Mastocytoma cell migration in vitro: inhibition by MIF-containing supernatants.

Supernatants with macrophage migration inhibition factor (MIF) activity were obtained from cultures of antigen-stimulated guinea pig and human lymphocytes, and from SV40-infected monkey kidney cells. The monkey and human but not guinea pig preparations were effective in inhibiting migration of mastocytoma cells as well as macrophages. This inhibition of migration was not associated with cytotoxicity and was reversible.     

Effect of C-reactive protein on peritoneal macrophages. I. Human C-reactive protein inhibits migration of guinea pig peritoneal macrophages

The effect of human C-reactive protein (CRP) isolated and purified from pooled patients' sera on macrophage function, especially on macrophage migration, was studied. Peritoneal exudate cells (PEC) from guinea pigs were used for macrophage migration inhibition (MMI) test of capillary method. Migration of either PEC or adherent purified macrophages exposed to CRP were inhibited dose-dependently. These findings indicate that CRP inhibits macrophage migration directly, not via activation of lymphocytes contained in PEC. As control, we examined the effect of normal human serum, anti C-polysaccharide antibodies isolated from patients' sera, and free endotoxin at the dose contaminated in CRP preparation on macrophage migration and found that none of them were effective. The effect of CRP on MMI of sensitized PEC exposed to antigen was also studied. Large amounts of CRP inhibited MMI induced by antigen, indicating the possibility that CRP may act on macrophages competitively with migration inhibitory factor (MIF) and may modulate MMI. CRP possesses MIF-like activity and may play a functional role at the site of tissue injury by causing the accumulation of macrophages.     

Macrophage migration in fibrin gel matrices. II. Effects of clotting factor XIII, fibronectin, and glycosaminoglycan content on cell migration

We investigated the migration of oil-induced, guinea pig peritoneal macrophages in three-dimensional fibrin matrices, with particular attention to variables which modified fibrin gel structure and/or its adhesive properties for cells. The variables studied were fibrin concentration, gel cross-linking, and fibronectin and glycosaminoglycan content. Macrophage migration was an inverse linear function of fibrinogen concentration. Little or no fibrinolysis accompanied macrophage migration; rather, macrophages migrated through fibrin gels by an active process associated with marked distortions of cell shape and specialized plasma membrane contacts with fibrin strands. Fibrin matrices prepared from fibrinogen that had been depleted of clotting factor XIII and/or fibronectin provided a superior matrix for macrophage migration. Both the number of migrating cells and distance of migration were reduced when the gel matrix included fibronectin and was cross-linked by factor XIII. A hexapeptide containing the fibronectin cell-binding RGDS sequence reversed this migration inhibition, suggesting that fibronectin immobilized by cross-linking to fibrin may have bound macrophages and restricted cell migration. Hyaluronic acid, heparin, and heparan sulfate inhibited macrophage migration in cross-linked fibrin-fibronectin gels over a range of concentrations. These data are relevant to an understanding of macrophage migration in vivo where cross-linked fibrin-fibronectin gels containing variable amounts of glycosaminoglycans are deposited in tissues in immunologic reactions and in many other types of pathology.     

Release of macrophage migration inhibitory factor(s) from lymphocytes stimulated by streptococcal preparations

Lymphocytes from apparently healthy subjects, incubated for 5 hours with cellular components or extracellular products of group A streptococci and then washed and reincubated, were found to release factor(s) capable of inhibiting guinea pig lung macrophage migration (“indirect method”). Inhibitition of macrophage migration was also obtained when the same preparations were tested directly on guinea pig lung cells, a macrophage-lymphocyte population (“direct method”). The guinea pigs had not been experimentally sensitized. The inhibition of migration appeared to depend on the presence of lymphocytes among the macrophages, since macrophages purified by repeatedly discarding nonadherent cells proved resistant to the migration inhibiting activity of the most active Streptococcal preparation, a 20 × concentrated filtrate. Reconstitution of the original lymphocyte-macrophage mixture reestablished the reactivity. The macrophage migration inhibition did not correlate with the age of the guinea pigs. It could not be obtained with preparations of group D streptococci or of Salmonella paratyphi. Group C streptococci did not inhibit the macrophage migration with the indirect method, but it did with the direct one.The factor(s) released into the medium on stimulation of apparently normal lymphocytes by Streptococcal preparations was relatively heat resistant, nondialyzable, and DNase and RNase resistant; its release was inhibited by puromycin. Pretreatment of the cells with trypsin prevented the absorption of the factor(s) and left migration unaffected. These characteristics are similar to those previously described for the migration inhibitory factor (MIF) produced by the interaction of sensitized lymphocytes and specific antigens. Whether or not these similarities indicate an identity remains to be determined.     

In Vitro Assay of Endotoxin by the Inhibition of Macrophage Migration

A quantitative in vitro technique was used to compare the ability of different endotoxins to inhibit the migration of macrophages from explants of rabbit spleen cultured in a coagulated plasma medium. The order of potency was different from that observed in chick embryo assays, and in assays with mice, of the same endotoxins. In general, however, the sensitivity of the macrophage inhibition test was comparable to that of other bioassay methods. A highly purified endotoxin from Salmonella enteritidis (Ribi) in a concentration of 0.004 mug/ml regularly inhibited macrophage migration. The in vitro method was used to detect a progressive loss of biological activity in fractions obtained during acid hydrolysis of the purified endotoxin. The selective toxicity of very low concentrations of endotoxin for mammalian macrophages was important in estimating the degree of specificity of the reaction. The pattern of cellular response in explant cultures made it possible to differentiate endotoxic damage from the specific cytotoxic action of antigen associated with delayed hypersensitivity.     

An acidic lymphokine distinct from interferon-gamma inhibits the replication of herpes simplex virus in human pulmonary macrophages

Supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells were fractionated by gel filtration and isoelectric focusing. A fraction with an isoelectric point of 2.2-3.3 containing macrophage migration inhibition factor activity inhibited the replication of herpes simplex virus type 1 in human pulmonary macrophages and U937 cells. This fraction did not inhibit the replication of herpes simplex virus in human fibroblasts. Moreover, the ability of this lymphokine fraction to inhibit viral growth in macrophages was not neutralized by antibody against interferon-gamma. These findings identify a macrophage specific antiviral lymphokine which is distinct biochemically and immunologically from interferon-gamma.     

Antigen isolation from Rickettsia prowazekii cultured in FL cells and its use in the macrophage migration inhibition reaction

An antigen obtained from R. prowazekii cultivated in hydrocortisone-treated FL-cells possesses activity determined in the complement fixation test and the macrophage migration inhibition test. Such antigen can be used in the macrophage migration inhibition test with macrophages obtained from animals immunized with the preparations of rickettsial egg cultures.     

The mechanism of action of soluble lymphocyte mediators. IV. Effect of migratio inhibitory factor (MIF) on macrophage cyclic AMP and on responsiveness to adenylate cyclase stimulators.

Intracellular levels of cyclic 3′,5′-adenosine monophosphate (cAMP) in purified guinea pig peritoneal macrophages were elevated following incubation with the adenylate cyclase stimulators prostaglandins E₁ and E₂ (PGE₁, PGE₂), isoproterenol, and cholera toxin. Exposure of macrophages to antigen-stimulated lymphocyte culture supernatants, containing migration inhibitory factor (MIF), resulted in a moderate but consistent decrease in the cAMP level, which was best expressed after 1–2 hr of incubation. Incubation of macrophages with MIF-containing supernatants or partially purified MIF for 1–2 hr resulted in reduced cAMP accumulation in response to PGE₁, PGE₂, isoproterenol, and cholera toxin (nonspecific refractoriness). These findings indicate that MIF-induced inhibition of macrophage migration is not due to an increase in the cellular level of cAMP and that the reduction in cAMP concentration, caused by MIF, is probably a secondary phenomenon unrelated to the inhibition of cellular motility.     

Inhibition of macrophage nitric oxide production by gangliosides derived from a spontaneous T cell lymphoma: the involved mechanisms.

Gangliosides (DLG) derived from a spontaneous T cell lymphoma (Dalton's lymphoma) have been shown to impair the ability of lipopolysaccharide-activated macrophages to produce nitric oxide (NO). However, the mechanism and nature of this effect is not known. In this investigation, we sought to (1) determine whether the inhibitory action of DLG on macrophages is through the modulation of inducible nitric oxide synthase (iNOS) expression and (2) identify the possible mechanisms and signal transduction events underlying the inhibitory action of DLG. Immunoblot analysis of DLG-treated macrophages showed a decrease in iNOS expression. DLG also inhibited the production of monokines interleukin-1 and tumor necrosis factor by macrophages. However, the DLG-induced inhibition was reversible in nature. Studies showed that DLG-induced inhibition of macrophage activation could be blocked by sodium orthovanadate, indicating a role of phosphatase activity in ganglioside-induced inhibition.     

Comparative evaluation of the neutrophilokine-inducing activity of Yersinia pestis EV and Escherichia coli lipopolysaccharides

As shown in this study, neutrophilokine-inducing capacity of Y. pestis EV lipopolysaccharide (LPS) was not inferior to, and in secondary immune response even exceeded, that of E. coli LPS. Neutrophilokines synthesized under the action of the former preparation produced greater influence on the inhibition of macrophage migration from the focus of infection, the phagocytic activity of these cells (in secondary immune response) and the labilization of the lysosomic membranes of macrophages than neutrophilokines induced by E. coli LPS. Only in primary immune response the digestive capacity of macrophages was more actively stimulated by neutrophilokines induced by E. coli LPS. Both preparations did not induce the secretion of neutrophilokines regulating the expression of Fc-receptors on the surface of macrophages.     

Possible role of macrophage glycolipids as receptors for migration inhibitory factor (MIF).

Guinea pig peritoneal exudate cells incubated with water soluble glycolipids obtained from macrophages show an enhanced response to migration inhibitory factor. Incorporation of these glycolipids into liposomes greatly facilitates their interaction with indicator cells. Enhancement of peritoneal exudate cell responsiveness to migration inhibitory factor was specific for glycolipids from guinea pig macrophages. Glycolipids extracted from guinea pig brain and polymorphonuclear leukocytes as well as several bovine and porcine glycolipids had no effect. Specificity of enhancement was not due merely to a preferential association of macrophage glycolipids with indicator cells. The possible role of macrophage glycolipids as receptors for MIF is discussed.     

The phenotypic expression of the genes controlling the yield of a factor suppressing macrophage migration to Candida albicans antigens and tuberculin in mice

The regulation of the synthesis of factor inhibiting the migration of macrophages in response to C. albicans antigen in CBA (H-2k) and C57BL/6 (H-2b) mice has been studied. The low level of macrophage migration inhibition factor in response to this antigen is due to the existence of cyclophosphamide-inhibited specific suppressors. Differences between various strains of mice ensue from different activity of suppressors of thymic origin, whose nature has been revealed as the result of the transfer of marrow cells treated with anti-Thy-1 serum.     

Modification of the agarose drop micromethod for studying mouse macrophage migration inhibition factor

A modification of the drop agarose micromethod for the study of the factor inhibiting the migration of mouse macrophages is proposed. The syngeneic system was used instead of type RPMI-1640 complex culture media and calf embryonal serum, commonly used in the work with mouse macrophages. The production of the factor inhibiting the migration of macrophages by sensitized lymphocytes from mice of different strains has been shown to occur in vitro under the influence of pertussis corpuscular antigen. The method of obtaining supernatant containing macrophage migration inhibiting factor, from immune mouse lymphocyte culture is proposed. This supernatant may be used for, the analysis of the above-mentioned factor and other lymphokins.     

Further characterization of the putative glycolipid receptor for mif: Role of fucose associated with an acidic glycolipid

Water soluble glycolipids were extracted from guinea pig macrophages. These glycolipids, when incubated with macrophages, augment the cells' response to migration inhibitory factor. The glycolipids were fractionated by diethylaminoethyl-Sephadex ion exchange chromatography into neutral and acidic fractions. Only the acidic glycolipid fraction was able to enhance the responsiveness of macrophages to migration inhibitory factor. Additional studies indicate that the enhancing activity of these glycolipid preparations can be abrogated by the removal of terminal fucose residues with α-L-fucosidase. The possibility that fucose functions as an essential component of a macrophage glycolipid receptor for migration inhibitory factor is discussed.     

Endotoxin and macrophage-migration inhibition

Endotoxin inhibits the in vitro migration of macrophages. Macrophages which have been stimulated by intraperitoneal oil (Marcol) are more sensitive to the endotoxin than are nonstimulated, normal macrophages. Other factors appear to affect the sensitivity of macrophages and there is great variation between individual animals. This effect is not due to toxicity since the macrophages remain viable. Furthermore, it can be reversed by the addition of polymyxin B. This action appears to be a direct effect on macrophages since it is still evident with viable, enriched populations. The action of endotoxin can be potentiated by exposure of macrophages to lymphocyte supernatants containing migration-inhibition factor. The action is not potentiated by periodate treatment. In this situation the two effects are additive. It is suggested that some of the variability in the migration-inhibition factor assay might be due to contaminating endotoxin. Endotoxin has been found to contaminate most biological materials. The degree of contamination might well influence the level of activation of the macrophage, and thus the responsiveness to migration-inhibition factor. This work supports the concept that the macrophage needs to be at a certain level of activation to respond to migration-inhibition factor. It is clear that the presence of contaminating endotoxin needs to be considered, and prevented, in all work on migration-inhibition factor.     

Oxidized LDL/CD36 interaction induces loss of cell polarity and inhibits macrophage locomotion

Cell polarization is essential for migration and the exploratory function of leukocytes. However, the mechanism by which cells maintain polarity or how cells revert to the immobilized state by gaining cellular symmetry is not clear. Previously we showed that interaction between oxidized low-density lipoprotein (oxLDL) and CD36 inhibits macrophage migration; in the current study we tested the hypothesis that oxLDL/CD36-induced inhibition of migration is the result of intracellular signals that regulate cell polarity. Live cell imaging of macrophages showed that oxLDL actuated retraction of macrophage front end lamellipodia and induced loss of cell polarity. Cd36 null and macrophages null for Vav, a guanine nucleotide exchange factor (GEF), did not show this effect. These findings were caused by Rac-mediated inhibition of nonmuscle myosin II, a cell polarity determinant. OxLDL induced dephosphorylation of myosin regulatory light chain (MRLC) by increasing the activity of Rac. Six-thioguanine triphosphate (6-thio-GTP), which inhibits Vav-mediated activation of Rac, abrogated the effect of oxLDL. Activation of the Vav-Rac-myosin II pathway by oxidant stress may induce trapping of macrophages at sites of chronic inflammation such as atherosclerotic plaque.     

Nitric oxide causes macrophage migration via the HIF-1-stimulated small GTPases Cdc42 and Rac1

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of tumor development. Recently, the tumor microenvironment, with the presence of tumor-associated macrophages (TAMs), has gained considerable interest. The mechanisms of macrophage/TAM migration as well as the role of HIF-1 in macrophages for tumor progression are still under debate. We present evidence that under normoxic conditions, nitric oxide (NO) promotes macrophage migration. The response was impaired in macrophages from leukocyte conditional HIF-1α^−/− mice. NO production and cell migration in response to cytokines were attenuated in macrophages from iNOS^−/− mice, suggesting that iNOS-derived NO transmits cytokine signaling toward cell migration. We further identified the small GTPases Cdc42 and Rac1 as effectors of the NO–HIF axis to drive macrophage migration by modulating the actin cytoskeleton. Our observations strengthen the role of HIF-1 in macrophages as a target of NO in facilitating functional responses such as migration.     

Antigen handling by guinea pig macrophages: further evidence for the sequestration of antigen relevant for activation of primed T lymphocytes.

Guinea pig macrophages can take up sufficient 2,4 dinitrophenyl guinea pig albumin during a brief in vitro exposure at 37 degrees C to trigger proliferation and lymphokine production with primed T lymphocytes on subsequent co-culture. Treatment of such antigen-bearing macrophages with trypsin, a procedure which removes surface antigen, does not alter the ability of such macrophage to initiate the release of migration inhibition factor from sensitized T lymphocytes. In addition, formation of antigen-specific rosettes between primed T cells and antigen-bearing macrophages is not blocked by high concentrations of antibody directed against the antigen mediating this interaction. Similarly, primed T lymphocyte DNA synthesis induced by antigen-bearing macrophages is not inhibited by specific antibody to that antigen. These data support the conclusion that the fraction of macrophage-associated antigen which is relevant to T lymphocyte activation does not reside on the macrophage surface but rather remains in a restricted compartment from which it is accessible to the T cell but unavailable to either blockade by specific antibody or removal by proteolytic enzymes.     

Insulin increases macrophage triglyceride accumulation under diabetic conditions through the down regulation of hormone sensitive lipase and adipose triglyceride lipase

Diabetes mellitus (DM) is a major risk factor for the development of atherosclerosis, and high-serum levels of insulin are strongly associated with type 2 DM. Atherosclerosis is characterized by lipid-laden macrophage foam cell formations, which contain substantial amount of cholesterol and triglycerides (TG). This study analyzed for the first time, the effects of insulin on TG metabolism in macrophages under normal and diabetic conditions. Mouse peritoneal macrophages from C57BL6 mice were cultured under normal (5 mM) or high (diabetic condition, 25 mM) glucose concentration, with or without insulin, followed by the assessment of TGs metabolism in these cells. Under diabetic condition, insulin increased TG accumulation in macrophages by 100%, decreased cellular TG degradation by 21%, and increased C-reactive protein levels in macrophages by 83%. Insulin decreased hormone-sensitive lipase mRNA and protein expression by 28 and 60%, respectively, and adipose TG lipase (ATGL) protein expression by 36%, with no significant reduction in ATGL mRNA levels. The inhibition of insulin-mediated phosphorylation, and the addition of cyclic adenosine 3'5'-monoposphate, abolished the insulin-mediated inhibition of TGs degradation in cells. Insulin increases macrophage TGs accumulation only under diabetic conditions, suggesting that impaired glycemic control in diabetic patients treated with insulin may contribute to foam cell formations and enhanced inflammation in macrophages.