Bone Marrow-derived Mononuclear Phagocytes Research Articles

FTY-720P Suppresses Osteoclast Formation by Regulating Expression of Interleukin-6 (IL-6), Interleukin-4 (IL-4), and Matrix Metalloproteinase 2 (MMP-2).

BackgroundOsteoclast formation is closely related to the immune system. FTY720, a new immunosuppressive agent, has some functions in immune regulation. Its main active ingredients become FTY-720P in vivo by phosphorylation modification. The objective of this study was to determine the effects of FTY-720 with various concentrations on osteoclasts in vitro.Material/MethodsRAW264.7 cells and bone marrow-derived mononuclear phagocytes (BMMs) were treated with RANKL to obtain osteoclasts in vitro. To investigate the role of FTY-720 in osteoclast formation, trap enzyme staining was performed and the number of osteoclasts was counted. Bone slices were stained with methylene blue, we counted the number of lacunae after bone slices were placed into dishes together with osteoclasts, and we observed the effect and function of FTY-720 in osteoclasts induced by RAW264.7 cells and BMMs. Then, we used a protein array kit to explore the effects of FTY-720P on osteoclasts.ResultsThe results of enzyme trap staining and F-actin staining experiments show that, with the increasing concentration of FTY-720P, the number of osteoclast induced by RAW264.7 cells and BMMs gradually decreased (P<0.05), especially when the FTY-720P concentration reached 1000 ng/ml, and the number of osteoclasts formed was the lowest (P<0.05). With bone lacuna toluidine blue staining, the results also show that, with the increasing concentration of FTY-720P, the number of bone lacuna gradually decreased (P<0.05), and the number of lacunae is lowest when the concentration reached 800 ng/ml. Finally, protein array results showed that IL-4, IL-6, IL-12, MMP-2, VEGF-C, GFR, basic FGF, MIP-2, and insulin proteins were regulated after FTY-720P treatment.ConclusionsFTY-720P can suppress osteoclast formation and function, and FTY-720P induces a series of cytokine changes.

Immunopharmacological in vitro effects of Eleutherococcus senticosus extracts.

The objective of these investigations was to further elucidate the immunopharmacological profile of fluid extracts of Eleutherococcus senticosus and to identify the specific role of its characteristic eleutherosides B and E. An ethanolic dry extract of Eleutherococcus senticosus was used as starting material for the isolation of the eleutherosides B and E. Immunopharmacological studies included expression of major histocompatibility complex class I and II molecules by rat bone marrow-derived mononuclear phagocytes, human lymphocyte marker flow cytometry, and in vitro testing of human lymphocyte functions. In contrast to the isolated eleutherosides B and eleutherosides E and the re-mixed eleutherosides B and E, the whole ethanolic fluid extract of Eleutherococcus senticosus was able to induce and enhance interleukin-1 and interleukin-6 but not interleukin-2 production in vitro. The effective concentration of the whole ethanolic extract ranged from 1.0-0.1 mg/ml for the enhancement of interleuking-1 alpha production and 1.0-0.03 mg/ml for the enhancement of interleukin-6 production. It is concluded that the observed enhancing immunopharmacological activities on acute phase response mediators are best exhibited by the induction with whole ethanolic extracts whereas the species-specific and characteristic eleutherosides B and E are not associated with these activities.

Microglia in the CNS: Immigrants from another world

Microglia are immunocompetent cells of the brain that continuously survey their environment with highly motile extensions. Although first described almost a century ago, the developmental origin of microglia is still debated. Besides early myeolomonocytic cell populations that colonize the developing neuroectoderm already during embryogenesis, newly migrated myeloid cells are considered important disease modulators in the adult brain. Thus, understanding the mechanisms of myeloid cell recruitment from the periphery and their development into bone marrow-derived phagocytes in the adult brain is pivotal for manipulating immune cell entry into the CNS and potentially reducing disease burden. A major question is whether bone marrow-derived mononuclear phagocytes have similar features and fates as their endogenous counterparts. Significant advances in our understanding of microglia biology under physiological as well as under pathological conditions have been made in the last few years, especially by combining novel gene-targeting techniques that allow cell manipulation and trafficking analysis. These new aspects will be discussed in the present review.

The macrophage response to bacteria. Modulation of macrophage functional activity by peptidoglycan from Moraxella (Branhamella) catarrhalis.

Moraxella (Branhamella) catarrhalis organisms have been shown to be particularly efficient in inducing in a pure population of bone marrow-derived mononuclear phagocytes secretory and cellular activities. In the present study, the ability of peptidoglycan from this Gram-negative organism to trigger a macrophage response was compared with that elicited by peptidoglycan from Staphylococcus aureus and Bacillus subtilis. The results show that the three peptidoglycans were similarly active in triggering the secretion of tumour necrosis factor and tumouricidal activity but differed considerably in their ability to induce the generation of nitrite in macrophages; in this respect, peptidoglycan from M. catarrhalis was particularly potent. The impressive capacity of M. catarrhalis peptidoglycan to induce in low concentration the secretion of tumour necrosis factor and nitrite and tumouricidal activity may, in addition to its lipopolysaccharide, contribute to the extraordinary potential of this organism to trigger the functional activities of macrophages.

Regulation of the differentiation and function of osteoclasts.

The osteoclast is the cell that resorbs bone. It has been known for many years that its formation and function are regulated by cells of the osteoblastic lineage. Recently the molecular basis for this regulation was identified; osteoblastic cells induce osteoclastic differentiation and resorptive activity through expression of tumour necrosis factor (TNF) activation-induced cytokine (TRANCE) (also known as RANKL, ODF, OPGL, and TNFSF11), a novel membrane-inserted member of the TNF superfamily. Osteoclastic regulation is assisted through secretion of an inhibitor, osteoprotegerin (OPG) (OCIF, TNFRSF11B), a soluble (decoy) receptor for TRANCE. Osteoclast formation and survival also depend on and are substantially enhanced by transforming growth factor-beta (TGF-beta), which is abundant in bone matrix. Surprisingly, not only TRANCE but also TNF-alpha can induce osteoclast formation in vitro from bone marrow-derived mononuclear phagocytes, especially in the presence of TGF-beta. Whether or not TNF-alpha does the same in vivo, its ability to generate osteoclasts in vitro has significant implications regarding the nature of osteoclasts and their relationship to other mononuclear phagocytes, and a possible wider role for TRANCE in macrophage pathobiology. A hypothesis is presented in which the osteoclast is a mononuclear phagocyte directed towards a debriding function by TGF-beta, activated for this function by TRANCE, and induced to become specifically osteoclastic by the characteristics of the substrate or signals from bone cells that betoken such characteristics.

Macrophage Response to Microbial Pathogens: Modulation of the Expression of Adhesion, CD14, and MHC Class II Molecules by Viruses, Bacteria, Protozoa and Fungi

The ability of inactivated viruses, bacteria, protozoa and fungi to modulate the expression of CD14, CD49d, CD49f, CD11a (LFA-1), and CD54 (ICAM-1) molecules in unprimed bone marrow-derived mononuclear phagocytes (BMM phi) was investigated by means of flow cytometry. Incubation with bacterial agents resulted in the large majority of experimental situations in enhanced expression of these macrophage surface molecules. In contrast, viruses and fungi down-regulated the expression of several adhesion receptors, especially integrins. Amplification of MHC class II expression triggered in macrophages by interferon gamma was clearly inhibited by viruses, bacteria, protozoa and fungi. The findings explain earlier results showing that, under the same experimental conditions, bacterial agents are, for the most part, potent stimulators of secretory and cell-mediated macrophage activities while viruses, protozoa and fungi are poor in this respect.

Coordinate Up- and Down-Modulation of Inducible Nitric Oxide Synthase, Nitric Oxide Production, and Tumoricidal Activity in Rat Bone Marrow-Derived Mononuclear Phagocytes by Lipopolysaccharide and Gram-Negative Bacteria

Simultaneous incubation of primary rat bone-marrow-derived mononuclear phagocytes (BMMø) and tumor cells with gram-negative agents triggers within 24 h interferon γ (IFNγ)- and tumor necrosis factor (TNFα)-independent tumoricidal activity. On the other hand, BMMø that had been incubated for 24 h with gram-negative agents prior to re-exposure to the same agent had largely lost their ability to generate tumoricidal activity, although their ability to bind lipopolysaccharide (LPS) was not diminished. Parallel measurements of the kinetics of inducible nitric oxide synthase (iNOS), nitrite secretion, and tumoricidal activity triggered in primary BMMø by LPS revealed that these parameters take a coordinate course, reaching a peak within 24 h and then rapidly decaying. Down-regulation of expression of NOS protein and iNOS activity could be attributed neither to down-regulation of LPS receptors nor to L-arginine depletion.

Macrophage Response to Viruses, Protozoa, and Fungi: Secretory and Cellular Activities Induced in Resting Unprimed Bone Marrow-Derived Mononuclear Phagocytes

The secretory (tumor necrosis factor, TNF-α; nitrite) and cellular response (mitochondrial respiration, TNF-α-independent tumoricidal activity) of a pure, lymphocyte-free population of resting, unprimed rat bone marrow-derived mononuclear phagocytes (BMMø) to direct interaction with viruses, protozoa, and fungi was assessed and compared with that triggered by bacterial agents and interferon-γ (IFN-γ). Viruses (herpes simplex, vaccinia, poliomyelitis, vesicular stomatitis, lymphocytic choriomeningitis, Sendai), protozoa (Trypanosoma brucei, Giardia lamblia), and fungi (Penicillium, Trichosporon, Fusarium, Rhizopus, Aspergillus, Geotrichum species) affected primarily the secretion of TNF-α and mitochondrial respiration of BMMø; their effects on the secretion of nitrite and on tumoricidal activity were at best marginal. Collectively, the macrophage response to viruses, protozoa, and fungi was less varied and less marked than that to bacterial agents (intact organisms, peptidoglycan, lipoteichoic acid, lipopolysaccharide) and IFN-γ.

Rapamycin but not FK506 inhibits the proliferation of mononuclear phagocytes induced by colony-stimulating factors.

FK506, CsA, and rapamycin are potent inhibitors of T lymphocyte activation; relatively little is known of their effects on cells of the monocyte/macrophage lineage. Studies were undertaken to determine the effects of these drugs on the proliferative response of bone marrow-derived mononuclear phagocytes (BMMP) to CSFs. Rapamycin inhibited the proliferation of BMMP cultured in the presence of 10% L cell-conditioned medium, used as a source of macrophage CSF. The inhibition by rapamycin was dose dependent and apparent at concentrations of 0.1 nM or greater. In a similar fashion, rapamycin inhibited the proliferation of BMMP stimulated by the recombinant forms of murine IL-3 and murine granulocyte-macrophage CSF, and human macrophage CSF. In contrast, neither FK506 nor CsA at concentrations as high as 1000 nM diminished the proliferation of BMMP cultured under identical conditions. FK506, but not CsA, blocked the inhibitory effects of rapamycin on the response of BMMP to CSFs. In summary, these data indicate that rapamycin inhibits the proliferation of BMMP in response to CSFs. These results imply that patients receiving rapamycin, but not FK506 or CsA, may have an impaired ability to generate a functional mononuclear phagocyte population.

Macrophage colony-stimulating factor and the enhanced migration of monocytes are essential in primary but not secondary host defenses to Listeria organisms.

Nonimmune mice infected with Listeria monocytogenes exhibited elevated expression of macrophage colony-stimulating factor (M-CSF) mRNA and the enhanced migration of Mac-1 antigen-positive bone marrow-derived mononuclear phagocytes (BMMP) to their livers. Treatment with monoclonal anti-M-CSF antibody diminished the traffic of BMMP and promoted the replication of listeriae. Immune animals infected with listeriae expressed significantly lower levels of M-CSF mRNA than did nonimmune animals. Moreover, listerial infections did not elicit the migration of BMMP to the livers of immune mice, nor did anti-M-CSF affect the capacity of immune animals to respond to infection. Adoptive immunization experiments suggest that T lymphocytes can mediate protective immunity to listeriae in the absence of M-CSF and migrating BMMP. These findings indicate that M-CSF and the enhanced migration of BMMP are critical factors in primary but not secondary host defenses to listerial infections.

Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids.

Lipoteichoic acids (LTAs) from various bacterial species, including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, and Listeria monocytogenes, were examined for the ability to induce secretory and cellular responses in a pure population of bone marrow-derived mononuclear phagocytes. Some of the highly purified LTAs, in particular LTAs from Bacillus subtilis, S. pyogenes, E. faecalis, and Enterococcus hirae, were able to affect each of the macrophage parameters measured, i.e., reductive capacity, secretion of tumor necrosis factor and nitrite, and tumoricidal activity. As after stimulation with whole organisms or other bacterial products, secretion of tumor necrosis factor induced by these LTAs reached its maximum within the first few hours of the interaction, while secretion of nitrite and tumoricidal activity required 24 to 36 h for full expression. Other purified LTAs, i.e., LTAs from Streptococcus sanguis, S. pneumoniae, and L. monocytogenes, as well as lipomannan from Micrococcus luteus affected only some of these parameters, while native LTA from S. aureus was inactive. There was no obvious correlation between biological activity and chain length, kind of glycosyl substituents, glycolipid structures, or fatty acid composition of LTAs. Deacylation of LTAs resulted in a complete loss of activity, and deacylated LTAs did not impair the activity of their acylated counterparts, suggesting that acyl chains may be essential for binding of LTA to the cell surface. The results demonstrate that some LTA species are potent inducers of macrophage secretory and cellular activities.

Induction of nitric oxide synthase is a necessary precondition for expression of tumor necrosis factor-independent tumoricidal activity by activated macrophages

Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuclear phagocytes (BMMø) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2-), the flow of L-arginine to citrulline, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO2- on the one hand and expression of tumoricidal activity were not always closely related; however, these parameters were suppressed in a dose-dependent manner by the flavoprotein inhibitor, diphenyleneiodonium (DPI) and the L-arginine analogue, NG-monomethyl-L-arginine (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor-independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the lytic principle from the effector to the target cell remain to be elucidated.

The interaction of macrophages and bacteria: Escherichia coli species, bacterial lipopolysaccharide, and lipid A differ in their ability to induce tumoricidal activity and the secretion of reactive nitrogen intermediates in macrophages

The ability of nine Escherichia coli strains, and of bacterial lipopolysaccharide (LPS) 3 3 Abbreviations used: BMMø, bone marrow-derived mononuclear phagocytes; FCS, fetal calf serum; IFN, Interferon; IMDM, Iscove's modified Dulbecco medium; KDO, 2-keto-3-deoxyoctulosonic acid; LPS, lipopolysaccharide; NMMA, N G-monomethyl- l-arginine; NO, nitric oxide; NO − 2 , nitrite; RNI, reactive nitrogen intermediates. and lipid A preparations, to elicit in a pure population of bone marrow-derived mononuclear phagocytes (BMMø) tumoricidal activity and/or the generation of reactive nitrogen intermediates (RNI) was compared. Generally, low concentrations of E. coli organisms were able to trigger the generation of RNI; however, for induction of tumoricidal activity, higher concentrations were required. Nonisogenic E. coli species exhibited different ability; isogenic E. coli organisms that differed only in the expression of K antigen exhibited similar ability to elicit the macrophage activities. LPS proved to be highly efficient in triggering the secretion of reactive nitrogen intermediates; lipid A was clearly less potent, but evidence is presented to suggest that this was due to the diminished solubility of these reagents. On the other hand, all LPS and lipid A samples were very poor inducers of tumoricidal activity. Although RNI secretion and expression of tumoricidal activity are both strongly dependent on l-arginine, various evidence suggests that the two functions are not closely correlated and are induced by different bacterial structures.

Modulation of tumoricidal activity, induced in bone‐marrow‐derived mononuclear phagocytes by interferon γ or Corynebacterium parvum, by interferon β, tumor necrosis factor, prostaglandin E2, and transforming growth factor β

Among a series of agents, including various interleukins and growth factors, only interferon gamma (IFN gamma) and heat-killed Corynebacterium parvum (CP) organisms were able to elicit, within 24 hr, tumoricidal activity in bone-marrow-derived mononuclear (BMM) phagocytes. In subsequent experiments, the abilities of interferon beta (IFN beta), tumor necrosis factor alpha (TNF alpha), prostaglandin E2 (PGE2), and transforming growth factor beta (TFG beta), alone or in combinations of 2, to modulate tumoricidal activity triggered in BMM phagocytes by IFN gamma or CP, were compared. In concentrations secreted by macrophages under physiological conditions, these agents proved potent in modulating induction and/or expression of tumoricidal activity. However, their ability to interfere with tumoricidal activity varied considerably, depending on the extent of macrophage differentiation and/or functional responsiveness, the pathway of macrophage activation, the type, concentration and combination of the macrophage secretory molecules, and on whether the agents were present during induction and expression or only during expression of tumoricidal activity. In showing that IFN beta and TNF alpha were mostly enhancing and TGF beta mostly suppressive, whereas PGE2 suppressed induction but enhanced expression of tumoricidal activity, our findings provide further support for the concept that these macrophage-derived molecules have a key role in autocrine regulation of macrophage functional activities.

The macrophage response to bacteria: Flow of L-arginine through the nitric oxide and urea pathways and induction of tumoricidal activity

The consequences of the interaction of heat-killed bacteria and lipopolysaccharide (LPS) with a pure population of bone marrow-derived mononuclear phagocytes (BMMø) were investigated, utilizing changes in the flow of L-arginine and expression of tumoricidal activity as parameters of macrophage (mø) function. Gram negative bacteria and LPS proved potent in inducing the flow of L-arginine through the nitric oxide and the urea path-ways but were mostly poor in eliciting tumoricidal activity. Gram positive bacteria affected the metabolism of L-arginine only little but were often efficient in triggering tumoricidal activity. The findings show that the mø response to bacteria, which may determine the outcome of their interaction with the host, may differ considerably depending on the type of bacteria.

The interaction of macrophages and bacteria: A comparative study of the induction of tumoricidal activity and of reactive nitrogen intermediates

The abilities of various bacteria to induce in a pure population of bone marrow-derived mononuclear phagocytes (BMMφ) tumoricidal activity and/or the generation of reactive nitrogen intermediates (RNI) were comparatively assessed. Interaction of (BMMφ) with bacteria led to expression of these functional activities, indicating that the organisms were recognized as foreign. As the majority of bacteria elicited in (BMMφ) either tumoricidal activity (that is maintained for days) or the production of RNI, measured by the release of nitrite (that is short-lived), it appears that the two functions are under separate control. However, both functions are inhibited or even abrogated by arginase or the l-arginine analogue, N G-monomethyl- l-arginine, suggesting that their expression is dependent on l-arginine.

Expression of cellular effector functions and production of reactive nitrogen intermediates: A comparative study including T lymphocytes, T-like cells, neutrophil granulocytes, and mononuclear phagocytes

The ability of various cell types to secrete reactive nitrogen intermediates (RNI) upon functional activation was comparatively assessed. Neither in T lymphocyte clones mediating MHC class I or class II antigen-restricted killing via α/β T cell receptor (TcR) or MHC-unrestricted killing via γ/δ TcR, nor in peripheral blood mononuclear cells expressing natural killer or lymphokine-activated killer activity, target cell lysis was associated with detectable RNI production. Also, activated neutrophil granulocytes did not secrete RNI. In contrast, bone marrow-derived mononuclear phagocytes, activated to express tumoricidal activity, secreted marked RNI activity.

Mechanisms of macrophage‐mediated tumor cell killing: A comparative analysis of the roles of reactive nitrogen intermediates and tumor necrosis factor

The roles of tumor necrosis factor (TNF alpha) and reactive nitrogen intermediates (RNI) as effectors of macrophage-mediated tumor cell killing were investigated in a variety of tumor cell lines. Three TNF alpha-sensitive tumor targets were also susceptible to resting bone-marrow-derived mononuclear phagocytes (BMMP). This macrophage lytic activity was markedly diminished or even abolished by anti-TNF alpha, indicating that TNF alpha is the major effector of macrophage-mediated killing of these targets. The other 21 tumor cell lines examined were resistant to TNF alpha but, in their large majority, were more or less susceptible to killing by interferon gamma (IFN gamma)- and Corynebacterium parvum (CP)-activated BMMP. Among the various analogues of L-arginine used to assess the role of L-arginine-derived RNI as mediators of macrophage tumoricidal activity, NG-monomethyl-L-arginine (NMMA) was most efficient in suppressing RNI secretion by activated macrophages. In some macrophage tumor-cell combinations, NMMA inhibited both the generation of RNI and the expression of tumoricidal activity in a dose-dependent manner, suggesting a central role for RNI as effectors. In other combinations, NMMA in concentrations that abolished secretion of RNI either affected tumor-cell killing only after its induction by IFN gamma, or not at all. The findings not only support the thesis that macrophages posses various means of coping with tumor cells but also suggest that the mechanism becoming operative is determined predominantly by the pathway of macrophage activation and the properties of the tumor-cell type.

Direct Stimulation of Cells Expressing Receptors for Macrophage Colony-Stimulating Factor (CSF-1) by a Plasma Membrane-Bound Precursor of Human CSF-1

Secreted forms of macrophage colony-stimulating factor (M-CSF or CSF-1) are generated by proteolytic cleavage of membrane-bound glycoprotein precursors. Alternatively spliced transcripts of the human CSF-1 gene encode at least two different transmembrane precursors that are differentially processed in mammalian expression systems. The larger precursor rapidly undergoes proteolysis to yield the secreted growth factor and does not give rise to forms of CSF-1 detected on the cell surface. By contrast, the smaller human CSF-1 precursor is stably expressed on the plasma membrane where it is inefficiently cleaved to release a soluble molecule. To determine whether the smaller precursor is biologically active on the cell surface, mouse NIH-3T3 fibroblasts expressing the different forms of human CSF-1 were killed by chemical fixation and tested for their ability to support the proliferation of cells that require this growth factor. Only fixed cells expressing human CSF-1 precursors on their surface stimulated the growth in vitro of a murine macrophage cell line or normal mouse bone marrow-derived mononuclear phagocytes. The ability of these nonviable fibroblasts to induce the proliferation of CSF-1-dependent cells was not mediated by release of soluble growth factor, required direct contact with the target cells, and was blocked by neutralizing antiserum to CSF-1. These results demonstrate that the cell surface form of the human CSF-1 precursor is biologically active and indicate that plasma membrane-bound growth factors can functionally interact with receptor-bearing targets by direct cell-cell contact.

Direct stimulation of cells expressing receptors for macrophage colony- stimulating factor (CSF-1) by a plasma membrane-bound precursor of human CSF-1

Secreted forms of macrophage colony-stimulating factor (M-CSF or CSF-1) are generated by proteolytic cleavage of membrane-bound glycoprotein precursors. Alternatively spliced transcripts of the human CSF-1 gene encode at least two different transmembrane precursors that are differentially processed in mammalian expression systems. The larger precursor rapidly undergoes proteolysis to yield the secreted growth factor and does not give rise to forms of CSF-1 detected on the cell surface. By contrast, the smaller human CSF-1 precursor is stably expressed on the plasma membrane where it is inefficiently cleaved to release a soluble molecule. To determine whether the smaller precursor is biologically active on the cell surface, mouse NIH-3T3 fibroblasts expressing the different forms of human CSF-1 were killed by chemical fixation and tested for their ability to support the proliferation of cells that require this growth factor. Only fixed cells expressing human CSF-1 precursors on their surface stimulated the growth in vitro of a murine macrophage cell line or normal mouse bone marrow-derived mononuclear phagocytes. The ability of these nonviable fibroblasts to induce the proliferation of CSF-1-dependent cells was not mediated by release of soluble growth factor, required direct contact with the target cells, and was blocked by neutralizing antiserum to CSF-1. These results demonstrate that the cell surface form of the human CSF-1 precursor is biologically active and indicate that plasma membrane- bound growth factors can functionally interact with receptor-bearing targets by direct cell-cell contact.