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.     

Human mesenchymal stem cells inhibit the differentiation and effector functions of monocytes

Although monocytes represent an essential part of the host defence system, their accumulation and prolonged stimulation could be detrimental and may aggravate chronic inflammatory diseases. The present study has explored the less-understood immunomodulatory effects of mesenchymal stem cells on monocyte functions. Isolated purified human monocytes were co-cultured with human umbilical cord-derived mesenchymal stem cells under appropriate culture conditions to assess monocytes’ vital functions. Based on the surface marker analysis, mesenchymal stem cells halted monocyte differentiation into dendritic cells and macrophages and reduced their phagocytosis functions, which rendered an inability to stimulate T-cell proliferation. The present study confers that mesenchymal stem cells exerted potent immunosuppressive activity on monocyte functions such as differentiation, phagocytosis and Ag presentation; hence, they promise a potential therapeutic role in down-regulating the unwanted monocyte-mediated immune responses in the context of chronic inflammatory diseases.     

Could a B-1 Cell Derived Phagocyte “Be One” of the Peritoneal Macrophages during LPS-Driven Inflammation?

The inflammatory response is driven by signals that recruit and elicit immune cells to areas of tissue damage or infection. The concept of a mononuclear phagocyte system postulates that monocytes circulating in the bloodstream are recruited to inflamed tissues where they give rise to macrophages. A recent publication demonstrated that the large increase in the macrophages observed during infection was the result of the multiplication of these cells rather than the recruitment of blood monocytes. We demonstrated previously that B-1 cells undergo differentiation to acquire a mononuclear phagocyte phenotype in vitro (B-1CDP), and we propose that B-1 cells could be an alternative origin for peritoneal macrophages. A number of recent studies that describe the phagocytic and microbicidal activity of B-1 cells in vitro and in vivo support this hypothesis. Based on these findings, we further investigated the differentiation of B-1 cells into phagocytes in vivo in response to LPS-induced inflammation. Therefore, we investigated the role of B-1 cells in the composition of the peritoneal macrophage population after LPS stimulation using osteopetrotic mice, BALB/Xid mice and the depletion of monocytes/macrophages by clodronate treatment. We show that peritoneal macrophages appear in op/op((-/-)) mice after LPS stimulation and exhibit the same Ig gene rearrangement (VH11) that is often found in B-1 cells. These results strongly suggest that op/op((-/-)) peritoneal "macrophages" are B-1CDP. Similarly, the LPS-induced increase in the macrophage population was observed even following monocyte/macrophage depletion by clodronate. After monocyte/macrophage depletion by clodronate, LPS-elicited macrophages were observed in BALB/Xid mice only following the transfer of B-1 cells. Based on these data, we confirmed that B-1 cell differentiation into phagocytes also occurs in vivo. In conclusion, the results strongly suggest that B-1 cell derived phagocytes are a component of the LPS-elicited peritoneal macrophage population.     

Inhibition of macrophage phagocytosis by methylation inhibitors. Lack of correlation of protein carboxymethylation and phospholipid methylation with phagocytosis

Adenosine (Ado), deoxyadenosine (dAdo), and adenine arabinoside (AraA) inhibit the phagocytosis of IgG-coated erythrocytes and zymosan by resident and thioglycollate-elicited macrophages (thio-macrophages) in a dose-dependent and reversible manner. 3-Deazaadenosine (3cAdo) and adenine (Ade) also inhibit the phagocytosis by resident macrophages. Homocysteine thiolactonate (Hcy) potentiates the inhibition by Ado and 3cAdo while erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) potentiates the inhibition by Ado, dAdo and AraA. This inhibition has a very rapid onset and the drugs do not interfere with the binding of IgG-coated erythrocytes to macrophages. The combination of Ado, Hcy and EHNA does not appreciably affect the intracellular level of ATP and S-adenosyl-L-methionine (AdoMet) in thio-macrophages but causes accumulations of Ado and S-adenosyl-L-homocysteine (AdoHcy) up to 135 and 145 nmol/mg of protein, respectively. During phagocytosis reversal, Ado is metabolized within 15 min while AdoHcy decreases log-arithmically with a half-life of 50 min. Carboxymethylation and phospholipid methylation, however, resume about 60-90 min after phagocytosis has recovered, and thus cannot function as transmembrane signals for phagocytosis. Other evidence showing the lack of correlation between phagocytosis and carboxymethylation inhibition include 1) Ado + Hcy inhibit carboxymethylation much better than Ado + EHNA (91 versus 75%) in thio-macrophage, but the two combinations show comparable phagocytosis inhibition potency; 2) Ado + Hcy inhibit carboxymethylation almost as well as Ado + Hcy + EHNA, but the latter is a much more effective drug combination for phagocytosis inhibition; 3) Ade and 3cAdo, although inhibiting resident macrophage phagocytosis as well as Ado + EHNA + Hcy, are much weaker carboxymethylation inhibitors; 4) dAdo and AraA potently inhibit phagocytosis but not carboxymethylation. The difference in the apparent methylation levels is not due to changes in the specific activities of AdoMet, which decrease with a half-life of 88 min. Interestingly, after the initial lag phase of about 90 min after the initiation of inhibition reversal, carboxymethylation and phagocytosis increase in parallel. In a log-log plot of carboxymethylation, phospholipid methylation, or phagocytosis versus the intracellular AdoHcy accumulation, a linear relationship is obtained. It is possible that AdoHcy accumulation is responsible for phagocytosis inhibition but inhibits by a mechanism other than interfering with protein and lipid methylations.     

Repression of alpha 2-macroglobulin and stimulation of alpha 1-proteinase inhibitor synthesis in human mononuclear phagocytes by endotoxin

Mononuclear phagocytes are a bone-marrow-derived subgroup of white blood cells which circulate as monocytes and, after differentiation into macrophages, become resident in many tissues. By synthesizing the important proteinase inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor mononuclear phagocytes contribute to the control of proteolysis both in blood and tissues. Applying a culture system which enables human blood monocytes to differentiate into macrophages in vitro, synthesis of alpha 2-macroglobulin and alpha 1-proteinase inhibitor was studied. The normal course of monocyte-macrophage maturation is accompanied by a strong increase of specific alpha 2-macroglobulin synthesis and a concomitant slight decrease of alpha 1-proteinase inhibitor. alpha 2-Macroglobulin can be designated as a marker protein of the monocyte/macrophage differentiation. Endotoxin (Salmonella typhi) in a concentration as low as 100 ng/ml strongly represses alpha 2-macroglobulin synthesis both in monocytes and macrophages. Furthermore, endotoxin completely abolishes the induction of alpha 2-macroglobulin synthesis during the course of normal monocyte in vitro cultivation, indicating that endotoxin is a strong inhibitor of the monocyte-macrophage maturation. In contrast to alpha 2-macroglobulin, alpha 1-proteinase inhibitor synthesis is strongly stimulated by endotoxin in monocytes as well as in macrophages.     

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.     

Transmembrane potential responses during HL-60 promyelocyte differentiation

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC₂-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by K_Ca channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl⁻ conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions. © 1996 Wiley-Liss, Inc.     

Endogenous peroxidase activity in mononuclear phagocytes

The diaminobenzidine (DAB) technique has been used to visualize the subcellular localization of peroxidatic enzymes in mononuclear phagocytes. The latter cells are part of the mononuclear phagocyte system (MPS), which includes the monocytes in the bone marrow and blood, their precursors in the bone marrow, and the resident macrophages in the tissues. The DAB cytochemistry has revealed distinct subcellular distribution patterns of peroxidase in the mononuclear phagocytes. Thus the technique facilitates the identification of the various phagocyte types: Promonocytes contain peroxidase reaction in the nuclear envelope, endoplasmic reticulum, Golgi apparatus, and cytoplasmic granules. Monocytes exhibit the reaction product only in cytoplasmic granules. Most resident macrophages show the activity only in the nuclear envelope and endoplasmic reticulum. Furthermore, new phagocyte types have been detected based on the peroxidase cytochemistry. Intermediate cells between monocytes and resident macrophages contain reaction product in the nuclear envelope, endoplasmic reticulum and cytoplasmic granules. The resident macrophages can be divided into two subtypes. Most of them exhibit the pattern noted above. Some, however, are totally devoid of peroxidase reaction. Most studies on peroxidase cytochemistry of monocytes and macrophages agree that the peroxidase patterns reflect differentiation or maturation stages of one cell line. Some authors, however, still interpret the patterns as invariable characteristics of separate cell lines. As to the function of the peroxidase in phagocytes, the cytochemical findings imply that two different peroxidatic enzymes exist in the latter cells: one peroxidase is synthesized in the endoplasmic reticulum of promonocytes and transported to granules via the Golgi apparatus. The synthesis ceases when the promonocyte matures to the monocyte. Upon phagocytosis the peroxidase is discharged into the phagosomes. Biochemical and functional studies have indicated that this peroxidase (myeloperoxidase) is part of a microbicidal system operating in host defence mechanisms. The other enzyme with peroxidatic activity is confined to the nuclear envelope and endoplasmic reticulum of resident macrophages in-situ and of monocytes at early stages in culture. As suggested by the subcellular distribution, the inhibition by peroxidase blockers, and the localization during phagocytosis studies, the latter peroxidase is functionally different from the myeloperoxidase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipid droplet: A functionally active organelle in monocyte to macrophage differentiation and its inflammatory properties

Lipid droplets (LDs) perform several important functions like inflammatory responses, membrane trafficking, acts as secondary messengers, etc. rather than simply working as an energy reservoir. LDs have been implicated as a controlling factor in the progression of atherosclerosis followed by foam cell formation that derives from macrophages during the differentiation process. However, the role of LDs in monocyte differentiation or its further immunological function is still an area that mandates in-depth investigation. We report that LD dynamics is important for differentiation of monocytes and is absolutely required for sustained and prolonged functional activity of differentiated macrophages. In THP-1 cell line model system, we elucidated that increase in total LD content in monocyte by external lipid supplements, can induce monocyte differentiation independent of classical stimuli, PMA. Differential expression of PLIN2 and ATGL during the event, together with abrogation of de novo lipogenesis further confirmed the fact. Besides, an increase in LD content by free fatty acid supplement was able to exert a synergistic effect with PMA on differentiation and phagocytic activity compared to when they are used alone. Additionally, we have shown Rab5a to play a vital role in LDs biosynthesis/maturation in monocytes and thereby directly affecting differentiation of monocytes into macrophages via AKT pathway. Thus our study reveals the multi-faceted function of LDs during the process of monocyte to macrophage differentiation and thereby helping to maintain the functional activity.     

Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages

Fas (CD95, APO-1) is regarded as the prototypical cell death receptor of the TNFR superfamily. Fas-induced apoptosis is generally considered to be a noninflammatory process, contributing to the silent resolution of immune and inflammatory responses. However, accumulating evidence indicates that Fas may also induce cellular activation signals. We hypothesized that Fas could activate proinflammatory cytokine responses by normal human monocytes and macrophages. Monocytes were isolated by negative immunoselection from the PBMC fraction of venous blood from healthy volunteers, and monocyte-derived macrophages were cultivated in vitro. Both monocytes and monocyte-derived macrophages released TNF-alpha and IL-8 following Fas ligation, and conditioned medium from Fas-activated monocytes and macrophages induced the directed migration of neutrophils in a chemotaxis assay. Fas-induced monocyte cytokine responses were associated with monocyte apoptosis, nuclear translocation of NF-kappaB, and cytokine gene expression and were blocked by caspase inhibition but not by inhibition of IL-1beta signaling. In contrast, Fas-induced macrophage cytokine responses occurred in the absence of apoptosis and were caspase independent, indicating maturation-dependent differences in the Fas signaling pathways that lead to proinflammatory cytokine induction. Rather than contributing to the resolution of inflammation, Fas ligation on circulating monocytes and tissue macrophages may induce proinflammatory cytokine responses that can initiate acute inflammatory responses and tissue injury.     

Uptake of lactoferrin by mononuclear phagocytes inhibits their ability to form hydroxyl radical and protects them from membrane autoperoxidation.

Human mononuclear phagocytes do not contain the iron-binding protein lactoferrin that we have previously demonstrated inhibits the potential for human neutrophils to generate hydroxyl radical in the presence of an exogenous iron catalyst of the Haber-Weiss reaction. Previous work by other investigators has suggested that mononuclear phagocytes (monocytes and monocyte-derived macrophages (MDM] have the capacity to bind exogenous lactoferrin via lactoferrin-specific membrane surface receptors. Accordingly, we examined the possibility that uptake of iron-free (apo) lactoferrin by human mononuclear phagocytes could play a role in limiting the potential for generation of hydroxyl radical during the monocyte/MDM respiratory burst. When monocytes or MDM were incubated in the presence of apo-lactoferrin, cell-associated lactoferrin increased in proportion to the concentration of lactoferrin provided. Similar results were obtained with iron-loaded (diferric) milk lactoferrin. Consistent with the in vivo importance of these findings, we found that lactoferrin was intimately associated with human alveolar macrophages obtained by bronchoalveolar lavage. The fucose polymer fucoidan inhibited lactoferrin uptake whereas exogenous transferrin or MDM exposure to IFN-gamma was without effect. Scatchard binding analysis confirmed the presence of a lactoferrin-specific receptor with a calculated kDa of 3.56 x 10(-6) M and 3.4 x 10(7) binding sites per cell. Subcellular fractionation studies indicated that twofold more of the lactoferrin which became cell-associated over the 1-h incubation time could be found in the cytoplasmic fraction compared to the plasma membrane-containing fraction, consistent with previous evidence by others for internalization of lactoferrin by mononuclear phagocytes. When lactoferrin-loaded monocytes/MDM were incubated in lactoferrin-free media, evidence for release of lactoferrin was obtained by SDS-PAGE and immunoblot analysis, suggesting the presence of a recyclable pool of cell-associated lactoferrin. To assess the impact of lactoferrin loading on monocyte/MDM hydroxyl radical formation, lactoferrin-loaded phagocytes were stimulated with PMA in the presence of catalytic iron. Hydroxyl radical generation by lactoferrin-loaded cells was decreased to about 50% of control cells. Similarly, monocytes that had been lactoferrin-loaded demonstrated a 28% decrease in autooxidation of their membrane when stimulated in the presence of catalytic iron. These data suggest that lactoferrin binding may play an important role in maintaining optimal mononuclear phagocyte function and protecting adjacent tissue from untoward phagocyte-associated hydroxyl radical generation.     

Time dependence of transmembrane potential changes and intracellular calcium flux in stimulated human monocytes

An important characteristic of the functional differentiation of the blood monocyte is the development of its capacity to recognize and respond to stimuli. This ability is mediated to a large extent by specific receptor glycoproteins located on the cell surface. Stimulation of mononuclear phagocytes via these receptors results in a rapid rise in intracellular Ca++ concentration, accompanied or followed by a change in membrane potential, generation of oxidative products, degranulation, and effector functions such as phagocytosis, aggregation, or locomotion. While the development of these characteristics is difficult to characterize in vivo, several investigators have demonstrated in vitro changes in these cells that correlate with the development of effector function. To examine the mechanisms of specific membrane-stimulus interactions of monocytes as they differentiate into macrophage-like cells, we studied the responses of human monocytes and of monocytes incubated in serum-containing medium for up to 96 hr to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Freshly isolated monocytes exhibited little change in transmembrane potential following stimulation with an optimal concentration of peptide and underwent a significant increase only after 48 hr in culture. While constant resting intracellular Ca++ concentrations were maintained during the culture period, intracellular Ca++ levels following fMLP stimulation increased with with incubation in serum, for up to 96 hr. In contrast, fMLP-induced respiratory burst activity increased from 0 to 24 hr in culture; it remained elevated at 48 hr but declined again by 96 hr. Incubation of the cells for 24 hr increased their random (unstimulated) motility in modified Boyden chambers but did not alter the cells' directed (chemotactic) response to fMLP in comparison to the response of freshly isolated monocytes. Peptide binding to the cells did not increase during the incubation period, indicating that an increase in receptor number or in affinity for fMLP was not responsible for the enhanced responsiveness to fMLP as incubation time increased. These studies indicate that incubation of monocytes in serum-containing medium leads to a complex, altered series of responses to fMLP that correlate with the differentiation of the original monocytes in vitro and may relate to the in vivo differentiation of monocytes to macrophages.     

Cyclic nucleotides promote monocyte differentiation toward a DC-SIGN+ (CD209) intermediate cell and impair differentiation into dendritic cells

Recruitment of monocytes into tissues and their differentiation into macrophages or dendritic cells (DCs) depend on the microenvironment of the inflammatory site. Although many factors affecting this process have been identified, the intracellular signaling pathways implicated are poorly understood. We found that cyclic nucleotides regulate certain steps of monocyte differentiation into DCs. Increased levels of the cyclic nucleotides, cAMP or cGMP, inhibit differentiation of CD14(+)/CD1a(low) monocytes into CD14(-)/CD1a(high) DCs. However, DC-specific ICAM-3-grabbing nonintegrin (CD209) up-regulation was not affected by cyclic nucleotides, indicating that DC development was not blocked at the monocyte stage. Interestingly, Ag-presenting function was increased by cyclic nucleotides, as measured by the higher expression of MHC class II, CD86, and an increased ability to stimulate CD4(+) T cell proliferation in allogeneic MLRs. Although cyclic nucleotides do not completely block DC differentiation, they do block the ability of DCs to be induced to mature by LPS. Treatment during DC differentiation with either cAMP or cGMP analogues hampered LPS-induced expression of CD83, DC-LAMP, and CCR7 and the ability of DCs to migrate toward CCL19/macrophage-inflammatory protein 3beta. Interestingly, the induction of a CD16(+) subpopulation of cells was also observed. Thus, signals causing an increase in either cAMP or cGMP levels during monocyte recruitment to inflammatory sites may restrain the activation of acquired immunity by blocking DC development and migration to lymph nodes. At the same time, these signals promote development of an active intermediate cell type having properties between those of macrophages and DCs, which might contribute to the innate immune response in the periphery.     

Influence of tumor necrosis factor-alpha on the ability of monocytes and lymphocytes to destroy intraerythrocytic Plasmodium falciparum in vitro

It has been shown that administration of TNF-alpha causes an increase of survival of plasmodium-infected mice. However, this anti-parasitic effect cannot be reproduced in vitro upon direct incubation of the cytokine with the parasite. This suggests that TNF-alpha may act through modulation of some plasmodicidal mechanism not yet clarified. We evaluated the effect of exogenous TNF-alpha on the phagocytosis of Plasmodium falciparum-infected erythrocytes by monocytes and its influence on the ability of monocytes and lymphocytes to inhibit parasite growth. The capacity of endogenous TNF-alpha to influence the ability of monocytes to inhibit the parasite was also verified. We found that addition of 33 ng TNF-alpha/mL to cultures of human monocytes and P. falciparum-infected erythrocytes increased the phagocytic index from 3.8 to 7.8 in the presence of serum containing P. falciparum antibody. TNF-alpha increased the capacity of monocyte plus lymphocyte to inhibit parasite growth by about 3 times at 0.5 and 5 ng/mL. Sera from severely ill P. falciparum-infected individuals inhibited the parasite growth, but addition of anti-TNF-alpha antibody was unable to modify this inhibition. These data show that TNF-alpha can increase the phagocytic capacity. This was probably due to an increased expression of Fc receptors on monocytes or to the modulation of Fc receptor signaling pathways by signals originating from the binding of TNF-alpha to its receptors. TNF-alpha also acted on lymphocytes plus monocytes by increasing the inhibition of P. falciparum by a mechanism not related to phagocytosis. These findings suggest that TNF-alpha has a pleiotropic anti-malaria effect and that this protective effect depends on the interplay of different factors, such as monocytes/macrophages, lymphocytes, and antibodies, in addition to other cells and molecules.     

Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages

It is known that histamine suppresses gene expression and synthesis of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in human peripheral blood mononuclear monocytes (HPM) or alveolar macrophages via histamine H2 receptors. We investigated the effect of histamine and differentiation in macrophages on the expression and secretion of TNF-alpha, TNF-alpha-converting enzyme (TACE), and histamine H1 and H2 receptors by use of a leukemia cell line, U937, and HPM. Differentiation of U937 and HPM cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the H1 receptor expression and rather suppressed the H2 receptor, resulting in up-regulation of the histamine-induced expression and secretion of TNF-alpha, modulated via TACE. Therefore, histamine failed to inhibit up-regulated expression of TNF-alpha induced by LPS in macrophages. The switch from H2 to H1 receptors during differentiation in the monocyte/macrophage lineage could participate in the pathogenic processes of atherosclerosis and inflammatory reactions in the arterial wall.     

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.     

LILRA2 selectively modulates LPS-mediated cytokine production and inhibits phagocytosis by monocytes

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

Fibronectin-enhanced phagocytosis of an alternative pathway activator by human culture-derived macrophages is mediated by the C4b/C3b complement receptor (CR1)

We examined the ability of human monocytes and culture-derived macrophages under serum-free conditions to phagocytose desialated sheep erythrocytes (E), an activator of the alternative pathway of human complement. Freshly derived monocytes ingested desialated erythrocytes, but the degree of phagocytosis varied among individual donors. However, exposing the phagocyte to intact plasma fibronectin (Fn) had no effect on monocyte phagocytosis. Macrophages derived from monocytes in culture were far more efficient at ingesting desialated E, and the extent of phagocytosis was proportional to the degree of desialation. Although exposure of macrophages to substrate-bound Fn or fluid-phase Fn enhanced the phagocytosis of desialated E, pretreatment of desialated E with Fn did not enhance phagocytosis, demonstrating that Fn acted through an interaction with the macrophages. Fn-enhanced phagocytosis of desialated E was inhibited by treating macrophages with a monoclonal antibody to the C4b/C3b receptor (CR1), but not with a monoclonal antibody to the receptor for C3bi (CR3). Addition of cobra venom factor (CVF) to the macrophages also inhibited Fn-enhanced phagocytosis of desialated E. Phagocytosis of IgG-sensitized E, either in the absence or in the presence of Fn, was not significantly affected by anti-CR1 or CVF, demonstrating that these reagents did not lead to a general inhibition of phagocytosis. These experiments suggest that macrophages may deposit enough C3b onto desialated E to cause CR1-mediated phagocytosis in the presence of Fn. The ability of macrophages to opsonize and ingest foreign particles that activate complement may be critically important in areas of inflammation where concentrations of serum-derived specific opsonins may be inadequate.     

The cyclic AMP-Epac1-Rap1 pathway is dissociated from regulation of effector functions in monocytes but acquires immunoregulatory function in mature macrophages

cAMP mediates its intracellular effects through activation of protein kinase A (PKA), nucleotide-gated ion channels, or exchange protein directly activated by cAMP (Epac). Although elevation of cAMP in lymphocytes leads to suppression of immune functions by a PKA-dependent mechanism, the effector mechanisms for cAMP regulation of immune functions in monocytes and macrophages are not fully understood. In this study, we demonstrate the presence of Epac1 in human peripheral blood monocytes and activation of Rap1 in response to cAMP. However, by using an Epac-specific cAMP analog (8-CPT-2'-O-Me-cAMP), we show that monocyte activation parameters such as synthesis and release of cytokines, stimulation of cell adhesion, chemotaxis, phagocytosis, and respiratory burst are not regulated by the Epac1-Rap1 pathway. In contrast, activation of PKA by a PKA-specific compound (6-Bnz-cAMP) or physiological cAMP-elevating stimuli like PGE(2) inhibits monocyte immune functions. Furthermore, we show that the level of Epac1 increases 3-fold during differentiation of monocytes into macrophages, and in monocyte-derived macrophages cAMP inhibits FcR-mediated phagocytosis via both PKA and the Epac1-Rap1 pathway. However, LPS-induced TNF-alpha production is only inhibited through the PKA pathway in these cells. In conclusion, the Epac1-Rap1 pathway is present in both monocytes and macrophages, but only regulates specific immune effector functions in macrophages.