Murine Mononuclear Phagocytes Research Articles

Activation of endothelial NO synthase and P2X7 receptor modification mediates the cholinergic control of ATP-induced interleukin-1β release by mononuclear phagocytes.

The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a central role in host defense against infections. High systemic IL-1β levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1β release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1β release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R). Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1β was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain. The inhibitory effect of nAChR agonists on the BzATP-induced IL-1β release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1β release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1β release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification. We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1β release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.

A flow cytometry approach reveals heterogeneity in conventional subsets of murine renal mononuclear phagocytes

Mononuclear phagocytes (MNPs) participate in inflammation and repair after kidney injury, reflecting their complex nature. Dissection into refined functional subunits has been challenging and would benefit understanding of renal pathologies. Flow cytometric approaches are limited to classifications of either different MNP subsets or functional state. We sought to combine these two dimensions in one protocol that considers functional heterogeneity in each MNP subset. We identified five distinct renal MNP subsets based on a previously described strategy. In vitro polarization of bone marrow-derived macrophages (BMDM) into M1- and M2-like cells suggested functional distinction of CD86 + MHCII + CD206- and CD206 + cells. Combination of both distinction methods identified CD86 + MHCII + CD206- and CD206 + cells in all five MNP subsets, revealing their heterologous nature. Our approach revealed that MNP composition and their functional segmentation varied between different mouse models of kidney injury and, moreover, was dynamically regulated in a time-dependent manner. CD206 + cells from three analyzed MNP subsets had a higher ex vivo phagocytic capacity than CD86 + MHCII + CD206- counterparts, indicating functional uniqueness of each subset. In conclusion, our novel flow cytometric approach refines insights into renal MNP heterogeneity and therefore could benefit mechanistic understanding of renal pathology.

Th17 immunity in the colon is controlled by two novel subsets of colon-specific mononuclear phagocytes

Abstract Intestinal immunity is coordinated by specialized populations of mononuclear phagocytes. Although the cell subsets that constitute these populations are thought uniform throughout the intestine, small and large intestine have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity is unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

Salt Transiently Inhibits Mitochondrial Energetics in Mononuclear Phagocytes.

Dietary high salt (HS) is a leading risk factor for mortality and morbidity. Serum sodium transiently increases postprandially but can also accumulate at sites of inflammation affecting differentiation and function of innate and adaptive immune cells. Here, we focus on how changes in extracellular sodium, mimicking alterations in the circulation and tissues, affect the early metabolic, transcriptional, and functional adaption of human and murine mononuclear phagocytes. Using Seahorse technology, pulsed stable isotope-resolved metabolomics, and enzyme activity assays, we characterize the central carbon metabolism and mitochondrial function of human and murine mononuclear phagocytes under HS in vitro. HS as well as pharmacological uncoupling of the electron transport chain under normal salt is used to analyze mitochondrial function on immune cell activation and function (as determined by Escherichiacoli killing and CD4+ T cell migration capacity). In 2 independent clinical studies, we analyze the effect of a HS diet during 2 weeks (URL: http://www.clinicaltrials.gov. Unique identifier: NCT02509962) and short-term salt challenge by a single meal (URL: http://www.clinicaltrials.gov. Unique identifier: NCT04175249) on mitochondrial function of human monocytes in vivo. Extracellular sodium was taken up into the intracellular compartment, followed by the inhibition of mitochondrial respiration in murine and human macrophages. Mechanistically, HS reduces mitochondrial membrane potential, electron transport chain complex II activity, oxygen consumption, and ATP production independently of the polarization status of macrophages. Subsequently, cell activation is altered with improved bactericidal function in HS-treated M1-like macrophages and diminished CD4+ T cell migration in HS-treated M2-like macrophages. Pharmacological uncoupling of the electron transport chain under normal salt phenocopies HS-induced transcriptional changes and bactericidal function of human and murine mononuclear phagocytes. Clinically, also in vivo, rise in plasma sodium concentration within the physiological range reversibly reduces mitochondrial function in human monocytes. In both a 14-day and single meal HS challenge, healthy volunteers displayed a plasma sodium increase of [Formula: see text] and [Formula: see text] respectively, that correlated with decreased monocytic mitochondrial oxygen consumption. Our data identify the disturbance of mitochondrial respiration as the initial step by which HS mechanistically influences immune cell function. Although these functional changes might help to resolve bacterial infections, a shift toward proinflammation could accelerate inflammatory cardiovascular disease.

Th17 Immunity in the Colon Is Controlled by Two Novel Subsets of Colon-Specific Mononuclear Phagocytes.

Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites.

Gamma-aminobutyric acid (GABA) serves diverse biological functions in prokaryotes and eukaryotes, including neurotransmission in vertebrates. Yet, the role of GABA in the immune system has remained elusive. Here, a comprehensive characterization of human and murine myeloid mononuclear phagocytes revealed the presence of a conserved and tightly regulated GABAergic machinery with expression of GABA metabolic enzymes and transporters, GABA-A receptors and regulators, and voltage-dependent calcium channels. Infection challenge with the common coccidian parasites Toxoplasma gondii and Neospora caninum activated GABAergic signaling in phagocytes. Using gene silencing and pharmacological modulators in vitro and in vivo in mice, we identify the functional determinants of GABAergic signaling in parasitized phagocytes and demonstrate a link to calcium responses and migratory activation. The findings reveal a regulatory role for a GABAergic signaling machinery in the host-pathogen interplay between phagocytes and invasive coccidian parasites. The co-option of GABA underlies colonization of the host by a Trojan horse mechanism.

Human and Mouse Transcriptome Profiling Identifies Cross-Species Homology in Pulmonary and Lymph Node Mononuclear Phagocytes

SUMMARYThe mononuclear phagocyte (MP) system consists of macrophages, monocytes, and dendritic cells (DCs). MP subtypes play distinct functional roles in steady-state and inflammatory conditions. Although murine MPs are well characterized, their pulmonary and lymph node (LN) human homologs remain poorly understood. To address this gap, we have created a gene expression compendium across 24 distinct human and murine lung and LN MPs, along with human blood and murine spleen MPs, to serve as validation datasets. In-depth RNA sequencing identifies corresponding human-mouse MP subtypes and determines marker genes shared and divergent across species. Unexpectedly, only 13%–23% of the top 1,000 marker genes(i.e., genes not shared across species-specific MP subtypes) overlap in corresponding human-mouse MP counterparts. Lastly, CD88 in both species helps distinguish monocytes/macrophages from DCs. Our cross-species expression compendium serves as a resource for future translational studies to investigate beforehand whether pursuing specific MP subtypes or genes will prove fruitful.

The macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell–mediated resistance to metastasis

The plasma membrane tetraspan molecule MS4A4A is selectively expressed by macrophage-lineage cells, but its function is unknown. Here we report that MS4A4A was restricted to murine and human mononuclear phagocytes and was induced during monocyte-to-macrophage differentiation in the presence of interleukin 4 or dexamethasone. Human MS4A4A was co-expressed with M2/M2-like molecules in subsets of normal tissue-resident macrophages, infiltrating macrophages from inflamed synovium and tumor-associated macrophages. MS4A4A interacted and colocalized with the β-glucan receptor dectin-1 in lipid rafts. In response to dectin-1 ligands, Ms4a4a-deficient macrophages showed defective signaling and defective production of effector molecules. In experimental models of tumor progression and metastasis, Ms4a4a deficiency in macrophages had no impact on primary tumor growth, but was essential for dectin-1-mediated activation of macrophages and natural killer (NK) cell-mediated metastasis control. Thus, MS4A4A is a tetraspan molecule selectively expressed in macrophages during differentiation and polarization, essential for dectin-1-dependent activation of NK cell-mediated resistance to metastasis.

Imbalance of Ly-6Chi and Ly-6Clo Monocytes/Macrophages Worsens Hyperoxia-Induced Lung Injury and Is Rescued by IFN-γ.

Inflammation in response to oxygen exposure is a major contributing factor in neonatal lung injury leading to bronchopulmonary dysplasia. Although increased levels of proinflammatory cytokines are seen in airway samples and blood from bronchopulmonary dysplasia patients, the innate immune responses in this common neonatal lung condition have not been well characterized. We previously reported that depletion of murine CD11b-expressing mononuclear phagocytes at birth led to severe acute hyperoxia-induced lung injury (HILI) and significant mortality. In this study, we further define the mononuclear phagocyte populations that are present in the neonatal lung and characterize their responses to hyperoxia exposure. We used myeloid depleter mice (CD11b-DTR and CCR2-DTR) to contrast the effects of depleting different monocyte/macrophage subpopulations on the innate immune response to hyperoxia. Using RNA sequencing and subsequent data analysis, we identified an IFN-γ-mediated role for interstitial monocytes/macrophages in acute HILI, in which decreased IFN-γ expression led to increased disease severity and increased Mmp9 mRNA expression. Importantly, intranasal administration of rIFN-γ largely rescued CD11b-DTR+ mice from severe HILI and decreased Mmp9 mRNA expression in Ly-6Clo and Ly-6Chi interstitial monocyte/macrophages. We conclude that the proinflammatory effects of hyperoxia exposure are, at least in part, because of the modulation of effectors downstream of IFN-γ by pulmonary monocytes/macrophages.

Comprehensive overview of murine epididymal mononuclear phagocytes and lymphocytes: Unexpected populations arise

Despite increasing evidence that epididymal immune disorders can lead to infertility, the cells and mechanisms underlying epididymal immunity remain poorly understood. In this study, we propose a rather exhaustive overview of innate and adaptive immune cells present in the murine caput and cauda epididymis. Using flow cytometry and a wide set of markers, we screened the broadest panel of immune cells ever, in this organ. For the first time, we unequivocally quantified the innate populations of monocytes, macrophages, and dendritic cells subtypes. We also revealed the presence of B cells, gamma delta T cells, and double negative T cells in the murine epididymis. They were localized by immunofluorescence stainings, and appeared to be all present in the interstitium and epithelium along the organ, but with respective preferential regional distribution. Altogether, these findings provide new insights on the actors and potential mechanisms involved in the immune responses against genital tract ascending pathogens and in the setting and maintenance of tolerance toward the sperm cells.

Fate Mapping Mononuclear Phagocytes using the CX3CR1 promoter (111.13)

Abstract The mononuclear phagocyte system comprises monocytes, dendritic cells (DCs) and macrophages. Collectively these cells play central roles in the maintenance of tissue integrity during development and after injury, as well as the initiation and resolution of innate and adaptive immunity. The chemokine receptor CX3CR1 is widely expressed in the mononuclear phagocyte system. Indeed mice harboring a GFP reporter in the CX3CR1 locus have been instrumental in identifying and defining clonotypic lineage precursors, dedicated classical DC precursors, as well as tissue-resident mononuclear phagocyte populations. Moreover, discrete expression levels of CX3CR1/GFP in CX3CR1gfp animals led to the identification of two monocyte subsets in mice as CX3CR1int Ly6C+ and CX3CR1hi Ly6C- cells. Here, we report a fate mapping study of the murine mononuclear phagocyte compartment using mice that harbour conditional and constitutive active Cre recombinase genes in their CX3CR1 loci. Our data obtained by side-by-side comparison of CX3CR1Cre and CX3CR1CreERT2 mice, as well as CX3CR1gfp mice suggest the existence of three distinct pathways yielding peripheral mononuclear phagocyte populations in the adult organism: (1) macrophages seeded by primitive macrophages and maintaining themselves through longevity and limited self-renewal; (2) macrophages seeded during embryonic development from definitive precursors and (3) macrophages that are continuously renewed from blood monocytes.

Defining the anatomical localisation of subsets of the murine mononuclear phagocyte system using integrin alpha X (Itgax, CD11c) and colony stimulating factor 1 receptor (Csf1r, CD115) expression fails to discriminate dendritic cells from macrophages

The murine mononuclear phagocyte (MNP) system comprises a diverse population of cells, including monocytes, dendritic cells (DC) and macrophages. Derived from the myeloid haematopoietic lineage, this group of cells express a variety of well characterized surface markers. Expression of the integrin alpha X (Itgax, CD11c) is commonly used to identify classical DC, and similarly expression of colony stimulating factor 1 receptor (Csf1r, CD115) to identify macrophages. We have characterized the expression of these markers using a variety of transgenic mouse models. We confirmed previous observations of Itgax expression in anatomically defined subsets of MNPs in secondary lymphoid organs, including all MNPs identified within the germinal centres. The majority of MNPs in the intestinal lamina propria and lung express Itgax. All mucosal Itgax expressing cells also express Csf1r suggesting Csf1-dependent haematopoietic derivation. This double-positive population included germinal centre MNPs. These data reveal that Itgax expression alone does not specifically define classical DC. These results suggest more cautious interpretation of Itgax-dependent experimentation and direct equation with uniquely DC-mediated activities, particularly in the functioning of non-lymphoid MNPs within the intestinal lamina propria.

Mechanisms of TNF induction by heat-killed Staphylococcus aureus differ upon the origin of mononuclear phagocytes

Mononuclear phagocytes are among the first immune cells activated after pathogens invasion. Although they all derive from the same progenitor in the bone marrow, their characteristics differ on the compartment from which they are derived. In this work, we investigated the contribution of phagocytosis for tumor necrosis factor (TNF) production by murine mononuclear phagocytes (monocytes, peritoneal and alveolar macrophages) in response to heat-killed Staphylococcus aureus (HKSA). Mononuclear phagocytes behaved differently, depending on their compartment of residence. Indeed, when bacterial uptake or phagosome maturation was blocked, activation through membrane receptors was sufficient for a maximal production of TNF and interleukin-10 by peritoneal macrophages. In contrast, monocytes, and to a lesser extent alveolar macrophages, required phagocytosis for optimal cytokine production. While investigating the different actors of signalization, we found that p38 kinase and phosphatidylinositol 3-kinase were playing an important role in HKSA phagocytosis and TNF production. Furthermore, blocking the α(5)β(1)-integrin significantly decreased TNF production in response to HKSA in all three cell types. Finally, using mononuclear phagocytes from NOD2 knockout mice, we observed that TNF production in response to HKSA was dependent on NOD2 for monocytes and peritoneal macrophages. In conclusion, we demonstrate that the mechanisms of activation leading to TNF production in response to HKSA are specific for each mononuclear phagocyte population and involve different recognition processes and signaling pathways. The influence of the compartments on cell properties and behavior should be taken into account, to better understand cell physiology and host-pathogen interaction, and to define efficient strategies to fight infection.

Francisella gains a survival advantage within mononuclear phagocytes by suppressing the host IFNγ response

Tularemia is a zoonotic disease caused by the Gram-negative intracellular pathogen Francisella tularensis. These bacteria evade phagolysosomal fusion, escape from the phagosome and replicate in the host cell cytoplasm. IFNγ has been shown to suppress the intra-macrophage growth of Francisella through both nitric oxide-dependent and -independent pathways. Since Francisella is known to subvert host immune responses, we hypothesized that this pathogen could interfere with IFNγ signaling. Here, we report that infection with Francisella suppresses IFNγ-induced STAT1 expression and phosphorylation in both human and murine mononuclear phagocytes. This suppressive effect of Francisella is independent of phagosomal escape or replication and is mediated by a heat-stable and constitutively expressed bacterial factor. An analysis of the molecular mechanism of STAT1 inhibition indicated that expression of SOCS3, an established negative regulator of IFNγ signaling, is highly up-regulated during infection and suppresses STAT1 phosphorylation. Functional analyses revealed that this interference with IFNγ signaling is accompanied by the suppression of IP-10 production and iNOS induction resulting in increased intracellular bacterial survival. Importantly, the suppressive effect on IFNγ-mediated host cell protection is most effective when IFNγ is added post infection, suggesting that the bacteria establish a permissive environment within the host cell.

Induction of Macrophage Glutamine: Fructose-6-Phosphate Amidotransferase Expression by Hypoxia and by Picolinic Acid

We studied the expression of glutamine: fructose-6-phosphate amidotransferase (GFAT), the rate limiting enzyme in the hexosamine biosynthetic pathway controlling protein glycosylation. We obtained the first evidence that the GFAT mRNA and protein are constitutively expressed in murine mononuclear phagocytes (Mf) and inducible by picolinic acid (PA), a catabolite of tryptophan, hypoxia and desferrioxamine (DFX). These stimuli share the property to transactivate gene expression through the Hypoxia Responsive Element (HRE). The promoter of GFAT contains the consensus sequence of HRE in position 74/-65 (GFAT-HRE), and we studied the role of HRE on the activation of the promoter utilizing appropriate expression vectors. We found that GFAT-HRE is essential for the response to hypoxia, PA or DFX and that Hypoxia Inducible Factor-1alpha (HIF-1alpha) can augment this response. Finally, we demonstrate that iron chelation is part of the mechanism by which PA and DFX activate GFAT expression. Our results provide the first indication that hypoxia, PA or DFX induce the transcription of GFAT gene in murine Mf cell lines and that the HRE of the promoter is essential for this response.

Mitf–PU.1 interactions with the tartrate-resistant acid phosphatase gene promoter during osteoclast differentiation

It has been postulated that the transcription factors micropthalmia associated factor (Mitf) and PU.1 interact with the tartrate-resistant acid phosphatase (TRAP) gene promoter and activate TRAP gene expression in osteoclasts. However, studies on the interaction of these factors with the TRAP promoter employing nuclear extracts from osteoclasts and osteoclast precursors have not been reported. We therefore treated murine mononuclear phagocyte cells with various cytokines to generate cultures of osteoclasts and macrophagic cells with high or low potential to form osteoclasts. The presence of Mitf and PU.1 in nuclear extracts from these cultures and the ability of these factors to bind to the TRAP promoter was then assessed. We demonstrate that Mitf and a related factor, TFE3, are present in nuclear extracts from all cultures and bind the TRAP promoter. While PU.1 is present in nuclear extracts from all cultures, it does not significantly interact with a putative binding site in the TRAP promoter. These results suggest Mitf and PU.1 interactions with the TRAP promoter are not responsible for the specific activation of TRAP gene expression in osteoclasts.

L-Arginine is not the limiting factor for nitric oxide synthesis by human alveolar macrophages in vitro.

Unlike murine mononuclear phagocytes, human macrophages do not release high amounts of nitric oxide (NO) in vitro despite the presence of nitric oxide synthase (NOS). To determine whether this limited NO synthesis in vitro is due to limited availability of the NOS substrate L-arginine, and putative NOS inhibiting factors present in foetal serum preparations, both alveolar macrophages (AM) and monocyte derived macrophages (MDM) were incubated in various circumstances. Nitrite production measured using stimulated AM was typically <5 pmol x min(-1) x 10(-6) cells. A range of stimuli were tested, but without result. Furthermore, incubation of MDMs with normal human serum or purified bovine serum albumin instead of foetal calf serum failed to enhance NO production. Moreover, neither the use of arginase inhibitors nor the addition of surplus L-arginine resulted in an increased NO synthesis. Interestingly, addition of the NOS intermediate Nomega-hydroxy-L-arginine (100 microM) to AM led to nitrite release, which was unaffected by the NOS inhibitor amino guanidine showing that this effect is NOS independent. It is concluded that the limited nitric oxide production of human macrophages in vitro can neither be explained by limited availability of L-arginine, nor by nitric oxide synthase inhibiting substances in foetal serum. Furthermore, it is shown that nitrite release from Nomega-hydroxy-L-arginine by alveolar macrophages is nitric oxide synthase independent.

Identification of specific recognition molecules on murine mononuclear phagocytes and B lymphocytes for Vi capsular polysaccharide: modulation of MHC class II expression on stimulation with the polysaccharide.

Vi bacterial polysaccharide is a homopolymer of alpha 1-4 N-acetyl polygalacturonic acid with variable O-acetylation at position C-3 and forms a capsule around many bacteria. It has been referred to as the virulence factor of Salmonella typhi and is also a candidate vaccine against typhoid fever. The present study reports the interaction of this polysaccharide with murine mononuclear phagocytes and lymphocytes, and with human monocytes. Vi showed a dose-dependent binding to the murine monocyte cell lines WEHI-274.1 and J774. This binding was abrogated if the polysaccharide was deacetylated, suggesting involvement of acetyl groups in this interaction. Vi also bound to the murine B-cell lymphoma line A20, to peritoneal exudate cells and to a lesser degree to spleen cells and thymocytes from BALB/c mice. The polysaccharide also interacted with the human histiocytic lymphoma line U937 but not with the human monocyte cell line THP-1. Stimulation with Vi led to up-regulation of surface major histocompatibility complex (MHC) class II expression on A20 cells. Immunoprecipitation of Vi-bound molecules from cell surface biotinylated A20 and WEHI-274.1 revealed two bands with MW of about 32,000 and 36,000. The study demonstrates that Vi capsular polysaccharide can interact with mononuclear phagocytes and lymphocytes through specific cell surface molecules and modulate MHC class II expression.

IL-4 and IL-13 Modulate IL-10 Release in Endotoxin-Stimulated Murine Peritoneal Mononuclear Phagocytes

Several recent reports presented conflicting data on the action of IL-4 and IL-13 in regulating the release of proinflammatory cytokines by human monocytes. Here we show that the regulation of cytokine release by IL-4 and IL-13 could be either inhibitory or stimulatory in LPS-treated murine peritoneal macrophages. When macrophages were treated with IL-13 or IL-4, between 6 and 24 hr prior to endotoxin challenge, TNFα and IL-6 levels were significantly augmented. On the other hand, when the cells were cotreated with LPS plus IL-13 or IL-4, the release of TNFα and IL-6 was inhibited. These effects of IL-4 and IL-13 were associated with the modulation of IL-10; pretreatment resulted in a decrease, whereas cotreatment gave rise to a dramatic increase in IL-10 levels. The inhibitory effect of IL-4 and IL-13 on the release of TNFα was partially reversed by neutralizing anti-IL10 antibody, and the inhibition of IL-6 release was completely reversed by the antibody. These data suggest that the mechanism of action of IL-13 and IL-4 in modulating macrophage TNFα and IL-6 release partially involves IL-10.

Impairment of the Murine Mononuclear Phagocyte System Function by Antigenic Stimulation

This study examined the mononuclear phagocyte system (MPS) capacity to eliminate IgG-sensitized syngeneic erythrocytes (EA) after antigenic challenge. Survival data of EA in normal and preimmunized mice showed that a single dose of T-dependent antigen was able to delay Fcγ,R-dependent clearance. This impairment in EA elimination was dependent on the dose of antigen injected. On the other hand, T-independent antigens, B cell mitogen, and the inflammatory agent Freund's incomplete adjuvant were ineffective in modulating MPS function. As expected, the liver and the spleen were the main sites of EA trapping, but the spleen of immunized mice sequestered significantly less EA than that of control mice. Impaired clearance capacity was observed as soon as 24 hr after immunization and was persistent up to the seventh day after antigenic stimulation. Moreover, mice decomplementation by cobra venom factor treatment did not prevent the impairment of MPS by antigenic stimulation, suggesting that it is strictly FcγR dependent. Our results indicate that stimulation of the immune system by T-dependent antigens can diminish the FcγR-mediated clearance capacity of the MPS. The possible mechanisms involved in this regulation are discussed.