Neuroendocrine control of thymic hormonal production. II. Stimulatory effects of endogenous opioids on thymulin production by cultured human and murine thymic epithelial cells

Data have now accumulated to strongly demonstrate that several neuropeptides, including endogenous opioids, can have immunomodulatory functions. Most of the studies have so far focused on the direct action of these substances on lymphocytes. We decided to investigate whether thymic epithelial cells (TEC) - the major component of the thymic microenvironment - could also be modulated by endogenous opioids. Primary cultures of human and murine TEC were subjected to several opioids (alpha-beta- or gamma-endorphins, as well as met- or leuenkephalins) applied in concentrations ranging from 10(-6) to 10(-9) M. On the following days we measured the levels of thymulin (a chemically-defined thymic hormone known to stimulate some steps of T-cell differentiation) in the culture supernatants, as well as the numbers of thymulin containing cells, evaluated by immunofluorescence with an anti-thymulin monoclonal antibody. After treatment of TEC cultures with beta-endorphin or leu-enkephalin a significant increase in the levels of thymulin in the culture media was observed, paralleled by a rise in the percentage of thymulin containing cells. In addition, this stimulatory effect was dose-dependent. Preincubation of the opioids with the specific antibodies abrogated the opioid-induced stimulatory effect on TEC. Moreover, naloxone, an opioid receptor antagonist, blocked the effect of beta-endorphin on thymulin production, suggesting that the effect of this neuropeptide on epithelial cells was mediated by an opioid receptor. Importantly, no effect on thymulin production was observed with the other opioids used, whatever the dose. These results suggest that, at least in vitro, beta-endorphin and leu-enkephalin stimulate the hormonal function of the thymic epithelium. These findings lead to the general concept that the modulatory role of endogenous opioids on the immune system is not restricted to lymphocytes but can also take place at the level of cells belonging to T-cell differentiating microenvironments.     

The single nucleotide polymorphism A118G alters functional properties of the human mu opioid receptor

The most common single nucleotide polymorphism in the coding region of the human mu opioid receptor gene is the A118G variant, an adenine to guanine transition at nucleotide position 118 of the coding sequence of the gene. This polymorphism codes for an asparagine to aspartic acid substitution at amino acid 40 in the amino-terminus, thereby removing a potential extracellular glycosylation site. Using in vitro cellular expression assays, this variant has been reported to change binding of the endogenous agonist beta-endorphin and signaling of the receptor following binding of beta-endorphin. Three clinical studies report that A118G genotype affects opioid antagonist-mediated increases in cortisol levels. These studies demonstrate a functional role of this variant in responses to endogenous and exogenous opioids. To further characterize function, we expressed the prototype and variant receptors in two types of cells (human 293 embryonic kidney cells and Syrian hamster adenovirus-12-induced tumor cells). Stable expression of variant and prototype receptors was characterized by differences in levels of cell surface binding capacity (B(max)), forskolin-induced cAMP accumulation, as well as agonist-induced accumulation of cAMP (EC(50)) for several agonists, but not for beta-endorphin. In contrast, transiently expressed variant receptors showed only a minor difference in cell surface binding capacity compared to the prototype, and no differences in cAMP EC(50) values.     

Platelet glycoprotein IIb-IIIa-like proteins mediate endothelial cell attachment to adhesive proteins and the extracellular matrix

The adherence of human umbilical vein endothelial (HUVE) cells to adhesive matrix proteins was examined to determine if cell attachment and spreading were mediated by the glycoprotein (GP) IIb-IIIa complex on endothelial cells. The HUVE cells adhered well to glass slides that had been coated with fibronectin, vitronectin, fibrinogen, or von Willebrand factor but failed to adhere to albumin-coated or to uncoated slides. The HUVE cell attachment and spreading on vitronectin, fibrinogen, and von Willebrand factor were greatly inhibited by a GP IIb-IIIa monoclonal antibody (7E3). In contrast, HUVE cell attachment to fibronectin was not inhibited by 7E3 but was inhibited by a fibronectin-receptor antibody (alpha GP140), which had no effect on cell attachment to the other adhesive proteins. The 7E3 antibody, but not alpha GP140, disrupted HUVE cell monolayers by detaching cells from their naturally occurring extracellular matrix. These data indicate that platelet GP IIb-IIIa-like proteins mediate the adherence of HUVE cells to specific adhesive proteins and to the extracellular matrix.     

Endogenous opioids and ventilatory responses to hypercapnia in normal humans

Though administration of opioid peptides depresses ventilation and ventilatory responsiveness, the role of endogenous opioid peptides in modulating ventilatory responsiveness is not clear. We studied the interaction of endogenous opioids and ventilatory responses in 12 adult male volunteers by relating hypercapnic responsiveness to plasma levels of immunoactive beta-endorphin and by administering the opiate antagonist naloxone. Ventilatory responsiveness to hypercapnia was not altered by pretreatment with naloxone, and this by itself suggests that endogenous opioids have no role in modulating this response. However, there was an inverse relationship between basal levels of immunoactive beta-endorphin in plasma and ventilatory responsiveness to CO2. Furthermore, plasma beta-endorphin levels rose after short-term hypercapnia but only when subjects had been pretreated with naloxone. We conclude that measurement of plasma endorphin levels suggests relationships between endogenous opioid peptides and ventilatory responses to CO2 that are not apparent in studies limited to assessing the effect of naloxone.     

Hypoxia does not increase CSF or plasma beta-endorphin activity

The ability of moderate (30-50 Torr arterial PO2) and severe (less than 30 Torr arterial PO2) hypoxia to generate endogenous opioids that modulate ventilation was studied in unanesthetized goats. Ventilation and its components, arterial blood gas tensions and pH, and plasma and cerebrospinal fluid (CSF) beta-endorphin activity were measured before and after 4 h of sustained moderate or severe hypoxia. Ventilation, as expected, increased with hypoxia. There were no significant changes in either plasma or CSF beta-endorphin activity after sustained hypoxia. To rule out elaboration of endogenous opioids other than beta-endorphin after hypoxia, naloxone or saline was administered to five of the seven goats exposed to 4 h of severe hypoxia, and their ventilatory responses were compared for 30 additional min of hypoxic breathing. No significant differences in ventilation occurred in the two treatment groups during this time period. We conclude that, unlike increases in airway resistance, moderate and severe hypoxia do not cause the elaboration of endogenous opioids that modify respiratory output in unanesthetized adult goats. The apparent ability of hypoxia to cause elaboration of endogenous opioids in the neonate may represent a maturational phenomenon.     

Expression of podocalyxin enhances the adherence, migration, and intercellular communication of cells

Podocalyxin (PODXL) is an anti-adhesive glycoprotein expressed abundantly in the epithelial cells of kidney glomeruli. In contrast, we report herein that expression of podocalyxin_GFP (PODXL_GFP) in CHO cells increased the adherence to immobilized fibronectin, spreading, and migration. The transient knockdown of PODXL or the expression of PODXL lacking the cytosolic carboxyterminal domain (PODXL-Δ₄₅₁) inhibited cell adherence. Moreover, the effect of PODXL was prevented by the ectodomain of podocalyxin (PODXL-Δ₄₂₉), by RGD peptides, or by inhibitors of the vitronectin receptor (αvβ3). CHO-PODXL_GFP also showed adherence to human vascular endothelial cells (HUVEC), exhibiting polarization of granular PODXL and emission of long and thin, spike-like, protrusions with PODXL granules progressing along. We found PODXL colocalized with β1 integrins at membrane ruffle regions on the leading edge of the cell and a blocking β1 mAb prevented the spreading of cells. PODXL was also associated with submembrane actin in lamellipodia ruffles, or with vinculin at cell protrusions. The proadhesive effects of PODXL were absent in sialic acid deficient O-glycomutant CHO cells. To conclude, we present evidence indicating that human PODXL enhances the adherence of cells to immobilized ligands and to vascular endothelial cells through a mechanism(s) dependent on the activity of integrins.     

Beta-endorphin and met-enkephalin stimulate human peripheral blood mononuclear cell chemotaxis

The neuropeptides beta-endorphin and met-enkephalin are potent analgesics and have a broad spectrum of biologic activities including the recently described alterations of lymphocyte proliferation and antibody production. The current study demonstrates that beta-endorphin and met-enkephalin stimulate human mononuclear cell chemotaxis, as measured by the in vitro leading front assay for migration. The response to both beta-endorphin and met-enkephalin was bimodal, with peak activities occurring at 10(-12) M and 10(-8) M. The distance migrated in response to optimal concentrations of beta-endorphin or met-enkephalin was approximately 80% of that obtained with 10(-8) M formyl-methionyl-leucyl-phenylalanine (f-MLP) and was blocked by prior incubation with 10(-8) M naloxone. Removal of glass adherent cells resulted in a loss of the response to beta-endorphin. Quantitation of the number of cells responding to beta-endorphin showed that only about 50% as many cells responded to beta-endorphin as compared with f-MLP. Human neutrophils showed some migration in response to beta-endorphin and met-enkephalin, although the average optimal migration was less than 30% of that observed with 10(-8) M f-MLP. Studies of the in vivo infusion of beta-endorphin into the cerebral ventricle of the rat resulted in the immigration of macrophage-like cells and are consistent with the in vitro evidence for a chemotactic effect of beta-endorphin.     

Lipopolysaccharide stimulates monocyte adherence by effects on both the monocyte and the endothelial cell

The effects of the LPS moiety of endotoxin on monocyte adherence to an endothelial cell surface were investigated over times before the development of well described LPS-induced endothelial cell surface adhesive molecules. In an in vitro microtiter adherence assay, LPS in concentrations of 10 ng/ml to 10 micrograms/ml incubated for 20 to 60 min with human monocytes significantly stimulated monocyte adherence to human umbilical vein endothelial cell monolayers (HUVEC) and serum-coated plastic surfaces. The time course and concentration dependence of LPS-stimulated monocyte adherence to glutaraldehyde-fixed HUVEC did not differ significantly from that to unfixed HUVEC or serum-coated plastic surfaces. Pretreatment studies suggested that LPS acted on the monocyte within 25 min to stimulate adherence to untreated endothelial cells but required a minimum of 1.5 to 2 h to render the endothelial cell more adhesive for untreated monocytes. The potential role of TNF-alpha, IL-1 alpha, and IL-1 beta in this system was assessed by determining the ability of these cytokines (+/- cytokine antibodies) to increase monocyte adherence. TNF, but neither IL-1, stimulated early monocyte adherence (1 h). This TNF-stimulated monocyte adherence was abrogated by coincubation with anti-rTNF-alpha polyclonal antibody. However, the anti-rTNF antibody had no effect on LPS-induced monocyte adherence to endothelial cells or serum-coated plastic surfaces. An early action of LPS on the monocyte to induce adherence to endothelial cell surfaces may contribute to the initial localization of peripheral blood monocytes in tissues during endotoxemia. The later effects of LPS on the endothelial cell to stimulate monocyte adherence may then amplify these initial monocyte-endothelial cell interactions to prolong and intensify monocyte adherence prior to migration into tissues.     

Chemotactic peptides modulate adherence of human polymorphonuclear leukocytes to monolayers of cultured endothelial cells

We have used a new centrifugation assay to examine the effects of highly purified human C5a and C5a des Arg, as well as effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP), on both the extent and strength of human polymorphonuclear leukocyte (PMN) adherence to monolayers of cultured human umbilical vein endothelial cells. At concentrations that were chemotactic for PMN, C5a (0.1 nM), C5a des Arg (5.0 nM), and FMLP (1.0 nM) significantly reduced the percentage of PMN that adhered to endothelial monolayers. Adherence also was reduced by C5a des Arg that was generated by incubating (37 degrees C, 30 min) fresh human serum with either zymosan or purified C5a. High concentrations of C5a (greater than 1.0 nM) and FMLP (greater than 50 nM) that diminished PMN chemotaxis significantly enhanced the percentage of PMN that adhered tightly to endothelial cells (adherent cells resisted a dislodgment force of 1200 X G). Tight adherence of PMN to endothelial cells also was increased by high concentrations of C5a that were added to human serum in which carboxypeptidase N activity was destroyed by heating (56 degrees C, 30 min), and by C5a that was generated by incubating (37 degrees C, 30 min) fresh human serum with zymosan in the presence of the carboxypeptidase N inhibitor, epsilon-aminocaproic acid. High concentrations of C5a des Arg (up to 80 nM) neither enhanced adherence of PMN to endothelial cells nor decreased PMN migration. Thus, a reciprocal relation exists between PMN migration and PMN adherence to endothelial cells in response to chemotactic factors. At concentrations that are chemotactic for human PMN, C5-derived peptides and FMLP reduce the adherence of PMN to endothelial monolayers. Only at concentrations that decrease PMN migration do C5a and FMLP augment PMN adherence.     

Modulation of human neuroblastoma transplanted into nude mice by endogenous opioid systems

The role of endogenous opioid systems (endogenous opioids and opioid receptors) in human cancer was explored using an opioid antagonist paradigm and neuroblastoma cells (SK-N-MC) transplanted into nude mice. Mice inoculated with 2.5 X 10(6) neuroblastoma cells received daily injections of either 0.1 or 10 mg/kg naltrexone (=0.1 and 10 NTX groups) which blocked the opioid receptor for 6-8 hr/day or the entire 24 hr/day, respectively, or sterile water. The latency for appearance of a measurable tumor (5 mm diameter) in the 0.1 NTX group was 27% longer than controls (11 days), and the first death in this group occurred 33% later than controls (day 27). Mice inoculated with tumor cells in the 10 NTX group had an acceleration (18%) in the latency of tumor appearance and, 2 weeks after cell inoculation, 70% of the mice in this group had tumors, in contrast to 10% of the controls. At the termination of the experiment (day 45), only 33% of the 10 NTX group were alive, in contrast to 90% of the controls. Receptor binding assays using DAGO, DADLE, or EKC revealed specific saturable binding only for DADLE and EKC. NTX administration resulted in a 148-186% increase in density for both binding sites, but no changes in binding affinity. Measures of opioid levels showed that tumor tissue levels of both beta-endorphin and methionine-enkephalin were elevated 2.5 to 6.5 fold from control values in both NTX groups, whereas plasma beta-endorphin was subnormal by 4 to 6 fold. These results indicate that endogenous opioid systems regulate human neuro-oncogenesis, with opioids being active inhibitors of growth. Opioid antagonists up-regulate receptors and increase tissue levels of endogenous opioids and, under conditions in which the opioid antagonist is short-acting (e.g., 0.1 NTX), can have an exaggerated antitumor effect during the interval when the antagonist is no longer present.     

A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling

Large scale sequencing of the human mu-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (beta-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, beta-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human mu-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo.     

Opioid regulation of food intake and body weight in humans

Relatively few studies of humans have evaluated the effects of opioids on food intake and body weight. Most have focused on the potential role of opioids in the etiology of obesity. Measurements of endogenous opioids in plasma or spinal fluid of humans reveal higher levels, particularly of beta-endorphin, in obese subjects. Opioid agonists such as methadone and butorphanol tartrate stimulate food intake, and all studies with naloxone, an opioid antagonist, demonstrate a reduction of short-term food intake in obese or lean humans. Long-term studies with naltrexone, an antagonist similar to naloxone, show no effect on food intake or body weight. Opioid agonists or antagonists have little effect on nutrient selection in humans. The effects on feeding-related hormones is equivocal. Further studies with more specific opioid receptor activities are needed.     

Vascular endothelial growth factor upregulates follistatin in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF), plays a key role in angiogenesis. Many endogenous factors can affect angiogenesis in endothelial cells. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during angiogenesis in endothelial cells. Searching for novel genes, involved in VEGF signaling during angiogenesis, we carried out differential display polymerase chain reaction on RNA from VEGF-stimulated human umbilical vein endothelial cells (HUVECs). In this study, follistatin (FS) differentially expressed in VEGF-treated HUVECs, compared with controls. Addition of VEGF (10 ng/mL) produced an approximately 11.8-fold increase of FS mRNA. FS or VEGF produced approximately 1.8- or 2.9-fold increases, respectively, in matrix metalloproteinase-2 (MMP-2) secretion for 12 h, compared to the addition of a control buffer. We suggest that VEGF may affect the angiogenic effect of HUVECs, through a combination of the direct effects of VEGF itself, and the indirect effects mediated via induction of FSin vitro.     

Antagonism of diazepam-induced feeding in rats by antisera to opioid peptides

Diazepam-induced feeding in rats is antagonized not only by the opiate antagonist naloxone but also intraventricular administration of specific antisera to the endogenous opioid peptides met-enkephalin or beta-endorphin. Pituitary beta-endorphin is probably not implicated in the diazepam effect since blockade with the glucocorticoid dexamethasone of the release of beta-endorphin from the anterior pituitary does not modify the diazepam-induced feeding, which is however prevented by TRH, a suggested physiological antagonist of some of the effects of opioid peptides. The possible central participation of both beta-endorphin and met-enkephalin in the ingestive behavior induced by diazepam gives further support to the postulated physiological role of endogenous opioids in appetite regulation.     

Correlated cholinomimetic-stimulated beta-endorphin and prolactin release in humans

Previous studies, both in animals and humans, have demonstrated that the intravenous or intraventricular administration of endogenous opioids and opiates produce dose dependent increases in plasma concentrations of prolactin. Notably, in humans, intravenous infusion of centrally active cholinomimetic drugs, such as physostigmine or arecoline, may produce significant increases in plasma concentrations of prolactin and beta-endorphin immunoreactivity. In three separate studies, conducted collaboratively between the National Institute of Mental Health and the University of California at San Diego, physostigmine and arecoline associated increases in plasma concentrations of beta-endorphin immunoreactivity were highly correlated with increases in plasma prolactin concentrations. These results are of interest because centrally active cholinomimetic drugs have been variously reported either to have no effect, to increase, or to inhibit anterior pituitary prolactin release. We propose that cholinergic stimulation of hypothalamic beta-endorphin may represent an interesting example of peptidergic modulation of primary neurochemical effects on hypothalamic-pituitary hormonal regulation.     

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl₄-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.     

Recombinant murine and human IL 1 alpha bind to human endothelial cells with an equal affinity, but have an unequal ability to induce endothelial cell adherence of lymphocytes

Consistent with the reports of others, we have demonstrated that human peripheral blood lymphocytes adhere to cultured human umbilical vein-derived endothelial cells (EC) in vitro. In our studies adherence was increased twofold to threefold by a 6-hr preincubation of the EC with IL 1. Recombinant human IL 1 alpha induced a maximal adherence response at less than 1 U per 2 X 10(4) EC. In contrast, recombinant murine IL 1 alpha was found to be 250- to 1250-fold less active in the adherence assay, based on units of IL 1 activity defined by the murine thymocyte proliferation assay. Moreover, when EC were preincubated with excess murine IL 1, no inhibition of the adherence-inducing effect of human IL 1 was noted. To characterize further this dichotomy of biological potency of murine and human IL 1 on the adherence assay, IL 1 binding studies were initiated. Recombinant human and murine IL 1 alpha were equally effective in inhibiting the binding of 125I-labeled human and murine IL 1, based on both micrograms of protein and units of IL 1 activity. The results of this study demonstrate that although human and murine IL 1 bind with equal affinity to receptors on human EC, human IL 1 is significantly more potent at inducing the increased EC adhesiveness for lymphocytes. The implications of these results for endothelial cell IL 1 receptor function are discussed.     

Pyk2/CAKbeta tyrosine kinase activity-mediated angiogenesis of pulmonary vascular endothelial cells.

Endothelial cell spreading, migration, and morphogenesis are essential for angiogenesis, the formation of new blood vessels. In the present study, we explored roles of tyrosine kinase Pyk2 in angiogenesis of pulmonary endothelial cells. We found that tyrosine kinase Pyk2 was particularly enriched in pulmonary vascular endothelial cells and lung, a major organ site for tumor metastasis. By using adenovirus-mediated expression of various Pyk2 mutants, we demonstrated that Pyk2 tyrosine kinase activity was essential for the pulmonary vascular endothelial cell spreading, migration, morphogenesis, as well as pulmonary vein and artery angiogenesis ex vivo. We further showed that Pyk2 kinase activity was required for the expression of focal adhesion kinase, p130Crk-associated substrate, and its homologue human enhancer of filamentation 1, thus regulating formation of focal adhesions and cytoskeletal reorganization. These results indicate that Pyk2 plays a crucial role in the pulmonary endothelial cell motility such as spreading and migration necessary for angiogenesis.     

Suppression of human lymphocyte chemotaxis and transendothelial migration by anti-LFA-1 antibody

The role of lymphocyte function-associated antigen 1 (LFA-1) in human T cell chemotaxis was investigated by using mAb specific to the beta-chain (TS1/18) (CD18) and alpha-chain (TS1/22) (CD11a) of LFA-1. T cell chemotaxis in response to IL-2 and to lymphocyte chemotactic factor (LCF) was markedly suppressed by the addition of TS1/18. TS1/22 was a less effective inhibitor than TS1/18 with only LCF stimulated responses showing significant inhibition when compared in seven different T cell preparations. Neither TS1/18 nor TS1/22 antibody inhibited random T cell migration. Control mAb to CD4 T cells failed to inhibit T cell random migration or chemotaxis. Additional studies to evaluate the adherence and migration of T cells through IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers showed that both TS1/22 and TS1/18 suppressed T cell migration through HUVEC, but failed to inhibit adherence of T cells to these cells. These studies indicate that LFA-1 plays a role in the migration of T cells through HUVEC and in the in vitro chemotactic response of T lymphocytes to IL-2 and LCF.     

Alterations in the functional characteristics of macrophages induced by hypercholesterolemia.

Intimal accumulation of monocyte-derived lipid-filled macrophages is an important early event in diet-induced hypercholesterolemia. To better understand the functional alterations in macrophages in hypercholesterolemia, we determined several variables in rat peritoneal macrophages putatively associated with atherogenesis including adhesion to, spreading and locomotion on an endothelial monolayer, migration and phagocytic capacities, and superoxide anion (O2-) production. Compared with macrophages from normal rats (NM0s), macrophages from hypercholesterolemic rats (HM0s) revealed a higher rate of adherence to endothelial cells (ECs) and plastic with more extensive cytoplasmic spreading. Towards a chemoattractant of zymosan-activated serum, HM0s exhibited greater chemotactic migration and more prominent aggregation than NM0s. A computerized film analysis using time-lapse cinemicrophotography disclosed that HM0s moved faster on ECs; the average speed of HM0s was almost twice that of NM0s. HM0 phagocytic activity of fluorescent latex beads was significantly heightened (P less than 0.01). By contrast, there was no significant difference in O2- production between the two groups. These results indicate that hypercholesterolemia may initiate and accelerate the atherosclerotic process, at least in part, by modifying a number of functional properties of macrophages.