Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

Cellular responses to excess phospholipid

Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase D-mediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A2.     

Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth

Once escaped from the quiescence niche, precursor cells interact with stromal components that support their survival, proliferation, and differentiation. We examined interplays between human myogenic precursor cells (mpc) and monocyte/macrophages (MP), the main stromal cell type observed at site of muscle regeneration. mpc selectively and specifically attracted monocytes in vitro after their release from quiescence, chemotaxis declining with differentiation. A DNA macroarray-based strategy identified five chemotactic factors accounting for 77% of chemotaxis: MP-derived chemokine, monocyte chemoattractant protein-1, fractalkine, VEGF, and the urokinase system. MP showed lower constitutive chemotactic activity than mpc, but attracted monocytes much strongly than mpc upon cross-stimulation, suggesting mpc-induced and predominantly MP-supported amplification of monocyte recruitment. Determination of [3H]thymidine incorporation, oligosomal DNA levels and annexin-V binding showed that MP stimulate mpc proliferation by soluble factors, and rescue mpc from apoptosis by direct contacts. We conclude that once activated, mpc, which are located close by capillaries, initiate monocyte recruitment and interplay with MP to amplify chemotaxis and enhance muscle growth.     

Lysophosphatidylcholine metabolism in Saccharomyces cerevisiae: the role of P-type ATPases in transport and a broad specificity acyltransferase in acylation

We recently described a new route for the synthesis of phosphatidylethanolamine (PtdEtn) from exogenous lyso-PtdEtn, which we have termed the exogenous lysolipid metabolism (ELM) pathway. The ELM pathway for lyso-PtdEtn requires the action of plasma membrane P-type ATPases Dnf1p and Dnf2p and their requisite beta-subunit, Lem3p, for the active uptake of lyso-PtdEtn. In addition, the acyl-CoA-dependent acyltransferase, Ale1p, mediates the acylation of the imported lysolipid to form PtdEtn. We now report that these components of the lyso-PtdEtn ELM pathway are also active with lyso-1-acyl-2-hydroxyl-sn-glycero-3-phosphocholine (PtdCho) as a substrate. Lyso-PtdCho supports the growth of a choline auxotrophic pem1Delta pem2Delta strain. Uptake of radiolabeled lyso-PtdCho was impaired by the dnf2Delta and lem3Delta mutations. Introduction of a lem3Delta mutation into a pem1Delta pem2Delta background impaired the ability of the resulting strain to grow with lyso-PtdCho as the sole precursor of PtdCho. After import of lyso-PtdCho, the recently characterized lyso-PtdEtn acyltransferase, Ale1p, functioned as the sole lyso-PtdCho acyltransferase in yeast. A pem1Delta pem2Delta ale1Delta strain grew with lyso-PtdCho as a substrate but showed a profound reduction in PtdCho content when lyso-PtdCho was the only precursor of PtdCho. Ale1p acylates lyso-PtdCho with a preference for monounsaturated acyl-CoA species, and the specific LPCAT activity of Ale1p in yeast membranes is >50-fold higher than the basal rate of de novo aminoglycerophospholipid biosynthesis from phosphatidylserine synthase activity. In addition to lyso-PtdCho, lyso-PtdEtn, and lyso-phosphatidic acid, Ale1p was also active with lysophosphatidylserine, lysophosphatidylglycerol, and lysophosphatidylinositol as substrates. These results establish a new pathway for the net synthesis of PtdCho in yeast and provide new tools for the study of PtdCho synthesis, transport, and remodeling.     

Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn-1 Diacylglycerol Lipase and a Monoacylglycerol Lipase

In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid [( 3H]AA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacylglycerols in unstimulated cultures revealed the presence of two primary [3H]AA-containing species, 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that AA release occurred through the successive actions of an sn-1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK-stimulated AA release in DRG cultures. Under our assay conditions there were no BK-stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with [3H]inositol, [3H]choline, or [3H]-ethanolamine, respectively.     

A quantitative and qualitative study of blood monocytes in patients with bronchogenic carcinoma

Summary Absolute circulating number and functions of blood monocytes (i.e., pinocytosis, phagocytosis, and chemotaxis) were studied in 25 patients with untreated bronchogenic carcinoma and in 28 control subjects. The absolute circulating monocyte count was increased in 20 (80%) of the patients. There was no difference in the pinocytic and phagocytic activity of patient and control monocytes. In contrast, patient monocytes showed depressed chemotactic responsiveness. This defect was more severe in small cell anaplastic carcinoma than in the other histologic types of bronchogenic carcinoma (P=0.001), and may explain the difference in macrophage infiltration seen in solid tumours of the lung. There was no correlation between chemotaxis and clinical stage. Depressed chemotaxis may be related to a plasma factor, since patient plasma inhibited the chemotaxis of control monocytes as well as the activity of chemotactic agents. The defective chemotaxis and the presence of plasma inhibitory activity may interfere with the ability of blood monocytes to accumulate as macrophages in tumour sites. Abbreviations used in this paper are: MCR, monocyte chemotactic response; SAC, small cell anaplastic bronchogenic carcinoma; OBC, non-small cell bronchogenic carcinoma MEM, Eagle's minimal essential medium; CFI, chemotactic factor inhibitor(s); HSA, human serum albumin     

Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading

The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases.     

Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca²⁺ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [³²P]P_i or [³H]glycerol into glycerolipids as well as on the redistribution of [¹⁴C]glycerol or [³H]arachidonic acid in [¹⁴C]glycerol- or [³H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [³²P]P_i incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [³²P]P_i into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [³²P]P_i incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [³H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [³H]glycerol into PtdA and PtdIns was greatly enhanced. But [³H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [³H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [¹⁴C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [¹⁴C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [³H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [³H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-³H]diacylglycerol and non-esterified [³H]-arachidonic acid was also enhanced, but the increase in [³H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-³H]PtdIns was not significantly affected. The activated formation of [arachidonate-³H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.     

Coexistence of a chemotactic factor and a retroviral P15E-related chemotaxis inhibitor in human tumor cell culture supernatants

Two sets of seemingly contradictory evidence have been reported concerning the effects of tumor cell products on the regulation of monocyte migration in vitro and presumably the extravasation of macrophages into tumors in vivo. The present study was designed to explore the relationship between chemotactic and anti-chemotactic products related to tumor cells: a tumor-derived chemotactic factor (TDCF) and retroviral P15E-related inhibitor(s) of chemotaxis. Culture supernatants of the human 8387 sarcoma and SW626 ovarian carcinoma were depleted of P15E-related antigens with immobilized anti-P15E monoclonal antibodies. This treatment produced a significant and consistent increase of the polarizing and chemotactic activity in the tumor cell supernatants. The material eluted from Sepharose-bound anti-P15E antibodies was devoid of chemotactic and polarizing activity and suppressed the polarization and migration of monocytes in response to chemoattractants. These results demonstrate the coexistence in culture supernatants of two human tumor cell lines of factors with opposite influences on monocyte chemotaxis. The data suggest that the entry of monocytes into neoplastic tissue may be regulated by the interplay of chemotactic and anti-chemotactic principals produced by tumor cells.     

Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.     

Chemotaxis of large granular lymphocytes

The hypothesis that large granular lymphocytes (LGL) are capable of directed locomotion (chemotaxis) was tested. A population of LGL isolated from discontinuous Percoll gradients migrated along concentration gradients of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and C5a, well known chemoattractants for polymorphonuclear leukocytes and monocytes, as well as interferon-beta and colony-stimulating factor. Interleukin 2, tuftsin, platelet-derived growth factor, and fibronectin were inactive. Migratory responses were greater in Percoll fractions with the highest lytic activity and HNK-1+ cells. The chemotactic response to f-MLP, casein, and C5a was always greater when the chemoattractant was present in greater concentration in the lower compartment of the Boyden chamber. Optimum chemotaxis was observed after a 1 hr incubation that made use of 12 micron nitrocellulose filters. LGL exhibited a high degree of nondirected locomotion when allowed to migrate for longer periods (greater than 2 hr), and when cultured in vitro for 24 to 72 hr in the presence or absence of IL 2 containing phytohemagluttinin-conditioned medium. The chemotactic LGL was HNK-1+, OKT11+ or HNK-1+, OKT11- on the basis of monoclonal antibody and complement depletion. They did not bear either T cell or monocyte cell surface markers, exhibiting an OKT3-, OKT4-, OKT8-, OKM1-, and MO2- phenotype, and did not form E rosettes at 29 degrees C, which is characteristic of lytic NK cells in contrast to T cells. Furthermore, a rat LGL leukemia (RNK) exhibited a chemotactic response to both f-MLP and casein. LGL chemotaxis to f-MLP could be inhibited in a dose-dependent manner by the inactive structural analog CBZ-phe-met, and the RNK tumor line specifically bound f-ML[3H]P, suggesting that LGL bear receptors for the chemotactic peptide.     

Prostaglandins and inflammation: enhancement of monocyte chemotactic responsiveness by prostaglandin E2.

The effects of prostaglandins on human monocyte chemotaxis were studied in vitro. None of the prostaglandins tested, including members of the A, B, E or F series, were chemotactic for monocytes. Prostaglandin E2 however, enhanced the chemotactic responsiveness of monocytes to complement - activated human serum by almost 200%. The enhancement of chemotaxis was not directly related to the ability of PGE2 to raise intracellular cyclic AMP levels. These studies support a role for prostaglandins as modulators of the inflammatory response.     

HIV-1 and its envelope glycoprotein down-regulate chemotactic ligand receptors and chemotactic function of peripheral blood monocytes

Peripheral blood monocytes from AIDS patients exhibit defective migratory responses to chemotactic stimuli in vitro and to inflammatory sites in vivo. In studies presented here, normal monocytes were infected with the HIV-1/HTLV-IIIBa-L isolate in vitro and evaluated for chemotactic responsiveness. Within 2 days after viral exposure, but before evidence of virus production in the monocytes, chemotactic activity was significantly impaired. Decreased chemotactic activity was associated with modulation of receptors for the chemotactic ligands, C5a and FMLP, on the monocyte cell surface. In addition to HIV-1, monocytes treated with purified HIV-1 envelope glycoprotein gp120 demonstrated a comparable modulation of chemotactic ligand receptors and migratory function. In addition, the HIV-1 or HIV-1 gp120-treated monocytes were induced to undergo differentiation as monitored by HLA-DR expression. Immunoprecipitation of the gp120 with a specific antibody reversed its effects on monocyte chemotaxis and HLA-DR expression. Taken together, these data indicate that the initial interaction of HIV-1 with the monocyte is not passive, but that the binding of HIV-1 and/or HIV-1 gp120 to the CD4R on monocytes transduces a signal leading to transient monocyte activation.     

Bronchial epithelial cells release monocyte chemotactic activity in response to smoke and endotoxin

An increase in mononuclear phagocytes occurs within the airways during airway inflammation. Bronchial epithelial cells could release monocyte chemotactic activity and contribute to this increase. To test this hypothesis, bovine bronchial epithelial cells were isolated and maintained in culture. Bronchial epithelial cell culture supernatant fluids were evaluated for monocyte chemotactic activity. Epithelial cell culture supernatant fluids attracted significantly greater numbers of monocytes compared to media alone and the number of monocytes attracted increased in a time dependent manner. Endotoxin and smoke extract induced a dose and time dependent release of monocyte chemotactic activity compared with cells cultured in media (52.5 +/- 2.6 (endotoxin), 30.5 +/- 2.3 (smoke) vs 20.5 +/- 2.2 cells/high power field (HPF) p less than 0.001). The released activity was chemotactic by checkerboard analysis. Stimulation of the epithelial cells by opsonized zymosan, calcium ionophore (A23187), and PMA also resulted in an increase in monocyte chemotactic activity (p less than 0.01). Because the release of activity was blocked by the lipoxygenase inhibitors, nordihydroguaiaretic acid and diethycarbamazine, epithelial cell monolayers were cultured with 3 microCi [3H]arachidonic acid for 24 h and then exposed to A23187, PMA, or both stimuli, for 4, 8, and 24 h. Analysis of the released 3H activity was performed with reverse-phase HPLC and revealed that the major lipoxygenase product was leukotriene B4. These data suggest that monocytes may be recruited into airways in response to chemotactic factors released by bronchial epithelial cells.     

A novel pathway for lipid biosynthesis: the direct acylation of glycerol.

The acylation of glycerol-3-phosphate by acyl-CoA is regarded as the first committed step for the synthesis of the lipoidal moiety in glycerolipids. The direct acylation of glycerol in mammalian tissues has not been demonstrated. In this study, lipid biosynthesis in myoblasts and hepatocytes was reassessed by conducting pulse-chase experiments with [1,3-(3)H]glycerol. The results demonstrated that a portion of labeled glycerol was directly acylated to form monoacylglycerol and, subsequently, diacylglycerol and triacylglycerol. The direct acylation of glycerol became more prominent when the glycerol-3-phosphate pathway was attenuated or when exogenous glycerol levels became elevated. Glycerol:acyl-CoA acyltransferase activity, which is responsible for the direct acylation of glycerol, was detected in the microsomal fraction of heart, liver, kidney, skeletal muscle, and brain tissues. The enzyme from pig heart microsomes displayed optimal activity at pH 6.0 and the preference for arachidonyl-CoA as the acyl donor. The apparent K(m) values for glycerol and arachidonyl-CoA were 1.1 mM and 0.17 mM, respectively. The present study demonstrates the existence of a novel lipid biosynthetic pathway that may be important during hyperglycerolemia produced in diabetes or other pathological conditions.     

Induction of monocyte migration by recombinant macrophage colony-stimulating factor

Human recombinant macrophage-CSF (M-CSF) induced migration across polycarbonate or nitrocellulose filters of human peripheral blood monocytes. Checkerboard analysis of M-CSF-induced migration, performed by seeding different cytokine concentrations above and below the filter, revealed that the locomotory response involved chemotaxis, though some gradient-independent augmentation of migration occurred. Polymixin B did not affect M-CSF chemotaxis and M-CSF was active on monocytes from the LPS-unresponsive mouse strain C3H/HeJ. These findings rule out a contribution of minute endotoxin contamination, below the sensitivity of the Limulus assay, in M-CSF chemotaxis. Rabbit anti-M-CSF antibodies inhibited the chemotactic activity of recombinant M-CSF, thus further indicating that the M-CSF molecule was indeed responsible for chemotaxis. M-CSF preparations encoded by 224 or 522 amino acid cDNA clones were equally effective in inducing monocyte migration. Recombinant M-CSF did not elicit a migratory response in large granular lymphocytes and in endothelial cells under conditions in which appropriate reference attractants were active. A modest stimulation of migration of polymorphonuclear leukocytes, inhibitable by antibodies, was observed at high cytokine concentrations (10 to 100 times higher than those required for monocyte locomotion). The maximal polymorphonuclear leukocytes response evoked by M-CSF was small compared to that evoked by reference chemoattractants or to that evoked by the same cytokine in monocytes. Hence, M-CSF is a potent chemoattractant for mononuclear phagocytes and exerts its action preferentially on cells of the monocyte-macrophage lineage. M-CSF, produced locally by activated macrophages, may play a role in the selective recruitment from the blood compartment of mononuclear phagocytes to amplify resistance against certain noxious agents.     

Secretagogue-induced diacylglycerol accumulation in isolated pancreatic islets. Mass spectrometric characterization of the fatty acyl content indicates multiple mechanisms of generation

Diacylglycerol accumulation has been examined in secretagogue-stimulated pancreatic islets with a newly developed negative ion chemical ionization mass spectrometric method. The muscarinic agonist carbachol induces islet accumulation of diacylglycerol rich in arachidonate and stearate, and a parallel accumulation of 3H-labeled diacylglycerol occurs in carbachol-stimulated islets that had been prelabeled with [3H]glycerol. Islets so labeled do not accumulate 3H-labeled diacylglycerol in response to D-glucose, but D-glucose does induce islet accumulation of diacylglycerol by mass. This material is rich in palmitate and oleate and contains much smaller amounts of arachidonate. Neither secretagogue influences triacylglycerol labeling, and neither induces release of [3H]choline or [3H]phosphocholine from islets prelabeled with [3H]choline. These observations indicate that the diacylglycerol that accumulates in islets in response to carbachol arises from hydrolysis of glycerolipids, probably including phosphoinositides. The bulk of the diacylglycerol which accumulates in response to glucose does not arise from glycerolipid hydrolysis and must therefore reflect de novo synthesis. The endogenous diacylglycerol which accumulates in secretagogue-stimulated islets may participate in insulin secretion because exogenous diacylglycerol induces insulin secretion from islets, and an inhibitor of diacylglycerol metabolism to phosphatidic acid augments glucose-induced insulin secretion.     

Chemotactic response of monocytes to thrombin

Human alpha-thrombin, the procoagulant activation product of prothrombin, elicits chemotaxis in human peripheral blood monocytes and several macrophagelike continuous cell lines, most notably J-774.2, but not in human peripheral blood granulocytes. alpha-Thrombin is effective in stimulating cell movement at concentrations ranging from 10(-10) to 10(-6) M but is optimally active at 10(-8) M. At the latter concentration, the degree of response is equivalent, on a molar basis, to that observed with the peptide formylmethionylleucylphenylalanine, (FMP). In contrast to thrombin, prothrombin produces a minimal chemotactic response in monocytes and J-774.2. Blockade of alpha- thrombin's active center with diisopropylfluorophosphate (DIP-F) or tryptic proteolysis of the procoagulant exosite (i.e., gamma-thrombin) fails to alter chemotactic activity. On the other hand, addition of equimolar amounts of antithrombin III (AT3) to alpha-thrombin reduces thrombin-mediated chemotaxis by 60%, and increased ratios of AT3 to enzyme completely suppress chemotaxis. We conclude that thrombin is a potent monocyte chemotaxin and that the domains in thrombin involved in stimulating cell movement are distinct from the catalytic site and the fibrin recognition exosite. These chemotactic domains appear to be sequestered in prothrombin and in the thrombin-AT3 complex and, as such, are unavailable to the chemotactic receptor on the monocyte cell membrane.     

Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma

Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.