Regulation of oxygen metabolism in human granulocytes: relationship between stimulus binding and oxidative response using plant lectins as probes

Abstract

To examine the relationship between binding of surface-active stimuli to human neutrophils and activation of the superoxide (02.) and peroxide (H202) forming oxidase(s) of these cells. oxygen (02) consumption and 02. and H202 release were measured following stimulation by plant lectins. Both concanavalin A (Con-A) and phytohemagglutinin (PHA) were more active than other lectins in stimulating the respiratory activity of neutrophils. When the cells were pretreated with cytochalasin B (Cyto-B). recovery of extracellular 02 and H202 was increased by 3-10-fold. as found previously with particulate stimuli. Whereas cytochalasin B reduced 02 consumption by cells triggered with particulate stimuli. cytochalasin B treatment of cells produced more than a threefold increase in 02 consumption by cells triggered with plant lectins. The stimulatory effect of cytochalasin B on the respiration of Con-A-or PHA-treated cells was rapid. with an enhancement observed within 60 sec. With Con-A-stimulated cells. it could be inhibited by alpha-methyl-D-mannoside (AMM). indicating that it was specifically due to an activity mediated by the lectin as opposed to some other action of cytochalasin B. To correlate these events with binding of the lectin to the cells. 3H-Con-A was employed. Pretreatment of cells with Cyto-B increased 3H-Con-A binding in direct parallel to the stimulation of respiration. Similar results were found when the Cyto-B was added in the middle of an incubation period. Both the increased binding and respiratory activity of the cytochalasin-B-treated cells could be inhibited by the simultaneous addition of AMM. These findings support the concept that the degree of activation of the neutrophil oxidase(s) is a direct function of the extent of binding of the initial stimulus. They also suggest that ongoing stimulus binding is a prerequisite for continued respiratory activity. The precise nature of the lectin binding sites ' 'uncovered" in Cyto-B-treated cells and their potential relationship to sites of oxidase activity or regulation remains to be determined.

Stimulation of human leukocytes by protein II+ gonococci is mediated by lectin-like gonococcal components

“…The presence of carbohydrate-containing receptors on phagocytes is well known. Isolated plant lectins such as ConA and wheat germ agglutinin have been used to define roles for mannose-, N-acetyl-glucosamine-, and sialic acidcontaining receptors in the stimulation of the oxidative burst of human phagocytes (7,32,34). Several gram-negative organisms associate with and are phagocytosed by eucaryotic cells by a mechanism that is mediated by mannose- [41][42][43].…”

“…They are soluble or membrane bound; possess the ability to precipitate glycoconjugates or agglutinate plant or animal cells (i.e., they have at least two carbohydrate-binding sites); and are defined in terms of the monosaccharide(s) or simple oligosaccharide(s) that inhibit lectininduced glycoprecipitation or agglutination (15). If one looks at the stimulation of human neutrophils by lectins such as concanavalin A (ConA) or wheat germ agglutinin, one can see a working model of this definition as it is applied to precipitation or agglutination of glycoconjugates (receptors) within the plane of the membrane of a eucaryotic cell (6,7,32,34,56). Such mechanisms might be involved in neutrophil stimulation by bacteria, such as that described in the present report, or in inhibition of phagocyte oxidative responses by pathogens, such as that which has been observed for Salmonella typhi (27), Brucella abortus (23), and Toxoplasma gondii (59), among others.…”

New Research on the Importance of Cystic Fibrosis Transmembrane Conductance Regulator Function for Optimal Neutrophil Activity

“…The burst of oxygen consumption upon phagocytosis and the absence of this process in CGD leading to impaired bacterial killing was understood to indicate that the oxygen consumed was converted to antimicrobial oxidants. By exposing neutrophils to opsonised bacteria, Cohen and colleagues demonstrated that 99% of oxygen consumed was converted to superoxide (O 2 -) [41] and the requirement of O 2 -as a precursor to hydrogen peroxide (H 2 O 2 ) was confirmed [42]. It is not known what concentration of H 2 O 2 is attained within the vacuole, with measurement from 0.01μM [42] to 100mM estimated, depending on the amount of phagocytosis and the intracellular pH [43].…”

Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human Neutrophils by Interaction with Mobilizable Receptors

“…Many plant lectins are toxic, some may aggregate erythrocytes of different blood groups (hemagglutination), and yet others function as mitogens of T and B lymphocytes (42,45,52). In relation to neutrophils, lectins with different carbohydrate specificities, e.g., concanavalin A binding to mannose and wheat germ agglutinin (WGA) binding to N-acetylglucosamine (GlcNAc) and sialic acid, have been shown to induce cellular responses (10,22,34,39).…”