Bernardo Mantovani

Latrunculin A is a potent inhibitor of phagocytosis by macrophages

We have found that latrunculin A, a Red Sea sponge toxin, is a potent inhibitor of immunological phagocytosis by mouse peritoneal macrophages, but does not block the binding (recognition) of the immune complexes (erythrocytes sensitized with IgG antibodies) to the cells. The inhibition begins to be appreciable around 12 nM latrunculin A, and is complete with a toxin concentration of 60 nM. This inhibitory effect does not interfere with the cell viability, and can be reversed when the macrophages are incubated in fresh medium. Since latrunculin A is a disrupting agent of microfilament organization, these results strengthen the evidence for the active participation of microfilaments in the mechanism of phagocytosis and at the same time provide a new tool for the investigation of phagocytosis at the molecular level.

Chronic cold stress in mice induces a regulatory phenotype in macrophages: correlation with increased 11β-hydroxysteroid dehydrogenase expression.

Susceptibility to infections, autoimmune disorders and tumor progression is strongly influenced by the activity of the endocrine and nervous systems in response to a stressful stimulus. When the adaptive system is switched on and off efficiently, the body is able to recover from the stress imposed. However, when the system is activated repeatedly or the activity is sustained, as during chronic or excessive stress, an allostatic load is generated, which can lead to disease over long periods of time. We investigated the effects of chronic cold stress in BALB/c mice (4°C/4 h daily for 7 days) on functions of macrophages. We found that chronic cold stress induced a regulatory phenotype in macrophages, characterized by diminished phagocytic ability, decreased TNF-α and IL-6 and increased IL-10 production. In addition, resting macrophages from mice exposed to cold stress stimulated spleen cells to produce regulatory cytokines, and an immunosuppressive state that impaired stressed mice to control Trypanosoma cruzi proliferation. These regulatory effects correlated with an increase in macrophage expression of 11β-hydroxysteroid dehydrogenase, an enzyme that converts inactive glucocorticoid into its active form. As stress is a common aspect of modern life and plays a role in the etiology of many diseases, the results of this study are important for improving knowledge regarding the neuro-immune-endocrine interactions that occur during stress and to highlight the role of macrophages in the immunosuppression induced by chronic stress.

A simple method to study the activity of natural compounds on the chemiluminescence of neutrophils upon stimulation by immune complexes

INTRODUCTION Neutrophils (PMNs) are the main effector cells involved in the immune response to microorganisms. However, in various noninfectious states, such as autoimmune and immune complex (ICs) diseases, ICs are found to be deposited in various organs, leading to recruitment and activation of PMNs at these sites of deposition. Consequently, reactive oxygen species (ROS) and lysosomal enzymes are extensively released by activated PMNs into the extracellular milieu, leading to host tissue injury. METHODS In the present study, we discuss some experimental conditions of a luminol-enhanced chemiluminescence (LECL) assay to study the effect of natural compounds on the production of ROS by rabbit PMNs stimulated with precipitated ICs. Moreover, we evaluated the activities of quercetin and 7-allyloxycoumarin on this ROS-producing system and their toxicity to PMNs. RESULTS Both compounds had concentration-dependent inhibitory effects on LECL. Quercetin at concentration of 5 micromol/l inhibited 94.5+/-1.0% of LECL, whereas 7-allyloxycoumarin at concentration of 200 micromol/l inhibited 53.8+/-2.4% of LECL. Neither compound was toxic to PMNs under the tested conditions. DISCUSSION The proposed method may be useful for the screening of nontoxic compounds that can modulate ROS production by IC-stimulated PMNs. Special attention should be devoted to natural compounds from higher plants, since their potential as sources of new drugs is still largely unexplored.

Effects of latrunculin A on immunological phagocytosis and macrophage spreading-associated changes in the F-actin/G-actin content of the cells.

Latrunculin A, a toxin from a Red Sea sponge, was shown to be a very potent inhibitor of immunological phagocytosis by normal and activated macrophages (obtained from mice injected i.p. with LPS), as well as by polymorphonuclear leukocytes. This toxin blocks the interiorization of the immune complexes but does not interfere with their binding to the phagocyte (recognition phase); activated macrophages were more susceptible to this inhibition than normal macrophages and polymorphonuclear leukocytes. The effect of the toxin on cellular spreading of macrophages was also studied using two kinds of substrate: glass, and glass covered with IgG immune complexes. Latrunculin A was able to impair the spreading of normal macrophages on glass covered with immune complexes, and could also completely reverse the spreading after it had occurred. Contrarily, in activated macrophages, this toxin could neither impede the spreading nor reverse it, a difference that might be a distinctive property of the activated state. We have also found that latrunculin A can reduce the percentage of F-actin in both normal and activated macrophages, the activated cells being more susceptible to this effect. Since latrunculin A is a blocking agent of actin polymerization in vitro, these results indicate that actin polymerization and assembly must be an essential component of the primary, active event of the engulfment phase of phagocytosis.

In vitro activation of mouse neutrophils by recombinant human interferon-gamma: Increased phagocytosis and release of reactive oxygen species and pro-inflammatory cytokines

Here, we investigated the ability of IFN-γ to modulate the functions of mouse neutrophils in vitro. Neutrophils incubated in the presence of IFN-γ showed enhanced phagocytosis in response to zymosan, opsonized zymosan or precipitated immune complexes of IgG and ovalbumin. The effect of IFN-γ was dose-dependent with an initial response at 10 U/ml and a maximal response at 150 U/ml; 2 h of incubation were required to reach the optimal response level. These stimuli can also induce IFN-γ-pretreated neutrophils to release reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and hypochlorous acid, as well as granule lysosomal enzymes and the pro-inflammatory cytokines TNF-α and IL-6. We found that increased expression of FcγR, dectin-1 and complement receptors (CRs) correlated with these effects in these cells. The enhancing effect of IFN-γ on the respiratory burst was found to be associated with up-regulation of the gp91(phox) and p47(phox) subunits of NADPH oxidase, as measured by their mRNA levels. The enhancing effect of IFN-γ on phagocytosis and ROS release may not only be relevant for the efficient killing of invading microorganisms, but may also produce oxidative stress on adjacent cells, resulting in a possible inflammatory role that could also be favored by the liberation of the pro-inflammatory cytokines TNF-α and IL-6.

Phagocytosis of in Vitro-aged erythrocytes—A sharp distinction between activated and normal macrophages

Phagocytosis experiments performed in vitro in the absence of serum with mouse peritoneal macrophages have shown that normal macrophages can bind to in vitro-aged sheep red blood cells without antibody or complement, but only activated macrophages are able to ingest these erythrocytes. This sharp qualitative functional difference may be an index for the characterization of the activated state of macrophages.

Effects of Cold Stress, Corticosterone and Catecholamines on Phagocytosis in Mice: Differences between Resting and Activated Macrophages

Objective: We subjected mice to acute cold stress and studied the effect on phagocytosis by peritoneal macrophages mediated by 3 types of phagocytic receptors: Fcγ, complement receptors 3 (CR3) and mannose and β-glucan receptors. Methods: Mice were subjected to a cold stress condition (4°C for 4 h), and then peritoneal macrophages were harvested and phagocytosis assays performed in vitro. Results: We found a striking difference between resting and lipopolysaccharide (LPS)-activated macrophages (by intraperitoneal injection of LPS 4 days before the stress experiment): for resting macrophages cold stress caused a decrease in phagocytosis mediated by Fcγ or mannose receptors, while for activated macrophages we observed an increase in phagocytosis by the 3 types of receptors. These effects were associated with an increase in plasma concentrations of corticosterone and catecholamines following the cold stress. In order to verify whether these hormone changes could account for the observed effects on phagocytosis, we performed in vitro assays by incubating macrophages harvested from nonstressed animals with these hormones for 4 h at 37°C and measuring their phagocytic capacity. The following experiments were done: (a) with resting (nonactivated) macrophages; (b) with macrophages previously activated in vitro by incubation with LPS; (c) with macrophages previously activated in vivo by intraperitoneal injection of mice with LPS, 4 days before harvesting the cells. We found that for resting macrophages, corticosterone decreased phagocytosis mediated by Fcγ and mannose and β-glucan receptors, but catecholamines had no effect. For macrophages activated either in vivo or in vitro, catecholamines caused an increase in phagocytosis (excluding mannose receptors) while corticosterone had no effect. Conclusion: The above findings suggest that stress can regulate phagocytosis in different ways, depending on the kind of phagocytic receptor involved, the level of stress hormones and the physiological state of the macrophages.

Effects of Acute Cold Stress on Phagocytosis of Apoptotic Cells: The Role of Corticosterone

Background and Aims: Stress can alter many aspects of the immune response, and many studies have been conducted on the effects of stress on inflammatory processes, but little is known about its influence on the resolution of inflammation in tissue homeostasis, which includes the clearance of apoptotic cells by macrophages in a non-phlogistic way. In the present study, we investigated the effect of acute cold stress on the phagocytosis of apoptotic cells by macrophages. Methods: Mice were submitted to acute cold stress (4°C for 4 h) and the capacity of peritoneal macrophages to phagocyte apoptotic thymocytes and to secrete anti-inflammatory cytokines was evaluated. Plasma corticosterone and catecholamine levels were investigated to assess their effect on the phagocytic capacity of macrophages in vitro. Results: We showed that acute cold stress decreases phagocytosis of apoptotic cells at the inflammatory site by lipopolysaccharide-activated macrophages but did not affect resting macrophages. The inhibitory effect on phagocytosis is accompanied by a reduced level of TGF-β and higher IL-10 secretion. After stress, plasma concentrations of corticosterone increased 6-fold, epinephrine 2-fold and norepinephrine 1.7-fold compared to control mice. In vitro experiments showed that the decrease in phagocytosis after stress could be attributed, at least in part, to the effects of corticosterone; epinephrine and norepinephrine had no effect. Conclusions: The current study shows that acute cold stress decreases phagocytosis of apoptotic cells from an inflammatory environment by macrophages, and this inhibition is mediated by the intracellular glucocorticoid receptor.

Latrunculin A is a potent inducer of aggregation of polymorphonuclear leukocytes.

We have shown that latrunculin A, a toxin from a Red Sea sponge which has striking effects in several phenomena dependent on the cytoskeleton, is a potent inducer of aggregation of rabbit polymorphonuclear leukocytes in a dose- and time-dependent manner. From 12 nM to 60 nM toxin the degree of aggregation (after 8 min) in nearly directly proportional to the toxin concentration; the aggregation effect is energy-dependent from the glycolytic pathway. It was also shown that 120 nM latrunculin A, after 5 min incubation, can reduce to more than half the F-actin percent of the leucocytes. These results may contribute to the study of the relations between the actin cytoskeleton of leukocytes and the process of aggregation which is involved in important physiological functions of these cells.

Avaliação bioquímica e ultraestrutural da interação de imunocomplexos de IgG com leucócitos polimorfonucleares: efeito de antioxidantes naturais

The generation of reactive oxygen species (EROs) by polymorphonuclear leukocytes (LPMNs) during phagocytosis plays a key role in the organism defense against invading microorganisms. However, in some diseases the immune complexes deposition occurs, that causes an increase in the production and release of EROs. Consequently, the host tissue is damaged. In this work we studied the effect of natural compounds (quercetin and 7-allyloxycoumarin) on EROs generation by rabbit (LPMNs) stimulated with immunecomplexes (IC) of chicken egg albumin (antigen) and class G immuneglobulins (IgG) - ICIgG. The EROs production was measured by chemiluminescence assay (QL) enhanced by luminol (QL lum) or by lucigenin (QL luc). The interaction between ICIgG (conjugated with colloidal gold) e LPMNs, mediated by Fcg membrane receptor, was observed by transmission electron microscopy. The results indicated that the studied compounds inhibited QL lum and QL luc. They also suggest that such effects are not related to phagocytosis inhibition, as observed by the electron microscopy assay.