Secretion of a nonspecific lipid transfer protein by hepatoma cells in culture

Nonspecific lipid transfer protein (sterol carrier protein2) has previously been proposed to function as (i) a catalyst for intracellular movement of newly synthesized phospholipid, (ii) a cofactor in the biosynthesis and metabolism of cholesterol, and (iii) a cofactor in the feedback inhibition of cholesterol synthesis. Each of these functions is based upon the premise that nonspecific lipid transfer protein (nsLTP) is cytosolic. However, evidence presented in this report suggests that, at least in the case of cultured hepatoma cells, nsLTP is secreted. This conclusion is supported by three observations. First, after culture of hepatoma cells for 10 h, 88% of the nsLTP (as judged by its phosphatidylethanolamine transfer activity) appears in the medium, whereas the cytosolic level of transfer activity remains unchanged. Furthermore, this is accompanied by the appearance in the medium of a polypeptide of Mr 12,200-12,500, which corresponds to the known molecular weight of nsLTP. Finally, it was observed that the appearance of both the activity and the polypeptide in the medium are inhibited by monensin, an inhibitor of secretion. Thus their appearance seems to represent secretion and not simply leakage from the cells. Further evidence that nsLTP does not play an important role in the cytosolic transport of phospholipid and sterol is provided by our observation that hepatoma cells containing a level of nsLTP only 10-15% of that found in liver nevertheless possess near-normal membrane phospholipid compositions and retain the ability to feedback-inhibit cholesterol biosynthesis.     

Plasma apolipoprotein secretion by human monocyte-derived macrophages

Apolipoprotein E has been demonstrated to be a major secretory protein of human monocyte macrophages. The synthesis of the other plasma apolipoproteins by these cells has not been documented. Human monocyte macrophages cultured for 17-76 days were preincubated for 24 h in RPMI 1640/0.2% bovine serum albumin with or without malondialdehyde-LDL (100 micrograms/ml), followed by an additional 24 h incubation in RPMI 1640/0.2% bovine serum albumin. The media from the two incubation periods were analyzed for apolipoproteins A-I, B, C-II, C-III and E by specific radioimmunoassays. No apolipoprotein B mass was detected with a specific radioimmunoassay capable of detecting 10 ng apolipoprotein B. No apolipoproteins A-I, C-II or C-III mass was detected, even though the radioimmunoassays for these apolipoproteins were as sensitive as that for apolipoprotein E (detection limit of 0.2 ng). In contrast, significant levels of macrophage-secreted apolipoprotein E were quantified. Baseline apolipoprotein E production ranged from 0.64 to 2.82 micrograms/mg cell protein per 24 h. Preincubation in the presence of malondialdehyde-LDL (100 micrograms/ml) stimulated a 1.6-3.0-fold increase in apolipoprotein E secretion. The identification of the immunoreactive material as apolipoprotein E was confirmed by labelling the cells with [35S]methionine, followed by fractionation of the 35S-labelled secretory products by anti-apolipoprotein E affinity chromatography and SDS-gel electrophoresis. We thus report the absence of synthesis of apolipoproteins A-I, B, C-II and C-III by cultured human monocyte macrophages. These cells, however, can synthesize microgram levels of apolipoprotein E on a per mg protein basis.     

Quantifying complement-mediated phagocytosis by human monocyte-derived macrophages

The present study demonstrates that SRBC can be opsonized with untreated human serum such that lysis by active complement components is minimal but sufficient opsonization occurs to permit high rates of complement-mediated phagocytosis. Phagocytosis of SRBC opsonized with 2% whole human serum by human monocyte-derived macrophages was quantified in a colourimetric assay. Ingestion of SRBC was shown to occur solely via complement receptors because no phagocytosis was observed when SRBC were coated with heat- inactivated human serum, phagocytosis was augmented by the phorbol ester, PMA, and phagocytosis was inhibited by a protein kinase C (PKC)-specific inhibitor RO 31-8220. This method was used to demonstrate directly that HIV-1 infection of human monocyte-derived macrophages inhibits complement-mediated phagocytosis and will provide a useful tool for pharmacological investigations on complement-mediated phagocytosis by adherent macrophages.     

The secretion of apolipoprotein E by human monocyte-derived macrophages

The secretion of newly synthesized apolipoprotein E (apo E) by human monocyte-derived macrophages (HMD macrophages) was measured in the medium of cells which had been incubated for 24 h with or without either native or acetylated low-density lipoproteins (LDL or AcLDL, respectively), and subsequently with [35S]methionine in the presence of high-density lipoproteins (HDL, 350 micrograms/ml) for 24 h, by isolating the lipoprotein fraction by centrifugation for 48 h at a density adjusted with KBr to 1.21 g/ml (d = 1.21). The d less than 1.21 medium was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or precipitation with trichloroacetic acid (TCA). Fluorography of the gels demonstrated that the d less than 1.21 fraction of the medium contained one major labeled band, which migrated with an apparent molecular weight of approximately 35,000. Immunoprecipitation of the d less than 1.21 fraction showed that the labeled band was precipitated by anti-apo E, but not by anti-HDL. As the apo E band appeared to be the only labeled band in the d less than 1.21 fraction, the amount of apo E secreted by the cells was quantitated by scintillation counting of the TCA-precipitable radioactivity in the d less than 1.21 fraction as compared with that in the whole medium. The proportion of secreted apo E to the total secreted protein was similar whether the cells had been in culture for 3 or 16 days, but was increased if the cells had been incubated with LDL or AcLDL. The proportion of apo E of the secreted proteins was always more than 6% and was as much as 16% when the cells were preincubated with lipoproteins, suggesting that the increased cholesterol influx induced apo E secretion.     

Efficient gene transfer into human monocyte-derived macrophages using defective lentiviral vectors.

Gene therapy is a promising approach for the treatment of neurological disorders. However, current approaches to gene transfer in the central nervous system (CNS) are limited by the lack of effective, but non-invasive methods to deliver transgenes across the blood-brain barrier (BBB). In an effort to begin to explore the use of migratory monocytes as vehicles for delivery of therapeutic and antiviral genes into the CNS, we have utilized three HIV-based transfer vectors encoding cis-acting elements but lacking either structural genes (gag/pol and env), most accessory genes (vif, vpr and nef) and/or rev. These defective lentiviral vectors (DLV) encode the green fluorescent protein (GFP), display potent antiviral activity in CD4+ lymphocytes and can be mobilized by wild-type HIV-1 DLV were generated by transient transfection of 293T cells. Vector titers ranged from 4.2-6.6 x 10(6) infectious units (IU)/ml prior to concentration (by ultracentrifugation) and were equal to or higher than 1 x 10(9) IU/ml after concentration. Primary human monocyte-derived macrophages (MDM) were exposed to DLV resulting in efficiencies of transduction ranging from 14 to 26%. GFP expression in transduced MDM remained stable for more than 8 weeks without apparent cytopathic effect. Given the previously reported antiviral activities of these DLV and their lack of cytopathic effects on primary MDM, it may be possible to use these vectors to inhibit HIV-1 replication within the CNS.     

Evaluation of nitrite production by human monocyte-derived macrophages.

Reactive nitrogen intermediates are important in the anti-tumor and anti-microbial activities of rodent macrophages, but it is not known whether this is the case for human macrophages. In the present study, nitrite concentrations in vitro were used as an indicator of reactive nitrogen intermediate production by mouse, rat, and human macrophages. Human macrophages derived by culturing peripheral blood monocytes did not consistently produce detectable nitrite levels in response to any stimulus examined. Human macrophages were viable and metabolically active as indicated by the MTT assay, and their respiratory burst response to phorbol myristate acetate was increased following incubation with Interferon-gamma, as expected for typical macrophages. In contrast, rat or mouse peritoneal macrophages produced nitrite concentrations of approximately 20-100 microM in response to lipopolysaccharide, Interferon-gamma, or both. These results demonstrate substantial differences in the production of nitrites by rodent and human macrophages. Because of the heterogeneity among macrophage populations, these findings may not be applicable to all human macrophage populations, but they suggest a need for caution in extrapolating from rodent studies regarding the role of reactive nitrogen intermediates in anti-tumor or anti-microbial functions of human macrophages.     

Immunoprecipitation of lipid transfer protein activity by an antibody against human plasma lipid transfer protein-I

Two lipid transfer proteins, designated lipid transfer protein-I (Mr 69 000) and lipid transfer protein-II (Mr 55 000), each of which facilitates the transfer of radiolabelled cholesteryl ester, triacylglycerol and phosphatidylcholine between plasma lipoproteins, were purified from human plasma. Immunoglobulin G was prepared from goat antiserum to human lipid transfer protein-I (i.e., anti-human LTP-I IgG). The progressive addition of anti-human LTP-I IgG to buffered solutions containing either a highly purified mixture of human lipid transfer protein-I and lipid transfer protein-II, or highly purified rabbit lipid transfer protein (Abbey, M., Calvert, G.D. and Barter, P.J. (1984) Biochim. Biophys. Acta 793, 471-480) resulted in specific immunoprecipitation and the removal of increasing amounts, up to 100%, of cholesteryl ester, triacylglycerol and phosphatidylcholine transfer activities. However, similar precipitation studies on human and rabbit lipoprotein-free plasma resulted in the progressive removal of all cholesteryl ester and triacylglycerol transfer activities but only 30% (human) or 20% (rabbit) of phosphatidylcholine transfer activity. In all cases more anti-human LTP-I IgG was required to precipitate rabbit lipid transfer activity than human lipid transfer activity. These results suggest that lipid transfer protein-I and lipid transfer protein-II have antigenic sites in common, allowing precipitation of both proteins by specific antibody to lipid transfer protein-I. Most plasma phosphatidylcholine transfer activity is mediated by a protein (or proteins) other than lipid transfer protein-I and lipid transfer protein-II. In lipoprotein-free plasma all cholesteryl ester and triacylglycerol transfer activity, and some phosphatidylcholine transfer activity, is mediated by lipid transfer protein-I (or lipid transfer protein-I and an antigenically similar protein, lipid transfer protein-II.     

Interaction of plasma-derived lipid transfer protein with macrophages in culture

This study investigates the ability of human plasma-derived lipid transfer protein to facilitate lipid transfer to and from intact viable cells in culture. Mouse peritoneal macrophages or J774 macrophages were preincubated with acetylated low density lipoprotein and [3H]oleate/albumin to promote the intracellular synthesis and accumulation of cholesteryl [3H]oleate and 3H-labeled triglyceride. The addition of partially purified lipid transfer protein to cultures of lipid-loaded macrophages resulted in a time and concentration-dependent transfer of radiolabeled cholesteryl ester and triglyceride from macrophages to the medium. At 48 hr, lipid transfer protein facilitated the net transfer of 16 and 11% of cellular cholesteryl ester and triglyceride radioactivity, respectively, to the medium; transfer in the absence of the lipid transfer protein was less than 2%. The transfer of cholesteryl ester radioactivity was accompanied by a similar decrease in cellular cholesteryl ester mass indicating a net transfer event. Lipid transfer from cells was not dependent on the presence of a lipoprotein acceptor in the medium; however, low and high density lipoproteins present at 200 micrograms cholesterol/ml did significantly stimulate the transfer protein-facilitated efflux of these lipids. Lipid transfer protein did not appear capable of transferring radiolabeled lipid from low density or high density lipoprotein to macrophages. Radiolabeled cholesteryl ester and triglyceride transferred from cells to the medium by lipid transfer protein were associated with large molecular weight (greater than 2 x 10(6)) components in the medium with an average density greater than 1.21 g/ml; these lipids were not associated with lipid transfer protein itself. However, these radiolabeled lipids were readily incorporated into low or high density lipoproteins when these lipoproteins were added to the medium either during or after its incubation with cells. It is concluded that lipid transfer protein can facilitate the net efflux of cholesteryl esters from intact, living macrophages. These studies suggest a novel and potentially antiatherogenic role for lipid transfer protein.     

Interaction of a human plasma lipid transfer protein complex with lipid monolayers

The interaction of a purified human plasma lipid transfer complex with cholesteryl ester, triacylglycerol and phosphatidylcholine in binary and ternary lipid monolayers was investigated. The lipid transfer complex, designated LTC, catalyzes the removal of cholesteryl oleate and triacylglycerol from phosphatidylcholine monolayers. Preincubation of LTC with p-chloromercuriphenyl sulfonate inhibits LTC-catalyzed removal of triacylglycerol; cholesteryl ester removal is not affected. The rate of LTC-facilitated removal of cholesteryl oleate from a phosphatidylcholine monolayer depends on the amount of LTC added to the subphase up to 100 μg protein. In addition, the rate of the LTC-catalyzed transfer of cholesteryl oleate to the subphase increases linearly as the amount of cholesteryl oleate in the monolayer increases to 6 mol%. LTC also removes cholesterol from phosphatidylcholine-cholesterol monolayers, albeit at a rate which is 15% of that for removal of cholesteryl oleate. The ability of LTC to facilitate triacylglycerol and cholesteryl ester removal depends on the composition of the monolayer. Phosphatidylcholine supports cholesteryl ester transfer whereas sphingomyelin-cholesteryl ester monolayers are almost refractory to LTC. In contrast, LTC removes triacylglycerol from either a phosphatidylcholine or a sphingomyelin monolayer. The results suggest the existence of at least two lipid transfer proteins, one of which catalyzes the removal of cholesteryl ester and the other triacylglycerol. The role of these proteins as they relate to lipoprotein metabolism is discussed.     

Tumor-stimulated release of tumor necrosis factor-alpha by human monocyte-derived macrophages.

Tumor necrosis factor-alpha (TNF) release by monocytes and macrophages may be an important determinant of the physiologic response of the host to neoplastic disease; however, the mechanisms which regulate TNF release by macrophages in hosts with neoplastic diseases are poorly understood. The purpose of this study was to determine if cell membranes and growth medium from human leukemia cell lines and solid tumor cell lines induced TNF release by cultured human blood monocyte-derived macrophages. The capacity for TNF release and direct tumor killing was highest in monocytes cultured for 7 to 11 days. Cell membranes and culture media from K562 erythroleukemia and several small cell lung carcinoma cell lines, including H82, induced the release of up to 1500 TNF units per 10(6) macrophages over 24 hr. By contrast, allogeneic peripheral blood lymphocytes, cell membranes from normal mixed donor peripheral blood leukocytes, or growth medium from normal embryonic lung fibroblasts induced the release of little or no TNF during culture up to 24 hr, suggesting that this macrophage response was specific for tumor cells. Release of TNF by tumor-stimulated macrophages was gradual, peaking 24 hr following the addition of stimuli. Induction of macrophage TNF release was concentration dependent, with half-maximal TNF levels induced by 12.5 and 25 micrograms/ml cell membranes prepared from K562 and H82, respectively. Pretreatment of tumor cell membranes with polymixin B, which inhibits many of the actions of endotoxin, failed to neutralize tumor induction of TNF, suggesting that endotoxin was not responsible for this activity. Depletion of macrophages by treatment with 3C10 monoclonal antibody and complement abrogated tumor-induced TNF release, indicating that macrophages were the source of the secreted TNF. HPLC analysis of H82 growth medium demonstrated a single peak of macrophage activating activity with approximate 40-kDa molecular weight. We have demonstrated that cell membranes and growth medium from some human leukemia and solid tumor cell lines, but not from normal human cells, induce human peripheral blood monocytes and monocyte-derived macrophages to release functionally active TNF. This process may contribute to the host response to some neoplastic diseases.     

Comparison of lipid-mediated and adenoviral gene transfer in human monocyte-derived macrophages and COS-7 cells

Lipid-mediated transfection was compared to adenoviral-mediated gene transfer in COS-7 cells as well as human monocyte-derived macrophages (HMDM). For this purpose, we monitored enhanced green fluorescent protein (EGFP) expression by fluorescence microscopy and quantified gene transfer by competitive PCR. Transfection of COS-7 cells with a novel lipid formulation for DNA transfer was highly effective in COS-7 cells. On average, 30% of the cells were fluorescent 48 h after transfection. In HMDM, the same formulation resulted in the expression of EGFP in less than 0.5% of cells. We measured plasmid DNA by quantitative PCR in lipid-transfected macrophages and found that each macrophage contained on average 2 fg of plasmid DNA 24 h after transfection, that is, more than 400 molecules of plasmid DNA entered each cell. Despite the high level of reporter DNA in lipid transfected cells, expression of the fluorescent protein was suppressed in more than 99.5% of the macrophages. We also used adenoviral gene transfer to introduce the foreign DNA into both COS-7 cells and HMDM. Even though the multiplicity of infection was less than 30, expression of EGFP was observed in nearly all COS-7 cells and in more than 80% of HMDM 48 h after transfection. Despite major advances in the field of lipid-mediated transfection of HMDM, the lipid formulations that are available commercially cannot compete with the efficiency of adenoviral gene transfer.     

Sequestration of acetylated LDL and cholesterol crystals by human monocyte-derived macrophages

Monocyte-derived macrophages accumulate and process cholesterol in atherosclerotic lesions. Because of the importance of this process, we examined the interaction of cholesterol crystals and acetylated low density lipoprotein (AcLDL) with human monocyte-macrophages in a combined chemical and morphological study. These two forms of cholesterol induced extensive compartmentalization of the macrophage cytoplasm. Unexpectedly, the compartments maintained a physical connection to the extracellular space as demonstrated with ruthenium red staining. The compartments formed through invagination of the top surface of the macrophage plasma membrane. Some cholesterol crystals and AcLDL were sequestered within these surface-connected compartments for up to five days in the case of the crystals and for one day in the case of AcLDL. Pulse-chase studies of fractionated macrophages indicated that [3H]cholesterol redistributed from the surface-connected compartments into lysosomes (where the cholesterol remained unesterified) and into lipid droplets (where the cholesterol was stored as cholesteryl ester). Intracellular uptake and esterification of cholesterol was blocked by cytochalasin D. However, once cholesterol was sequestered in the surface-connected compartments, subsequent esterification of the cholesterol could not be inhibited by cytochalasin D. Apolipoprotein E was localized within the surface- connected compartments by immunogold labeling suggesting a possible function for this protein in the processing of lipid taken up through the sequestration pathway. Removal of microcrystalline cholesterol from the medium resulted in release of most of the accumulated cholesterol microcrystals from the macrophages, as well as disappearance of the surface-connected compartments. Thus, sequestration is a novel endocytic mechanism in which endocytic compartments remain connected to the extracellular space. This differs from phagocytosis where endocytic vacuoles rapidly pinch off from the plasma membrane. Sequestration provides a means for macrophages to remove substances from the extracellular space and later release them.     

Enhancement of the human plasma lipid transfer protein reaction by apolipoproteins

Transfer of cholesteryl ester between triacylglycerol/phospholipid microemulsions catalyzed by human plasma lipid transfer protein was investigated with a pyrene-containing analogue of which fluorescent properties depend on its concentration in the core of the microemulsions. The transfer of pyrene-cholesteryl ester between the emulsions was increased by the transfer protein linearly with its concentration, but maximally only to the extent of twice as much as spontaneous transfer in the given experimental conditions. When human apolipoproteins A-I or A-II are present in the reaction mixture enough to saturate the surface of the emulsion, the enhancement of the pyrene-cholesteryl ester transfer reaction by the transfer protein was 7.5-times more than in the absence of the apolipoproteins while the rate of spontaneous transfer was not affected significantly by the apolipoproteins. Bovine serum albumin did not have such an effect. Furthermore, the enhancement of the lipid transfer protein reaction by apolipoprotein A-I was linearly proportional to the percent saturation of the surface of the microemulsion with the apolipoprotein.     

Salmonella typhimurium induces apoptosis in human monocyte-derived macrophages

Salmonella species represent a leading cause of gastroenteritis worldwide. More recently, they have been proposed as putative vaccine delivery vehicles in humans. Oral infection with Salmonella leads to invasion of the intestinal epithelial barrier and subsequent interaction with mucosal macrophages. In this study, we investigated the fate of Salmonella typhimurium-infected human macrophages differentiated from blood monocytes by GM-CSF. Wild type S. typhimurium strain SL1344 induced macrophage surface blebbing and caused the release of host cytoplasmic lactate dehydrogenase beginning 30 min post-infection. Three hours later more than 80% of the macrophages in the culture were killed. In contrast, during the same period, macrophages infected with the non-invasive S. typhimurium strain BJ66 remained viable. Chromatin fragmentation is a hallmark of cells undergoing apoptosis. Using TUNEL analysis, we observed chromatin fragmentation in macrophages infected with SL1344 but not in BJ66 infected cells. Consistent with this observation, we found that pretreatment of human macrophages with an inhibitor of caspase-3, a member of the pro-apoptotic enzyme family shown to be involved in S. typhimurium-induced killing of mouse macrophages, reduced SL1344-mediated cytotoxicity by 40%. Our study provides the first evidence that invasive S. typhimurium induces apoptosis in human macrophages that were differentiated from blood monocytes by GM-CSF, and that cell death is a caspase-dependent phenomenon.     

Enhancement of cholesteryl ester metabolism in cultured human monocyte-derived macrophages by verapamil

The effect of the Ca2+ entry blocker, verapamil, on the biosynthesis of cholesterol and the metabolism of low-density lipoprotein (LDL) was studied in cultured human monocyte-derived macrophages. Addition of verapamil (50 microM) of monocyte-derived macrophages enhanced 125I-LDL and 125I-labelled acetyl-LDL binding and internalization, and increased [2-14C]acetate incorporation into cholesterol. Since higher levels of LDL and modified lipoproteins may be implicated in atherogenesis, the more efficient processing of these lipoproteins by monocyte-derived macrophages in the presence of Ca2+ blocker warrants further assessment for its potential as an antiatherogenic agent.     

Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages

The aim of this work was to investigate the possible mechanisms for uptake by human monocyte-derived macrophages (HMDM) of low density lipoprotein (LDL) pretreated with human arterial chondroitin-6-SO4-rich proteoglycan (LDL-PG). HMDM were incubated with 125I-labeled tyramine cellobiose-labeled LDL-PG, native LDL, and acetylated LDL (Ac-LDL). The results showed that two to four times more LDL-PG than LDL was bound and internalized by the HMDM. Competition experiments showed that LDL-PG competed with native LDL for the apoB,E (LDL) receptor, but not for the Ac-LDL scavenger receptor. Both the LDL and LDL-PG uptake were reduced after preincubation of the macrophages with unlabeled native LDL, though to a lesser extent with LDL-PG. The specific binding of 125I-labeled LDL and 125I-labeled LDL-PG at 4 degrees C was both saturable and concentration-dependent. The dissociation constant (Kd) for binding was 8.6 x 10(-9) M for LDL and 9.4 x 10(-9) M for LDL-PG, but the maximum binding (Bmax) was 1.5-times higher for LDL-PG. Cholesterol derived from LDL-PG was less effective than native LDL in suppressing HMG-CoA reductase activity. The results indicate that the uptake of LDL-PG is mediated not only by the LDL-receptor, but also by another unspecific pathway, which may not be subjected to regulation. These results provide further support for the hypothesis that LDL modifications induced by arterial PG may contribute to the formation of foam cells.     

Cholesterol esterification in human monocyte-derived macrophages is inhibited by protein kinase C with dual roles for mitogen activated protein kinases

The possible role of protein kinase C (PKC) and mitogen activated protein (MAP) kinases in the stimulation of cholesterol esterification by acetylated low density lipoprotein (acLDL) in human monocyte-derived macrophages (HMDM) was studied. Cholesterol esterification, as assessed by the rate of incorporation of [3H]-oleate into cholesteryl ester, was markedly higher in HMDM incubated with acLDL as compared to native LDL (nLDL). In the presence of the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 100 nM), however, the rate of incorporation was reduced by about 50% and 85% in incubations with nLDL and acLDL, respectively. Thus, the difference in the rate of cholesteryl esterification induced by the two types of lipoprotein was abolished by PMA, indicating that PKC activation inhibits the process, and this was confirmed by the finding that the PKC inhibitor calphostin C reversed the PMA-induced inhibition of cholesterol esterification. Incubation of HMDM with PMA was found to cause a considerable increase in the activation of p42/44 extracellular signal-regulated MAP kinases (ERK) and p38 MAP kinases, reaching a maximum at 30 min. In the presence of acLDL, the ERK inhibitor PD98059 decreased cholesterol esterification in HMDM by about 35%. In contrast, the p38 inhibitor SB203580 had no effect. However, when PMA was present in addition to SB203580, esterification was reduced to a level lower than that observed with PMA alone. These findings suggest that activation of ERK, but not p38, MAP kinases is involved in the induction of cholesterol esterification by acLDL in HMDM, while p38 MAP kinases may modulate the inhibitory effect of PKC, and thus provide evidence that MAP kinases play a role in the regulation of foam cell formation in human macrophages.     

Intracellular regulation of lipoprotein lipase in human monocyte-derived macrophages

The intracellular pathway of lipoprotein lipase (LPL) has been examined in human monocyte-derived macrophages in culture. These cells were previously shown to synthesize and constitutively secrete LPL. The secretion is dependent on new enzyme synthesis. 6-d-old human monocytes have stores of mRNA for linear release of LPL up to 24 h. Enzyme activity in cells and in culture medium was almost completely inhibited by 24 h treatment with tunicamycin, an inhibitor of glycosylation. In monensin-treated cells a pronounced increase in enzyme activity was found, whereas the secreted activity was markedly reduced. This indicates that LPL in human monocytes is processed through a pH sensitive part of the Golgi complex and that the terminal glycosylation is not needed for the expression of its catalytic activity. Our results suggest that lysosomal function is not important in secretion of the enzyme, whereas vesicular transport seem to be involved in regulating LPL in human monocyte-derived macrophages in culture.