lactoferrin and Granulomatous-Disease--Chronic

Chronic Granulomatous Disease (CGD) is a rare disorder caused by mutations in the NADPH oxidase. The CGD phenotype includes granuloma formation and susceptibility to infection with microorganisms including Aspergillus. The immune adjuvant interferon-gamma and the antifungal agent itraconazole have reduced the incidence of infections in CGD. Studies using CGD phagocytes have shown that reactive oxygen species (ROS), products of the NAPDH oxidase, are critical for killing Aspergillus hyphae. But despite lack of ROS production, CGD patients generally only get infected with Aspergillus after heavy exposure. To study why CGD patients are not infected with Aspergillus more frequently we studied host defense against this ubiquitous mold further. We found that neutrophil lactoferrin is fungistatic for Aspergillus fumigatus spores by chelation of iron, an essential growth factor. Thus, the neutrophil employs both nonoxidative (lactoferrin) and oxidative (hydrogen peroxide) defense mechanisms against A. fumigatus spores and hyphae, respectively.

Topics: Antifungal Agents; Aspergillosis; Aspergillus; Granulomatous Disease, Chronic; Humans; Iron Chelating Agents; Lactoferrin; Phagocytes; Reactive Oxygen Species

The initiation of both the normal and pathologic responses of human neutrophils to surface stimulation and the ensuing biochemical and physiologic events are elucidated. This knowledge has contributed to an understanding of the controlling mechanisms that may account for impaired phagocytic function in several clinical disorders associated with recurrent bacterial infections.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Blood Bactericidal Activity; Calcium; Cell Adhesion; Chediak-Higashi Syndrome; Chemotaxis, Leukocyte; Child; Cyclic AMP; Cytoplasmic Granules; Granulomatous Disease, Chronic; Guinea Pigs; Humans; Ion Channels; Lactoferrin; Male; Membrane Potentials; Neutrophils; Opsonin Proteins; Oxygen Consumption; Phagocyte Bactericidal Dysfunction; Phagocytosis; Rabbits

Antimicrobial action of polymorphonuclear leukocytes depends on an array of substances carried in their cell membranes and cytoplasmic granules. These substances mediate killing in several systems. Some depend on molecular oxygen while others are independent of it. Some of the systems that depend on oxygen also require myeloperoxidase. The different systems seem able to act in primary or in reserve capacities backing each other up in times of stress or failure. Thus a deficiency in one system does not necessarily leave the polymorphs completely incapable of antimicrobial action although impairment may be severe as in chronic granulomatous disease. In general a poorly functioning polymorph is better than no polymorph at all.

Topics: Animals; Bacteria; Bacterial Physiological Phenomena; Blood Bactericidal Activity; Blood Proteins; Cations; Cytoplasmic Granules; Escherichia coli; Granulocytes; Granulomatous Disease, Chronic; Humans; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydroxides; Hydroxyl Radical; Iodine; Lactates; Lactic Acid; Lactoferrin; Leukocytes; Models, Biological; Muramidase; Neutrophils; Opsonin Proteins; Oxygen Consumption; Peptide Hydrolases; Peroxidase; Phagocytosis; Superoxides

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.

Topics: Antifungal Agents; Aspergillus fumigatus; Cell Degranulation; Cells, Cultured; Cilia; Granulomatous Disease, Chronic; Humans; Iron; Lactoferrin; Mucus; Neutrophils; Spores, Fungal

Human PMN and monocytes both possess a mechanism for amplifying Fc receptor-mediated phagocytic function, which is dependent on activation of the respiratory burst. The pathway for augmentation of phagocytosis requires superoxide anion, hydrogen peroxide, and lactoferrin and is independent of the hydrogen peroxide-MPO-halide system. In neither cell type is this mechanism induced upon exposure to the opsonized target. PMN require an additional signal for stimulation of the respiratory burst; this is not true of monocytes. On the other hand, monocytes require an exogenous source of lactoferrin in order to activate this pathway for enhanced ingestion. The dependence of this pathway for both PMN and monocytes on superoxide anion, hydrogen peroxide, and cell-bound lactoferrin is consistent with a role for locally generated reactive oxygen metabolites, possibly hydroxyl radicals, in phagocytosis amplification. Patients with chronic granulomatous disease, who are genetically deficient in the ability to activate the respiratory burst, are unable to amplify Fc receptor-mediated phagocytosis. Thus, these patients may have a previously unrecognized defect in the recruitment of phagocytic function at inflammatory sites.

Topics: Amphotericin B; Benzoates; Benzoic Acid; Biological Factors; Catalase; Cytokines; Free Radicals; Granulomatous Disease, Chronic; Humans; Lactoferrin; Male; Monocytes; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Opsonin Proteins; Oxygen Consumption; Phagocytosis; Phorbol 12,13-Dibutyrate; Receptors, Fc; Zymosan

The mechanism by which polymorphonuclear leukocytes (PMNs) kill ingested Bacteroides fragilis was examined using PMNs from patients with chronic granulomatous disease (CGD) which is an inherited disease characterized by the defect of their PMNs in oxygen-radical generation. The phagocytosis of B. fragilis by PMNs from CGD patients was comparable to that by normal PMNs. Although CGD cells killed B. fragilis to some extent, they did so less effectively than the normal PMNS. B. fragilis was killed by a xanthine oxidase system that generates oxygen radicals. When PMNs were incubated with opsonized B. fragilis, B. fragilis triggered the release of O2- and H2O2 from normal PMNs. Thus, normal PMNs appear to kill B. fragilis by both oxygen-dependent and oxygen-independent mechanisms.

Topics: Bacteroides fragilis; Free Radicals; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Lactoferrin; Neutrophils; Oxygen; Peroxidase; Phagocytosis; Superoxides; Xanthine Oxidase

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.

Topics: Cell Aggregation; Deoxyglucose; Egtazic Acid; Ethylmaleimide; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Lactoferrin; Methylmannosides; Neutrophils; Superoxides; Tosylphenylalanyl Chloromethyl Ketone

A technique has been developed for the kinetic measurement on the same blood sample of a variety of functions of phagocytic cells. Adherence to glass, the clearance of a mixture of microorganisms and their subsequent solubilisation, and the secretion of granule contents from the cells were determined. The numbers of residual viable intracellular staphylococci were measured at the completion of the study. These methods were used to investigate phagocyte function in 33 normal subjects and 6 patients with chronic granulomatous disease.

Topics: Adolescent; Adult; Blood; Blood Bactericidal Activity; Blood Platelets; Cell Adhesion; Female; Granulomatous Disease, Chronic; Humans; Kinetics; Lactoferrin; Leukocyte Count; Male; Monocytes; Neutrophils; Phagocytes; Staphylococcus aureus

Studies were performed to elucidate further the phenomenon of secretagogue-mediated enhancement in the binding of the chemoattractant f-met-leu-[3H]phe to human neutrophils (PMN). Specific f-met-leu-[3H]phe binding to unstimulated PMN reached maximum levels after 10 to 15 min of incubation at 0 degrees C with a saturating concentration of peptide, and consisted of a readily displaceable and a nondisplaceable component. PMN, preexposed to A23187 (2.5 X 10(-8) M) or PMA (0.5 ng/ml) for 30 min at 37 degrees C to stimulate limited and preferential release of specific (secondary) granules (10 to 20% of total lysozyme, no beta-glucuronidase), demonstrated an approximate doubling in the displaceable component of f-met-leu-["3H]phe binding, accompanied by an increasing nondisplaceable component that could not be explained by bulk pinocytosis of extracellular fluid (assessed by [3H]sucrose uptake). The increase in f-met-leu-[3H]phe binding was not affected by inhibitors of protein synthesis, could not be attributed to the secreted products lysosyme or lactoferrin acting on the cell, and, on the basis of studies with PMN from patients with chronic granulomatous disease, could not be attributed to the effects of reactive oxygen species generated in low concentration during stimulation. Functional studies on PMN indicated that preexposure to secretagogues at concentrations demonstrated to increase receptor availability also enhanced subsequent f-met-leu-phe-mediated superoxide and hydrogen peroxide generation. The present data demonstrate that secretagogues may activate PMN to enhance their subsequent responses in f-met-leu-phe-mediated processes, and, combined with previous reports, support the concept that specific granules provide a source of preformed membrane and receptor material that is translocated to the cell surface during the secretion associated with directed locomotion.

Topics: 1-Butanol; Butanols; Calcimycin; Calcium; Chemotaxis, Leukocyte; Cycloheximide; Cytoplasmic Granules; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Lactoferrin; Methionine; Muramidase; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Pinocytosis; Puromycin; Receptors, Immunologic; Superoxides; Tetradecanoylphorbol Acetate