Distinct differentiation potential of blood monocyte subsets in the lung

Peripheral blood monocytes are a population of circulating mononuclear phagocytes that harbor potential to differentiate into macrophages and dendritic cells. As in humans, monocytes in the mouse comprise two phenotypically distinct subsets that are Gr1(high)CX(3)CR1(int) and Gr1(low)CX(3)CR1(high), respectively. The question remains whether these populations contribute differentially to the generation of peripheral mononuclear phagocytes. In this study, we track the fate of adoptively transferred, fractionated monocyte subsets in the lung of recipient mice. We show that under inflammatory and noninflammatory conditions, both monocyte subsets give rise to pulmonary dendritic cells. In contrast, under the conditions studied, only Gr1(low)CX(3)CR1(high) monocytes, but not Gr1(high)CX(3)CR1(int) cells, had the potential to differentiate into lung macrophages. However, Gr1(high)CX(3)CR1(int) monocytes could acquire this potential upon conversion into Gr1(low)CX(3)CR1(high) cells. Our results therefore indicate an intrinsic dichotomy in the differentiation potential of the two main blood monocyte subsets.     

Effect of mononuclear phagocytes on the differentiation of syngeneic, allogeneic and xenogeneic hematopoietic stem cells

The colony formation in spleen of lethally irradiated syngeneic or hybrid recipients was studied after transplantation of bone marrow cells, with or without macrophages from lymph nodules or from peritoneal cavity of mice, cells of macrophage-like cell line J-774, and monocytes from peripheral blood of healthy donors. The direction of stem cell differentiations in the presence of all the types of mononuclear phagocytes was seen to change from mainly erythroid to mainly myeloid one. The ratio of erythroid to myeloid colonies became equal to 0.5-0.9 instead of 2.0, when bone marrow cells were injected with equivalent quantity of mononuclear phagocytes. This new regulatory function of mononuclear phagocytes is discussed.     

Killing of Escherichia coli by mononuclear phagocytes and neutrophils stimulated in vitro with beta-1,3-D-polyglucose derivatives.

Human monocytes, human peritoneal macrophages, mouse peritoneal macrophages and human peripheral neutrophils pretreated with beta-1,3-D-polyglucose derivatives showed pronounced bactericidal capacity to Escherichia coli compared to control cells. The increased bactericidal capacity was detectable in mononuclear phagocytes over a wide range of concentrations of bacteria. Granulocytes, however, showed bactericidal capacity only at low concentrations of bacteria. The pretreated mononuclear phagocytes released significant amounts of IL-1 and PGE2. However, there was no significant release of tumor necrosis factor (TNF). By incubating unstimulated cells with purified IL-1 and TNF, the bactericidal activity of neutrophils and mononuclear phagocytes was enhanced. Our data indicate that the inability of neutrophils stimulated with beta-1,3-D-polyglucose derivatives to kill large numbers of bacteria could be overcome by a combined treatment with purified IL-1 or TNF in addition to beta-1,3-D-polyglucose derivatives. By incubating unstimulated cells with medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, the bactericidal activity of the cells was enhanced to the same extent as cells pretreated with purified TNF and IL-1. Cells incubated with IL-1-depleted medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, showed reduced bactericidal activity compared to cells incubated with undepleted medium. These studies demonstrate that beta-1,3-D-polyglucose-treated mononuclear phagocytes and neutrophils show enhanced bactericidal activity. The enhanced activity is partly caused by stimulation of the cells with IL-1 released from mononuclear phagocytes and partly by other unknown effects of beta-1,3-D-polyglucose derivatives on both mononuclear phagocytes and neutrophils.     

Transepithelial pathogen uptake into the small intestinal lamina propria

The lamina propria that underlies and stabilizes the gut lining epithelium is densely populated with strategically located mononuclear phagocytes. Collectively, these lamina propria macrophages and dendritic cells (DC) are believed to be crucial for tissue homeostasis as well as the innate and adaptive host defense. Lamina propria DC were recently shown to gain direct access to the intestinal lumen by virtue of epithelium-penetrating dendrites. However, the role of these structures in pathogen uptake remains under debate. In this study, we report that entry of a noninvasive model pathogen (Aspergillus fumigatus conidia) into the murine small intestinal lamina propria persists in the absence of either transepithelial dendrites or lamina propria DC and macrophages. Our results suggest the existence of multiple pathogen entry pathways and point at the importance of villus M cells in the uptake of gut lumen Ags. Interestingly, transepithelial dendrites seem altogether absent from the small intestine of BALB/c mice suggesting that the function of lamina propria DC extensions resides in their potential selectivity for luminal Ags, rather than in general uptake or gut homeostasis.     

Macrophage growth inhibitors derived from the murine peritoneal cavity

Summary The murine peritoneal cavity contains factors that inhibit the in vitro growth and colony formation of macrophages. The inhibition of macrophage growth is not due to cell death. In the presence of inhibitors, the growth of colony-forming macrophages is suppressed, and small clusters are formed as a result of limited proliferation. The more mature mono-nuclear phagocytes (blood monocytes and peritoneal exudate macrophages) are more sensitive to the overall inhibitory effect of the peritoneal inhibitors than the less mature bone marrow mononuclear phagocytes. Furthermore, using dialysis and Amicon ultrafiltration, at least two inhibitors with differential inhibitory effects can be demonstrated. The colony formation of bone marrow mononuclear phagocytes is suppressed mainly by a protease-resistant, small molecular weight (<1,000) dialyzable inhibitor. In contrast, peritoneal exudate macrophages are sensitive to both the small molecular weight inhibitor and a protease-sensitive, large molecular weight (>12,000), nondialyzable inhibitor. The data suggest a possible existence of a dual inhibitor control on the proliferation of mononuclear phagocytes in vivo. In addition, the in vitro cultured peritoneal exudate cells are capable of producing inhibitors that mimic the activity of the in vivo inhibitors. This investigation was supported by Grants CA 09 11(SY) and AI15563(CCS) from the National Institutes of Health, Bethesda, MD     

In situ effector pathways of allograft destruction. 3. Plasminogen activator activity in rat renal allografts

The question of which cell components in a rejecting rat renal allograft secrete plasminogen activator (PA) has been analyzed. Although normal renal parenchymal cells also secreted PA, most of the PA in a renal allograft (and to a lesser extent also in an autograft) was produced by the inflammatory leukocytes. Fractionation at 1 g demonstrated that the inflammatory cell population responsible for the PA production in the allograft sedimented together with the large mononuclear phagocytes (macrophages). Fractions purified for small blast cells and large lymphocytes did not contain any PA activity but they were able to induce resting peritoneal macrophages to produce PA when cocultured in vitro. The results demonstrate that the allograft-infiltrating mononuclear phagocytes are "activated" in the sense that they secrete PA and that the activation of mononuclear phagocytes at the site of inflammation may be partially regulated by the inflammatory lymphoid cells.     

TRAIL-mediated apoptosis in HIV-1-infected macrophages is dependent on the inhibition of Akt-1 phosphorylation

HIV-1 uses mononuclear phagocytes (monocytes, tissue macrophages, and dendritic cells) as a vehicle for its own dissemination and as a reservoir for continuous viral replication. The mechanism by which the host immune system clears HIV-1-infected macrophages is not understood. TRAIL may play a role in this process. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The plasma level of TRAIL is increased in HIV-1-infected patients, particularly those with high viral loads. To study the effect of elevated TRAIL on mononuclear phagocytes, we used recombinant human (rh) TRAIL and human monocyte-derived macrophages (MDM) as an in vitro model. Our results demonstrated rhTRAIL-induced apoptosis in HIV-1-infected MDM and inhibited viral replication, while having a reduced effect on uninfected MDM. HIV-1 infection significantly decreased Akt-1 phosphorylation; rhTRAIL exposure further decreased Akt-1 phosphorylation. Infection with a dominant-negative Akt-1 adenovirus potentiated rhTRAIL-induced apoptosis, while constitutively active Akt-1 blocked rhTRAIL-induced apoptosis in HIV-1-infected MDM. From this data we conclude the death ligand TRAIL preferentially provokes apoptosis of HIV-1-infected MDM, and the mechanism is reliant upon the inhibition of Akt-1 phosphorylation. Understanding this mechanism may facilitate the elimination of HIV-1-infected macrophages and lead to new therapeutic avenues for treatment of HIV-1 infection.     

Transfer Agent of Immunity

An almost pure population of mononuclear phagocytes (macrophages) was obtained by repeated replacement of the culture medium. When treated in vitro with an immune ribonucleic acid (RNA) preparation extracted from the spleens of mice immunized with horse red blood cells (H-RBC), the rosette forming cells against H-RBC were demonstrated in some of the cultured macrophages but not against calf red blood cells. According to both microscopic observations and phagocytic activity, almost all of the rosette formers in this population were found to be large mononuclear phagocytes. These results support our view that large mononuclear phagocytes of mesenchymal origin constitute another cell line responsible for antibody formation in addition to the plasma and lymphocytic cell lines.     

Cytochemical localization of glucose-6-phosphatase in rabbit mononuclear phagocytes during differentiation

Cytochemical and biochemical investigations have revealed glucose-6-phosphatase (G-6-Pase) activity in Kupffer cells of the liver. To determine whether other mononuclear phagocytes are also reactive for G-6-Pase, rabbit bone marrow, blood, and alveolar macrophages were tested for G-6-Pase by a modified Wachstein-Meisel method and prepared for electron microscopy. Some mononuclear phagocytes from all three tissues were intensely reactive; others were unreactive. In promonocytes, monocytes, and alveolar macrophages, reaction product for the enzyme was localized throughout all cisternae of the endoplasmic reticulum (ER) and the perinuclear cisternae, but it was absent from the Golgi complex, lysosomes, and occasional smooth tubular channels. These results indicate that mononuclear phagocytes at all stages of development contain cytochemically demonstrable G-6-Pase and that the distribution of the enzyme is not altered during their differentiation from immature cells in the bone marrow to mature macrophages in the lung.     

Macrophage maturation: differences in complement secretion by marrow, monocyte, and tissue macrophages detected with an improved hemolytic plaque assay

In order to examine one function of mononuclear phagocytes during maturation from bone marrow precursors to tissue macrophages, an improved hemolytic plaque assay for the detection of synthesis of the second (C2) and fourth (C4) components of C by single cells was developed. With this method, production of C2 and C4 was assessed in cell populations derived from bone marrow, blood, lung, peritoneum, and spleen. The proportion of cells producing C2 and C4 in each population varied. Approximately 10% of bone marrow cells produced C4, but not detectable C2 plaque-forming cells (PFC) were detected. Circulating monocytes yielded about 10% PFC each for C2 and C4. The proportion of C2-producing cells in tissue macrophages varied from approximately 2% in bronchoalveolar macrophages to about 45% in peritoneal and splenic macrophage populations, whereas C4 production by macrophages from lung, peritoneum, and spleen were all approximately 45%. These data suggest that differences in C biosynthesis characterize mononuclear phagocytes at different stages of maturation.     

Effects of differentiation in vivo and of lymphokine treatment in vitro on the mobility of C3 receptors of human and mouse mononuclear phagocytes

The C3 receptors of human peripheral blood monocytes are able to move laterally within the plasma membranes of the cells and remain mobile even when the cells develop into "macrophages" in vitro. In contrast, the C3 receptors of mouse peritoneal macrophages are immobile. To determine whether these differences are species differences or differences between cells of different stages of differentiation, we assessed the mobility of C3 receptors of mouse peripheral blood monocytes and of human pulmonary alveolar and peritoneal macrophages. The C3 receptors of mouse monocytes were mobile, whereas the C3 receptors of human tissue macrophages were immobile. The C3 receptors of macrophages mediate avid particle binding but do not normally promote ingestion. We have described a unique lymphokine that activates mouse peritoneal macrophage C3 receptors for phagocytosis by freeing them from their plasma membrane anchors. In the present experiments, we found that the lymphokine also freed the C3 receptors of human macrophages and activated them for phagocytosis. We conclude that the immobilization of C3 receptors appears to be a marker for the differentiation of human and mouse mononuclear phagocytes, that the differentiation of mononuclear phagocytes is influenced by the milieu in which the cells develop, that in vitro-differentiated macrophages may not accurately represent tissue macrophages, and that a lymphokine activates the C3 receptors of both human and mouse macrophages for phagocytosis by allowing the receptors lateral mobility within the cell plasma membrane.     

Direct visualization of peptide/MHC complexes at the surface and in the intracellular compartments of cells infected in vivo by Leishmania major

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.     

Characterization of Small,Mononuclear Blood Cells from Salmon Having High Phagocytic Capacity and Ability to Differentiate into Dendritic like Cells

Phagocytes are the principal component of the innate immune system, playing a key role in the clearance of foreign particles that include potential pathogens. In vertebrates, both neutrophils and mononuclear cells like monocytes, macrophages and dendritic cells are all professional phagocytes. In teleosts, B-lymphocytes also have potent phagocytic ability. We have isolated a population of small (<5 µm), mononuclear blood cells from Atlantic salmon (Salmo salar L.) not previously characterized. In order to identify them, we have performed morphological, gene expression, flow cytometry, cytochemical, ultrastructural and functional analyses. Interestingly, they highly express the gene encoding CD83, the most characteristic cell surface marker for dendritic cells in mammals, and MHC class II limited to professional antigen presenting cells. They did not express genes nor did they have cell markers for B-cells, T-cells, monocytes/macrophages or neutrophils as shown by qRT-PCR, flow cytometry and immunoblotting. A remarkable feature of these cells is their potent phagocytic capacity. Their oxygen-independent killing mechanism, as shown by intense acid phosphatase staining, is supported by lack of respiratory burst and myeloperoxidase activity and the acid phosphatase''s sensitivity to tartrate. They show a high level of morphological plasticity, as, upon stimulation with mitogens, they change morphology and obtain branching protrusions similarly to dendritic cells. We suggest, based on our findings, that the small, round cells described here are progenitor cells with potential to differentiate into dendritic like cells, although we can not exclude the possibility that they represent a novel cell type.     

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.     

Identification of genetic loci implicated in the survival of Mycobacterium smegmatis in human mononuclear phagocytes

A luminescence-based procedure that permits the rapid evaluation of the survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte-derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstrated impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the gene interrupted by the transposition event was amplified by ligand-mediated PCR and sequenced. In all cases, the existence of homologous genes of as yet unknown function were identified in the Mycobacteium tuberculosis genome. Complementation of the mutant mycobacterial strains with cosmids containing the homologous loci from M. tuberculosis restored normal intracellular growth in three of the four mutants tested, supporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of directly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mononuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents.     

The anatomy of peripheral lymphoid organs with emphasis on accessory cells: light-microscopic immunocytochemical studies of mouse spleen, lymph node, and Peyer's patch

Antigen, lymphocytes, and accessory cells interact within peripheral lymphoid organs to generate immunity. Two cell types have been studied for accessory function in culture: mononuclear phagocytes and nonphagocytic Ia-rich dendritic cells. The monoclonal antibodies which have been used to study isolated murine macrophages (M phi) and dendritic cells (DC) include alpha-macrophage (F4/80, M1/70), alpha-dendritic cell (33D1), alpha-Fc receptor (2.4G2), and alpha-Ia (B21-2) reagents. In this paper, the antibodies have been used to stain accessory cells in cryostat sections of mouse spleen, lymph node, and Peyer's patch. Each organ is known to contain subregions that are rich in either macrophages, B cells, or T cells. We found that the accessory cells in each subregion had a different phenotype. 1) Macrophage-rich regions: Macrophages that lined the site of antigen delivery (marginal zone of spleen, around afferent lymphatics of node, and below the epithelium of Peyer's patch) were stained with M1/70 but not with F4/80. F4/80 was abundant on macrophages in other sites: spleen red pulp, node medulla, and around Peyer's patch efferent lymphatics. 2) B-lymphocyte-rich follicles: Follicular dendritic cells, which retain immune complexes extracellularly, are concentrated on the outer aspect of the germinal center. This region stained strongly with alpha-Fc receptor antibody 2.4G2, but not with M1/70, F4/80, or 33D1. 3) T areas: The interdigitating cells of T areas have been linked to isolated dendritic cells. Irregular Ia-rich cells were distributed uniformly in the T areas of each organ. However, staining with 33D1 was not detected and was restricted to foci of nonphagocytic cells at the spleen red/white pulp junction. F4/80, M1/70 or 2.4G2 also did not stain the T area, except for the region close to splenic central arteries. Therefore the principal surface markers and locations of the candidate accessory cells in murine lymphoid organs are M1/70+ macrophages at the site of antigen entry; F4/80+ macrophages around regions of lymphocyte efflux; germinal center dendritic cells, which may be rich in 2.4G2; and Ia-rich interdigiting cells in the T area.     

DCIR3 and DCIR4 are widely expressed among tissue-resident macrophages with the exception of microglia and alveolar macrophages

Dendritic cell inhibitory receptor 3 (DCIR3, Clec4a3) and dendritic cell inhibitory receptor 4 (DCIR4, Clec4a1) are C-type lectin receptors that belong to mouse dendritic cell immunoreceptor (DCIR) family. We recently showed that DCIR3 and DCIR4 are co-expressed on inflammatory and patrolling monocytes. In this study, we investigated the expression of DCIR3 and DCIR4 on tissue-resident macrophages. We found that spleen red pulp macrophages, liver Kupffer cells, large and small peritoneal macrophages and small intestinal macrophages expressed both DCIR3 and DCIR4. By contrast, lung alveolar macrophages expressed DCIR3 but not DCIR4 and brain microglia expressed neither DCIR3 nor DCIR4. Considerable part of tissue-resident macrophages are derived from embryonic precursors. We, therefore, examined the expression of DCIR3 and DCIR4 on the embryonic precursors. Yolk-sac macrophages from embryonic day (E) 8.5 embryos expressed both DCIR3 and DCIR4, while DCIR3 and DCIR4 were expressed on subpopulations of fetal liver monocytes from E14.5 embryos. Our results, together with previous data, indicate that the expression of DCIR3 and DCIR4 is widely shared by mononuclear phagocytes, including monocytes and macrophages, and that the expression of DCIR3 and DCIR4 on the embryonic precursors are not always retained by their progenies, suggesting that expression of DCIR3 and DCIR4 on tissue-resident macrophages might be regulated by environment of the tissues where the embryonic precursors differentiate into macrophages.