperoxidase, β-glucuronidase) and 40–80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H2O2. Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.

A Domain of p47phox That Interacts with Human Neutrophil Flavocytochrome b558

The NADPH-dependent oxidase of human neutrophils is a multicomponent system including cytosolic and membrane proteins. Activation requires translocation of cytosolic proteins p47phox, p67phox, and Rac2 to the plasma membrane and association with the membrane flavocytochrome b to assemble a functioning oxidase. We report the location of a region in p47phox that mediates its interaction with flavocytochrome b. From a random peptide phage display library, we used biopanning with purified flavocytochrome b to select phage peptides that mimicked potential p47phoxbinding residues. Using this approach, we identified a region of p47phox from residue 323 to 342 as a site of interaction with flavocytochrome b. Synthetic peptides 315SRKRLSQDAYRRNS328, 323AYRRNSVRFL332, and 334QRRRQARPGPQSPG347 inhibited superoxide (O∸2) production in the broken cell system with IC50 of 18, 57, and 15 μM, respectively. 323AYRRNSVRFL332 and its derivative peptides inhibited phosphorylation of p47phox. However, the functional importance of this peptide was independent of its effects on phosphorylation, since 323AYRRNAVRFL332 inhibited O∸2 production, but had no effect on phosphorylation. None of the peptides blocked O∸2 production when added after enzyme activation, suggesting that they inhibited the assembly, rather than the activity, of the oxidase. Furthermore these peptides inhibited membrane association of p47phox in the broken cell translocation assay and O∸2 production by electropermeabilized neutrophils, thereby supporting the interpretation that this region of p47phox interacts with flavocytochrome b.

The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils

Studies were performed to examine the lateral organization of the NADPH oxidase system in the plasma membrane of human neutrophils. Analysis of the subcellular fractionation of human neutrophils by isopycnic sedimentation of cavitated cell lysates suggested that there may be more than one population of plasma membrane vesicles formed upon cell disruption. One population (30-32% sucrose) contained surface accessible wheat germ agglutinin binding sites, alkaline phosphatase activity, and cytochrome b. Another population (34-36% sucrose) contained membrane-bound flavin and, when the cells were prestimulated with phorbol myristate acetate (PMA), NADPH-dependent superoxide generating activity. Approximately 25% of the neutrophil cytochrome b cosedimented with the heavy population, confirming our previous hypothesis (Parkos et al. (1985) J. Biol. Chem. 260, 6541-6547) that only a fraction of the total cellular cytochrome b is involved in superoxide production. The heavy plasma membrane fraction was also enriched in membrane associated actin and fodrin as detected by Western blot analysis. After extraction of the plasma membrane vesicles with detergent cocktails, the majority of superoxide generating activity remained associated with the detergent insoluble pellet. Western blot analysis demonstrated that the pellets were also enriched in actin. Further analysis of these pellets using rate-zonal detergent-containing sucrose density gradients indicated that the superoxide generating complex had an approximate sedimentation coefficient of 80 S, suggesting that the neutrophil superoxide generating system may form a complex on the plasma membrane which is associated with or somehow organized by the membrane skeletal matrix. This organization may be of functional relevance not only to the actual production of superoxide, but also to the targeting of microbicidal oxidants.

The molecular basis of oxidative damage by leukocytes

Preface and Acknowledgements. Meeting Summary (A.J. Jesaitis, M. Quinn, G. Mukherjee, P.A. Ward, and E.A. Dratz). FEATURED PRESENTATIONS. Molecular Mechanism of Activation of Leukocyte Superoxide Production (L. McPhail). Molecular Dissection of the Leukocyte Superoxide Generating System (R.A. Clark). The Flavoprotein Component of the Superoxide Generating NADPH Oxidase (A.R. Cross). The Structure of the Human Neutrophil Plasma Membrane B-Type Cytochrome Involved in Superoxide Production (C.A. Parkos, M.T. Quinn, S. Sheets, and A.J. Jesaitis). Structural Control of Electron Transfer in Proteins (D.N. Beratan). Molecular Associations of Human Neutrophil Cytochrome B (M.T. Quinn). Role of CD11/CD18 Integrins and cAMP in TNF-Induced Respiratory Burst of Human Neutrophil (M. Fuortes and C. Nathan). Organization of the Leukocyte Plasma Membrane Components of Superoxide Production (A.J. Jesaitis). Perturbation of Arachidonic Metabolism and Membrane Function by Oxidants (H. J. Forman, T.W. Robison, D.C. Skelton, and D.P. Duncan). Influence of Myeloperoxidase on Oxidant Production and Lipid Oxidation by Neutrophils (C.C. Winterbourn and A.J. Kettle). The Chemistry and Biology of Superoxide (02-): Central Concepts and Residual Problems (I. Fridovich). The Mechanism of Inactivation of Glyceraldehyde 3-Phosphate Dehydrogenase by Hydrogen Peroxide In Vitro: A Study on a Molecular Aspect of Oxidant Mediated Tissue Injury (P.A. Hyslop, W.A. Halsey Jr., D.B. Hinshaw, I.U. Shraufstatter, R.D. Sauerheber and C.G. Cochrane). Leukocyte Oxygen Products and Tissue Damage (P.A. Ward, and M.S. Mulligan). Mechanisms of Cell Damage by Oxidants (C.G. Cochrane). The Role of Neutrophils in Reperfusion Injury (B.J. Zimmerman, F.E. Landreneau, and D.N. Granger). Ischemia-Reperfusion of the Myocardium: Mechanisms of Oxidative Injury and Defense (L.S. Terada, J.M. Brown, and J.E. Repine). Electron Paramagnetic Resonance Measurements of Free Radical Generation in Isolated Cells and Whole Tissues (J.L. Zweier, P. Kuppusamy, S.M.L. Shandelya, S. Thompson-Gorman, H.F. Weisman, D.T. Fearon, and M.L. Weisfeldt). Clinical Application of Free Radical Ablation for the Prevention of Reperfusion Injury (H.J. Schiller and G.B. Bulkley). Superoxide Production by Human Phagocytes: Insights from Chronic Granulomatous Disease (J.T. Curnutte). Superoxide Production and Human Disease (J.M. McCord). POSTER PRESENTATIONS. Changes in Plasma Membrane Microheterogeneity of Polymophonuclear Leukocytes During the Activation of the Respiratory Burst (A. Kantar, P.L. Giorgi, G. Curatola, and R. Fiorini). The Respiratory Burst Oxidase and its Relationship with the Cytoskeleton of Human Neutrophils (R.C. Woodman, J.M. Ruedi, A.J. Jesaitis, N. Okamura, M.T. Quinn, R.M. Smith, J.T. Curnutte, and B.M. Babior). Superoxide Production by Murine Tumour-Associated Macrophages in Inhibited by a Species Acting on the Signal Transduction Pathway (S.J. Eason and B.M. Hannigan). Correlations and Dissociations Between Superoxide Production and Adhesion Function of Human Neutrophils (P. Bellavite, S. Chirumbolo, G. Poli, and C. Mansoldo). Nitric Oxide Medulates Leukocyte Adhesion to Vascular Endothelium (P. Kubes, J. Russell, M. Suzuki, and D.N. Granger). Biological Surfaces Differentially Affect 02- Production by Human Neutrophils (NEU) and Eosinophils (EOS) (P. Dri, R. Cramer, P. Spessotto, M. Romano, and P. Patriarca). Differential Regulation of Reactive Nitrogen and Reactive Oxygen Intermediate Production by Hepatic Macrophages and Endothelial Cells (C.R. Gardner, D.E. Heck, L.S. Feder, T.W. McCloskey, J.D. Laskin, and D.L. Laskin). Activation of Carcinogens by the Systems Simulating Oxidants Generated by Polymorphonuclear Leukocytes (D. Malejka-Giganti, C.L. Ritter , and R.W. Decker). Electron Microscopic Immunocytochemical Localization of Lipid Peroxidation Products in Phagocytosing Human Neutrophils (J.G. Linner, E.S. Buescher, D. Siemsen, E.A. Dratz, M.T. Quinn, and A.J. Jesaitis). Generation of Reactive Oxygen Intermediates by Leukocytes Treated with Psoralens and Ultraviolet Light (D.E. Heck, E. Bisaccia, S. Armus, and J.D. Laskin). Secretion of Enzymatically Active Human Recombinant Myeloperoxidase by Chinese Hamster Ovary Cells in Culture (N. Moguilevsky, L. Garcia-Quintana, A. Jacquet, C. Tournay, L. Fabry, L. Pierard, and A. Bollen). Myeloperoxidase-Dependent Formation of Benzo(a)pyrene-7,8-DIOL DNA Adducts by Stimulated Pulmonary Neutrophils (J.M. Petruska, D.R. Mosebrook, G.J. Jakab, and M.A. Trush). Modulation of Endothelial Cell Injury All-Trans Retinoic Acid (RA): Role in the Anti-Inflammatory Effects of RA (J. Varani and K.J. Johnson). Transgenic Expression of Glutathione Peroxidases in Human Breast Cells: Increased Resistance to Clastogenic Oxidants (M.-E. Mirault, A. Tremblay, L. Lavoie, M. Tremblay, and N. Beaudoin). Comparison of Oxidative Damage and Inhibition of FE Uptake in HL60 Cells from a Mono or BIS Thiosemicarbazone Copper Complex (W.E. Antholine, J. Narasimhan, C.R. Chitambar, and D.H. Petering). Expression of Superoxide Dismutase During Endothelial Cell Stress (N.V. Ketis and J. Jones). Manganese Superoxide Dismutase Appears to be Associated with the Neutrophil Plasma Membrane (M.L. McCormick, L.W. Oberley, T.D. Oberley, and B.E. Britigan). Increased Neutrophil Radical Production, Increased Lipid Peroxidation and Increased Consumption of Antioxidants in HIV-Infected Patients (C. Jarstrand, O. Rasool, B. Akerlund, and B. Lindeke). Constitutive Extracellular Release of Phosphatidylcholine Preferring Phospholipase C (PC-PLC) by Human Fibroblasts and Smooth Muscle Cells (L.I. Gordon, D. Gutstein, S. Prachand and S.A. Weitzman). Cardiovasculatr Effects of Activated Polymorphonuclear Leukocytes (K. Prasad, J. Kalra, and D. Debnath). Oxidative Damage of Normal and Neoplastic Epidermal Cells by Stimulated Polymorphonuclear Leukocytes in Man (R.E. Schopf, C. Ziegler, M. Rehder, and B. Morsches). The Role of Oxidants in Influenza-Induced Airway Hyperactivity in Rats (J.S. Tepper, J.R. Lehmann, G. Dupont, J.R. Hoidal, D.L. Costa, G.R. Burleson, and T.P. Kennedy). The Role of Neutrophils in Hepatic Ischemia-Reperfusion Injury (H. Jaeschke, C.W. Smith, H. Hughes, and A. Farhood).

Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex

The integral membrane protein p22phox forms a heterodimeric enzyme complex with NADPH oxidases (Noxs) and is required for their catalytic activity. Nox4, a Nox linked to cardiovascular disease, angiogenesis, and insulin signaling, is unique in its ability to produce hydrogen peroxide constitutively. To date, p22phox constitutes the only identified regulatory component for Nox4 function. To delineate structural elements in p22phox essential for formation and localization of the Nox4-p22phox complex and its enzymatic function, truncation and point mutagenesis was used. Human lung carcinoma cells served as a heterologous expression system, since this cell type is p22phox-deficient and promotes cell surface expression of the Nox4-p22phox heterodimer. Expression of p22phox truncation mutants indicates that the dual tryptophan motif contained in the N-terminal amino acids 6-11 is essential, whereas the C terminus (amino acids 130-195) is dispensable for Nox4 activity. Introduction of charged residues in domains predicted to be extracellular by topology modeling was mostly tolerated, whereas the exchange of amino acids in predicted membrane-spanning domains caused loss of function or showed distinct differences in p22phox interaction with various Noxs. For example, the substitution of tyrosine 121 with histidine in p22phox, which abolished Nox2 and Nox3 function in vivo, preserved Nox4 activity when expressed in lung cancer cells. Many of the examined p22phox mutations inhibiting Nox1 to -3 maturation did not alter Nox4-p22phox association, further accenting the differences between Noxs. These studies highlight the distinct interaction of the key regulatory p22phox subunit with Nox4, a feature which could provide the basis for selective inhibitor development.

Formyl Peptide Receptor-1 Activation Enhances Intestinal Epithelial Cell Restitution through Phosphatidylinositol 3-Kinase-Dependent Activation of Rac1 and Cdc42

Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K-dependent activation of Rac1 and Cdc42.

IDENTIFICATION OF A LIGAND BINDING SITE IN THE HUMAN NEUTROPHIL FORMYL PEPTIDE RECEPTOR USING A SITE-SPECIFIC FLUORESCENT PHOTOAFFINITY LABEL AND MASS SPECTROMETRY

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-l-phenylalanine-Phe-Tyr-Lys-ε-N-fluorescein (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83–87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83–85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83–85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol.159, 4045–4054) and the photochemical cross-linking data.

Antigen–antibody interface properties: Composition, residue interactions, and features of 53 non-redundant structures

The structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag-Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag-Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS>ASN>GLU>ASP>ARG. This examination encompasses a large, diverse set of unique Ag-Ab crystal structures that help explain the biological range and specificity of Ag-Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.

Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes☆

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83000 X g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40-60% were achieved with a 20-70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g X cm-3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimented with specific granules (p = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg2+-ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg2+-ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.

Site-Specific Inhibitors of NADPH Oxidase Activity and Structural Probes of Flavocytochrome b: Characterization of Six Monoclonal Antibodies to the p22phox Subunit

The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the human phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species important in the elimination of infectious agents. This study reports the generation and characterization of six mAbs (NS1, NS2, NS5, CS6, CS8, and CS9) that recognize the p22phox subunit of the Cyt b heterodimer. Each of the mAbs specifically detected p22phox by Western blot analysis but did not react with intact neutrophils in FACS studies. Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9. Fluorescence resonance energy transfer experiments indicated that mAbs CS6 and CS8 efficiently compete with Cascade Blue-labeled mAb 44.1 (a previously characterized, p22phox-specific mAb) for binding to Cyt b, supporting phage display results suggesting that all three Abs recognize a common region of p22phox. Energy transfer experiments also suggested the spatial proximity of the mAb CS9 and mAb NS1 binding sites to the mAb 44.1 epitope, while indicating a more distant proximity between the mAb NS5 and mAb 44.1 epitopes. Cell-free oxidase assays demonstrated the ability of mAb CS9 to markedly inhibit superoxide production in a concentration-dependent manner, with more moderate levels of inhibition observed for mAbs NS1, NS5, CS6, and CS8. A combination of computational predictions, available experimental data, and results obtained with the mAbs reported in this study was used to generate a novel topology model of p22phox.