Mannoprotein fractions of Candida albicans were assayed for their effects on the anticandidal activity of human polymorphonuclear leukocytes (PMNL). One fraction, MP-F2, enhanced PMNL inhibition of candidal growth in vitro as potently as bacterial lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-8. MP-F2-mediated PMNL activation was manifested on yeast and mycelial forms of the fungus, required the integrity of the mannan, and was due to an increase in the actual number of phagocytic PMNL rather than to a greater ingestion of fungal cells by each individual neutrophil. While not inducing augmented O2 production or degranulation of azurophilic granules, MP-F2 strongly stimulated the release of lactoferrin. Lactoferrin inhibited candidal growth in the absence of PMNL, and anti-lactoferrin antibodies reversed both this inhibition and the PMNL activation by MP-F2, GM-CSF, and LPS. Thus, PMNL may be activated by relevant candidal mannoproteins, and release of lactoferrin may add to other antimicrobial mechanisms of PMNL for the control of candidal infections.
Evidence is presented that human polymorphonuclear neutrophils (PMN) can be induced to produce tumor necrosis factor (TNF). Other investigators have previously reported that TNF has been induced from macrophages by bacteria and, more recently, from natural killer cells by certain tumor cells. Our laboratory has reported that the opportunistic fungi, Candida albicans, can induce TNF, not only from human monocytes, but also from Percoll-fractionated large granular lymphocytes. We now report that incubation of PMN with C albicans for 3 hours was sufficient for detection of TNF release, and peak induction was observed at 8 to 18 hours. This release was inhibitable by actinomycin D, an inhibitor of RNA synthesis, as well as by emetine and cycloheximide, which block protein synthesis. The TNF produced by PMN was neutralized by specific monoclonal antibodies against human TNF. These results represent an important finding that TNF production is a normal response of PMN to stimulation by fungi such as C albicans and suggest that the release of TNF may be related to autocrine activation of PMN effector function to control Candida growth.
Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and SabnoneUa typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro. Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells. LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C. albicans in vitro. Antibodies against lactoferrin effectively and specifically reduced the anti-C. albicans activity of both LPS-stimulated and unstimulated PMN.Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 13, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS. The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide. These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN. These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C. albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN. Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunologic cytotoxic effector mechanism that is dependent on the cooperative interaction of humoral and cellular effector elements. This unit describes an assay of ADCC activity that can be used as a test for immunocompetence in effector cells or to test the activity of a monoclonal antibody to mediate ADCC. In this form of cytotoxicity, effector cells with receptors for the Fc portion of immunoglobulin produce target cell lysis by attachment to the Fc portion of antibodies that are bound to target cells via their antigen-combining sites. Therefore, an ADCC assay involves three essential components: labeled target cells, antibodies with specificity for target-cell surface antigens, and effector-cell populations. The basic protocol describes a method of measuring ADCC effector activity in lymphoid cells (peripheral blood mononuclear cells, or PBMC) that employs (51)Cr-labeled target cells. The three components are mixed in microtiter-plate wells and lysis of the target cells is detected by measuring the release of radioactivity into the cell supernatant. Support protocols describe procedures for preparing anti-target cell antiserum and (51)Cr-labeled target cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.