CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation

The macrophage fusion receptor (MFR), also called P84/BIT/SIRPalpha/SHPS-1, is a transmembrane glycoprotein that belongs to the superfamily of immunoglobulins. Previously, we showed that MFR expression is highly induced at the onset of fusion in macrophages, and that MFR appears to play a role in macrophage-macrophage adhesion/fusion leading to multinucleation. The recent finding that IAP/CD47 acts as a ligand for MFR led us to hypothesize that it interacts with CD47 at the onset of cell-cell fusion. CD47 is a transmembrane glycoprotein, which, like MFR, belongs to the superfamily of immunoglobulins. We show that macrophages express the hemopoietic form of CD47, the expression of which is induced at the onset of fusion, but to a lower level than MFR. A glutathione S-transferase CD47 fusion protein engineered to contain the extracellular domain of CD47, binds macrophages, associates with MFR, and prevents multinucleation. CD47 and MFR associate via their amino-terminal immunoglobulin variable domain. Of the nine monoclonal antibodies raised against the extracellular domain of CD47, three block fusion, as well as MFR-CD47 interaction, whereas the others have no effect. Together, these data suggest that CD47 is involved in macrophage multinucleation by virtue of interacting with MFR during adhesion/fusion.     

Species differences of macrophage very low-density-lipoprotein (VLDL) receptor protein expression

Triglyceride-rich lipoproteins (TGRLs) and low-density-lipoprotein (LDL) cholesterol are independent risk factors for coronary artery disease. We have previously proposed that the very low-density-lipoprotein (VLDL) receptor is one of the receptors required for foam cell formation by TGRLs in human macrophages. However, the VLDL receptor proteins have not been detected in atherosclerotic lesions of several animal models. Here we showed no VLDL receptor protein was detected in mouse macrophage cell lines (Raw264.7 and J774.2) or in mouse peritoneal macrophages in vitro. Furthermore, no VLDL receptor protein was detected in macrophages in atherosclerotic lesions of chow-fed apolipoprotein E-deficient or cholesterol-fed LDL receptor-deficient mice in vivo. In contrast, macrophage VLDL receptor protein was clearly detected in human macrophages in vitro and in atherosclerotic lesions in myocardial infarction-prone Watanabe-heritable hyperlipidemic (WHHLMI) rabbits in vivo. There are species differences in the localization of VLDL receptor protein in vitro and in vivo. Since VLDL receptor is expressed on macrophages in atheromatous plaques of both rabbit and human but not in mouse models, the mechanisms of atherogenesis and/or growth of atherosclerotic lesions in mouse models may be partly different from those of humans and rabbits.     

Macrophage fusion: are somatic and cancer cells possible partners?

Macrophages are present in all tissues and can fuse with themselves to differentiate into multinucleate osteoclasts or giant cells that play a central role in osteoporosis and chronic inflammatory diseases, respectively. Yet, the mechanism by which they fuse remains uncharacterized. The macrophage fusion receptor (MFR) and its ligand CD47 might mediate homotypic fusion of macrophages and allow for their recognition as 'self' before fusion. Although a novel process and controversial idea, macrophages might exploit a similar mechanism for fusion with somatic cells or tumor cells, with resultant organ repair or metastasis, respectively. Hence, macrophages might be the 'double-edged swords' of tissues.     

Expression of the transferrin receptor on murine peritoneal macrophages is modulated by in vitro treatment with interferon gamma

The expression of transferrin receptors on murine peritoneal macrophages has been shown to be down regulated during functional activation in vivo. This observation suggested that the level of transferrin receptor expression varies in response to discrete extracellular signals known to induce macrophage activation. We have tested this concept directly and have shown that decreased transferrin receptor expression can be reproduced in vitro by treatment of inflammatory macrophages with preparations of interferon gamma derived from a T cell hybridoma supernatant. The ability of this agent to down regulate the expression of the transferrin receptor exhibited dose and time dependencies similar to those required for development of other macrophage functions in vitro. The addition of LPS produced no further decrease in receptor expression. Furthermore, murine gamma interferon, produced by recombinant DNA technology also caused a downshift in transferrin receptor expression at doses similar to those which have been shown previously to induce activation. The changes in receptor activity were the result of altered numbers of binding sites and the receptor:ligand affinity remained unaffected. These results indicate that altered expression of the transferrin receptor is one element of the pleiotypic change which macrophages undergo in response to IFN gamma. This system may, therefore, provide a useful model in which to study the biochemical basis of IFN gamma action in mononuclear phagocytes.     

Evidence that Leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism

The pathogenic protozoan Leishmania donovani must gain entrance into mononuclear phagocytes to successfully parasitize man. The parasite's extra-cellular promastigote stage is ingested by human peripheral blood monocytes or monocyte-derived macrophages in the absence of serum, in a manner characteristic of receptor-mediated endocytosis. We have found remarkable similarities between the macrophage receptor(s) for promastigotes and a previously characterized eucaryotic receptor system, the mannose/fucose receptor (MFR), that mediates the binding of zymosan particles and mannose- or fucose-terminal glycoconjugates to macrophages. Ingestion of promastigotes by monocyte-derived macrophages was inhibited by several MFR ligands. Mannan (2.5 mg/ml) decreased ingestion by 63.7% (p less than 0.001), and the neoglycoproteins mannose-BSA and fucose-BSA (20 micrograms/ml) inhibited parasite ingestion by 46.5% and 39.6%, respectively (p less than 0.04). In contrast, promastigote ingestion by monocytes was unaffected by MFR ligands. These results are consistent with reports that MFR activity is present in monocyte-derived macrophages but not in monocytes. Furthermore, attachment of promastigotes to macrophages, assessed by using cytochalasin D to prevent phagocytosis, was reduced 49.8% by mannan. Reorientation of the MFR to the ventral surface of the cell was achieved by plating macrophages onto mannan-coated coverslips, reducing MFR activity on the exposed cell surface by 94% as assessed by binding of 125I-mannose-BSA. Under these conditions, ingestion of promastigotes was inhibited by 71.4% (p less than 0.006). Internalization of the MFR by exposure of macrophages to zymosan before infection with promastigotes resulted in a 62.3% decrease in parasite ingestion (p less than 0.006). Additionally NH4Cl, a weak lysosomotropic base that impairs MFR recycling, decreased macrophage ingestion of promastigotes by 38.2% (p less than 0.03, 30 mM NH4Cl). Subinhibitory concentrations of NH4Cl (10 mM) and of mannan (0.25 mg/ml) together inhibited parasite ingestion by 76.4% (p less than 0.002). These studies suggest that L. donovani promastigotes may utilize a receptor system on human monocyte-derived macrophages, the MFR, to efficiently parasitize the human host.     

Neuropeptide Y and its receptor subtypes specifically modulate rat peritoneal macrophage functions in vitro: counter regulation through Y1 and Y2/5 receptors

It is well documented that neuropeptide Y (NPY) exerts a wide range of biological functions through at least five NPY Y receptor subtypes (Y1-Y5), but its immunological effects only recently came into focus. Using NPY family peptides and NPY-related receptor-specific peptides as well as Y1 and Y2 receptor antagonists, we have tested which NPY Y receptors are involved in NPY-induced modulation of rat peritoneal macrophage function in vitro. NPY and PYY increased oxidative burst in phorbol myristate acetate (PMA)-stimulated macrophages involving activation of protein kinase C (PKC), and decreased it in zymosan-stimulated cells resembling inhibition of signaling pathways subsequent to binding of zymosan particles for the iC3b fragment receptor on macrophages. The combined treatment with NPY and NPY Y receptor antagonists revealed that NPY-induced potentiation of oxidative burst in PMA-stimulated cells is mediated through Y1 and Y2 receptors, while NPY-induced suppression in zymosan-stimulated cells is mediated through Y2 receptors only. NPY-related peptides differently modulated macrophage function, confirming involvement of NPY Y2 receptor in both potentiation and suppression of oxidative burst in these cells. Additionally, it was shown that NPY Y5 receptor mediated suppression of oxidative burst in PMA- and zymosan-stimulated macrophages. Taken together, the present data reveal an NPY Y1 and Y2/Y5 receptor interaction in NPY-induced modulation of macrophage functions related to inflammation.     

Recent advances in understanding the mechanisms of osteoclast precursor fusion

Bone marrow macrophages fuse on the bone surface to form multinucleated osteoclasts that then organize to efficiently resorb bone. Many, if not all, of the stages of macrophage fusion involve cytoskeletal components that reorganize the cells. Recruitment may involve chemotactic responses to bone matrix protein and calcium ion gradients and/or chemokine production by bone forming osteoblasts. The roles of integrins vary, depending on the particular subunits with some interfering with fusion and others having a participatory role. RANKL is essential for fusion and many identified modulators of fusion influence RANKL signaling pathways. Tetraspanins have been implicated in fusion of macrophages and myoblasts, but differences in impacts exist between these two cell types. Macrophage recruitment to apoptotic cells prior to their engulfment is driven by the exposed phospholipids on the external surface of the apoptotic cells and there is evidence that this same identification mechanism is employed in macrophage fusion. Because loss of cadherin or ADAM family members suppresses macrophage fusion, a crucial role for these membrane glycoproteins is evident. The Ig membrane glycoprotein superfamily members CD200 and MFR/SIRPα are involved in macrophage fusion, although their influences are unresolved. Differential screenings have identified the structurally related membrane proteins DC‐STAMP and OC‐STAMP as required components for fusion and the contributions to fusion remain active areas of investigation. While many of the key components involved in these processes have been identified, a great deal of work remains in resolving the precise processes involved and the interactions between key contributors to multinucleated osteoclast formation. J. Cell. Biochem. 110: 1058–1062, 2010. Published 2010 Wiley‐Liss, Inc.     

Augmentation of macrophage complement receptor function in vitro. V. Studies on the mechanisms of ligation of macrophage Fc receptors required to trigger macrophages to signal T lymphocytes to elaborate the lymphokine that activates macrophage C3 receptors for phagocytosis

We previously described a unique lymphokine that activates macrophage C3 receptors for phagocytosis. The lymphokine is generated when T lymphocytes receive a signal from macrophages that have ingested IgG-coated material. In the present work, we examined the mechanisms by which macrophage Fc receptors must be engaged for macrophages to signal lymphocytes to elaborate the lymphokine. We found that ingestion mediated by any of the three classes of murine macrophage Fc receptors was sufficient to trigger macrophages, and that engagement of macrophage Fc receptors by immobilized immune complexes was effective as well. We also found that ligation of Fc receptors by an anti-Fc receptor IgG antibody or by its F(ab')2 or Fab fragments also triggered macrophages. The ability of monovalent ligation of the receptor to elicit biologic activity suggests that this system may be of value in elucidating general mechanisms by which ligand binding of receptors is transduced into biologic effects.     

Secretion of ATP-utilizing enzymes,nucleoside diphosphate kinase and ATPase,by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?

Mycobacterium bovis BCG secretes two ATP-scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, during growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unless proteins such as bovine serum albumin (BSA), ovalbumin or extracts of macrophages are added to the medium. There is a gradient of activity among various proteins in triggering the induction of secretion of these two enzymes. Other mycobacteria, such as M. smegmatis, primarily secrete Ndk, while M. chelonae does not appear to secrete either of these two enzymes. Purification of the enzymes from the culture filtrate of 7H9-grown M. bovis BCG cells and determination of the N-terminal amino-acid sequence have demonstrated a high level of sequence identity of one of the ATPases with DnaK, a heat shock chaperone, of M. tuberculosis and M. leprae, while that of Ndk shows significant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological significance of the secretion of these two ATP-utilizing enzymes was explored. External ATP is important in the activation of macrophage surface-associated P2Z receptors, whose activation has been postulated to allow phagosome-lysosome fusion and macrophage cell death. We demonstrate that the presence of the filtrate containing these enzymes prevents ATP-induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase. In vitro complexation studies with purified Ndk/ATPase and hyperproduced P2Z receptor protein will demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome-lysosome fusion or macrophage apoptotic death.     

Leptin requires canonical migratory signaling pathways for induction of monocyte and macrophage chemotaxis

The growing worldwide obesity epidemic is frequently linked to an increased risk of developing diseases such as diabetes, cardiovascular disease, and cancer. These diseases are associated with the infiltration of macrophages in white adipose tissue (WAT), the artery wall, and tumors, respectively; and these macrophages likely contribute to disease progression and pathogenesis. Abdominal WAT, adipose tissue surrounding the heart and artery wall, as well as carcinoma cells, secrete many factors that could induce macrophage infiltration. Leptin is an adipocyte-secreted hormone, and deficiency of either leptin or its receptor has been shown to cause morbid obesity in animals and in humans. However, what is more commonly noted in human obesity is the presence of central leptin resistance leading to hyperleptinemia. As leptin receptors are present on macrophages, we hypothesized that leptin could act as a monocyte/macrophage chemoattractant. Our current study demonstrates: 1) leptin is a potent chemoattractant for monocytes and macrophages, inducing maximal chemotactic responses at 1 ng/ml; 2) leptin-mediated chemotaxis requires the presence of full-length leptin receptors on migrating cells; 3) leptin causes increased influx of intracellular calcium in macrophages; and 4) activation of janus kinase/signal transducers and activators of transduction (JAK/STAT), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) pathways are all necessary for leptin-induced macrophage migration. Taken together, these data demonstrate that leptin is a potent monocyte/macrophage chemoattractant in vitro and that canonical cell motility machinery is activated upon macrophage exposure to leptin. These data have implications for the impact of hyperleptinemia on obesity-related pathophysiological conditions such as diabetes, cardiovascular disease, and cancer.     

High concentrations of bacterial lipopolysaccharide, but not microbial infection-induced inflammation, activate macrophage C3 receptors for phagocytosis

Macrophage C3 receptors are normally immobilized in the plane of the cells' plasma membrane and are unable to promote phagocytosis even though they promote avid particle binding. We have previously identified a lymphokine that activates macrophage C3 receptors for phagocytosis both in vitro and in vivo, and others have found that certain types of nonimmunologically mediated inflammation are also able to activate mononuclear phagocyte C3 receptors. These findings raised the possibility that macrophage C3 receptor activation is a universal consequence of inflammation. We sought in the present experiments to determine whether or not inflammation induced by microbial infection in a nonimmune host resulted in activation of macrophage C3 receptors. We injected mice i.p. with either viable microorganisms, microbe-containing immune complexes, or bacterial LPS. Macrophages were harvested by peritoneal lavage 4 days later; nearly all lavage fluids grew the microorganism with which the mouse had been injected, indicating that an infection had been established. Monolayers of macrophages were established and their interaction with sheep E coated with C3 (EIgMC) was determined. All macrophages bound EIgMC, but only macrophages from mice injected with either very high concentrations of LPS or microbe-containing immune complexes ingested them. C3 receptors of macrophages that ingested EIgMC were mobile; others were not. Thus, inflammation induced by microbial infection does not commonly, if at all, activate macrophage C3 receptors; microbe-containing immune complexes and high concentrations of LPS do. The mechanism of receptor activation in each case is C3 receptor mobilization, which is probably mediated by a lymphokine.     

TRAIL (Apo2 ligand) and TWEAK (Apo3 ligand) mediate CD4+ T cell killing of antigen-presenting macrophages

The human marrow produces approximately 1010 monocytes daily, and this production must be balanced by a similar rate of destruction. Monocytes/macrophages can undergo apoptosis after activating CD4+ T cells, suggesting one mechanism that may contribute to macrophage homeostasis. Previous reports indicate that Fas-Fas ligand interactions are the principle molecules mediating this response. However, D10, an Iak-restricted cloned Th2 line, will similarly induce apoptosis in Ag-presenting macrophages, and D10 cells lack Fas ligand. To confirm that D10 cells kill macrophages through Fas-independent pathways, D10 cells were shown to kill MRL lpr/lpr (Iak) macrophages in an Ag-dependent fashion, indicating additional mechanisms. Recent reports demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), interacting with Apo2, and TNF-like weak inducer of apoptosis (TWEAK), interacting with Apo3, will induce apoptosis in some cells. Using Abs to TRAIL and an Apo3-IgG Fc fusion protein, we demonstrated that D10 cells express both TRAIL and TWEAK. The Apo3 fusion protein, but not human IgG, inhibited D10-induced macrophage apoptosis, as did anti-TRAIL. Further studies demonstrated that AE7, a cloned Th1 line, and splenic T cells express TWEAK, TRAIL, and Fas ligand, and inhibiting these molecules also inhibited macrophage killing. These results indicate that D10 cells induce macrophage apoptosis through TRAIL- and TWEAK-dependent pathways. Because normal T cells also express these molecules, these results support the concept that T cells have multiple pathways by which to induce macrophage apoptosis. These pathways may be important in immune processes such as macrophage homeostasis as well as in down-regulation of immune responses and elimination of macrophages infected with intracellular organisms.     

Macrophage Mannosyl Fucosyl Receptor: Its Role in Invasion of Virulent and Avirulent L. Donovani Promastigotes

The interaction of leishmania parasites with macrophages is known to be receptor mediated. Previous study from this laboratory (J. Parasitol. 82:632, 1996) showed the significant involvement of LPG and gp63 receptors in the recognition of virulent strains onto the macrophages. The role of carbohydrate receptors--the other major receptors besides LPG and gp63 receptors, in the recognition of both virulent (strains AG83 and GE1) and avirulent (strain UR6) leishmania onto the host macrophages has been the major focus of the present investigation. Various neoglycoproteins were used as efficient ligands to preblock the carbohydrate receptors on the macrophage surface. Similarly, various sugar specific lectins were used to preblock the corresponding carbohydrate ligands on the parasite surface. When these preblocked macrophages or parasites were used to study their mode of recognition, it was obvious from the findings that avirulent leishmania promastigotes possibly use the mannosyl fucosyl receptors (MFR) more avidly for their initial attachment and subsequent internalization into the macrophages whereas the virulent leishmania exhibits limited use of this receptor. When a macrophage-like cell line (J774), lacking in MFR, was purposely selected to test the previous findings, as expected, the attachment of avirulent promastigotes (UR6) onto the cell line was found to be negligible when compared to the peritoneal macrophages. Thus, it appears that avirulent leishmania promastigotes probably utilize MFR significantly for their initial recognition and subsequent internalization by macrophages.     

Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression.

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.     

Soluble extracellular domains of human SIRPα and CD47 expressed in Escherichia coli enhances the phagocytosis of leukemia cells by macrophages in vitro

Signal regulatory protein (SIRP) α, a transmembrane protein belonging to the immunoglobulin superfamily, is a receptor for CD47. The interaction between SIRPα and CD47 plays an important role in regulating the phagocytosis of leukemia cells and leukemia stem cells (LSCs) by macrophages. Blocking antibodies against CD47 have been shown to promote phagocytosis of LSCs by macrophages. Here, we consider an alternative way to interrupt the interaction between CD47 and SIRPα. We expressed the extracellular domains of the human SIRPα (hSIRP(ext)) and the human CD47 (hCD47(ext)) in Escherichia coli as Trx fusion proteins, and purified them by using affinity chromatography. We show that the purified fusion protein Trx-SIRP(ext) could interact in vitro with Trx-hCD47(ext). Moreover, Trx-SIRP(ext) could effectively bind to Jurkat T-ALL cells, which expressed CD47 at a high level. CD47(ext), on the other hand, bound to human macrophages. In vitro phagocytosis assay showed that these fusion proteins could enhance the phagocytosis of Jurkat cells by macrophage, with Trx-hSIRP(ext) showed a higher efficiency than Trx-CD47(ext). These results indicated that the soluble Trx-hSIRP(ext) and Trx-CD47(ext) polypeptides could be alternative molecules to interrupt CD47-SIRPα interaction between leukemia cells and macrophages, and might be potentially useful for the targeted therapy of leukemia.     

Macrophage Fusion Is Controlled by the Cytoplasmic Protein Tyrosine Phosphatase PTP-PEST/PTPN12

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.     

OxLDL-mediated survival of macrophages does not require LDL internalization or signalling by major pattern recognition receptors

Macrophages play a key role in the pathogenesis of atherosclerosis, in part by destabilizing plaques. We and others have shown that low concentrations of oxidized LDL (oxLDL) inhibit macrophage apoptosis. As oxLDL is present in lesions, this may be a mechanism by which macrophage populations in the intima are expanded. We have previously shown that oxLDL activates prosurvival signalling pathways such as the phosphoinositide 3-kinase (PI3K) pathway in bone marrow derived macrophages (BMDMs). However, little is known about more upstream signalling events especially at the receptor level. The endocytic pattern recognition receptors (PRRs), scavenger receptor A (SR-A) and CD36, are the main receptors on macrophages for uptake of oxLDL and are therefore important in foam cell formation. The signalling PRRs such as toll-like receptor (TLR) 2 and 4 also bind some types of oxLDL. This study was done to determine if any of the known PRRs are required for the anti-apoptotic effects of oxLDL in BMDMs. To do this, we tested the effect of oxLDL on viability of BMDMs lacking both SR-A and CD36 or lacking TLR2, TLR4, CD14, FcγRIIb, or RAGE. Our results indicate that none of these receptors are essential for activating the oxLDL prosurvival pathway. Furthermore, we show that the anti-apoptotic effect is not dependent on the uptake of oxLDL.     

PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically implicated in lung homeostasis in the GM-CSF knockout mouse model. These animals develop an isolated lung lesion reminiscent of pulmonary alveolar proteinosis (PAP) seen in humans. The development of the adult form of human alveolar proteinosis is not due to the absence of a GM-CSF gene or receptor defect but to the development of an anti-GM-CSF autoimmunity. The role of GM-CSF in the development of PAP is unknown. Studies in the GM-CSF knockout mouse have shown that lack of PU.1 protein expression in alveolar macrophages is correlated with decreased maturation, differentiation, and surfactant catabolism. This study investigates PU.1 expression in vitro and in vivo in human PAP alveolar macrophages as well as the regulation of PU.1 by GM-CSF. We show for the first time that PU.1 mRNA expression in PAP bronchoalveolar lavage cells is deficient compared with healthy controls. PU.1-dependent terminal differentiation markers CD32 (FCgammaII), mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR) are decreased in PAP alveolar macrophages. In vitro studies demonstrate that exogenous GMCSF treatment upregulated PU.1 and M-CSFR gene expression in PAP alveolar macrophages. Finally, in vivo studies showed that PAP patients treated with GM-CSF therapy have higher levels of PU.1 and M-CSFR expression in alveolar macrophages compared with healthy control and PAP patients before GM-CSF therapy. These observations suggest that PU.1 is critical in the terminal differentiation of human alveolar macrophages.