Motion of spin-labeled fatty acids in murine macrophages relation to cellular phagocytic activity

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels.Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings ($\text{2T}{}_{\mathrm{|}}$) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives.Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 °C, or by fixing them with 0.25 % glutaraldehyde.No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with $\text{cis-}$ or $\text{trans-}\text{unsaturated}$ fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.     

Adaptation to the UV-induced suppression of phagocytic activity in murine peritoneal macrophages following chronic exposure to solar simulated radiation.

Exposure of certain strains of mice to ultraviolet radiation (UVR) is known to suppress both local and systemic immune responses, including a reduction in the phagocytic activity of peritoneal macrophages. However, in many instances, the immunological effects have been observed following a single or a limited number of doses of UVR from sources containing a higher proportion of UVB than that emitted by the sun. The first aim of the present study was to establish whether a single exposure of C3H/HeN mice to solar simulated radiation (SSR) suppressed the ability of the peritoneal macrophages to phagocytose opsonised sheep red blood cells. The mice were irradiated with SSR from Cleo Natural lamps and a single dose of 31.9 J cm(-2) was found to be the minimal dose for significant suppression of macrophage phagocytic activity. Such a dose did not modulate the surface expression of I-A(k), CD11b, CD86 or FcgammaRII/III (CD32/16) on the macrophages. The second aim was to assess whether repeated SSR exposures with a dose below the minimal immunosuppressive dose affected macrophage activity and, if so, to test for photoadaptation by repeated exposures followed by a single, normally immunosuppressive dose of SSR, and then assaying the macrophage activity. Groups of mice were irradiated on each of 2, 10 and 30 days with 14.9 J cm(-2) SSR, followed in some instances by a single additional exposure of 31.9 J cm(-2) on the same day as the last irradiation. The phagocytic activity of the peritoneal macrophages was tested 24 h later. It was reduced by 32%, 18% and 4% respectively after 2, 10 and 30 repeated exposures to SSR, and by 39%, 21% and 7% respectively after 2, 10 and 30 repeated exposures plus the additional higher dose at the end. Thus, although the macrophage activity was initially suppressed by the SSR, photoadaptation of this immune parameter occurred following repeated exposures.     

Binding of spin-labeled fatty acids and lysophospholipids to hydrophobic region of calmodulin.

In the presence of bovine brain calmodulin activated by calcium, the sharp triplet electron spin resonance (ESR) lines of free doxyl stearic acids decreased, and the broad resonance lines increased concomitantly, suggesting that the doxyl stearic acids bound to calmodulin calcium-dependently. The bound molecules were displaced by a calmodulin inhibitor, W-7, whereas their nitroxide radicals were hardly reduced by ascorbic acid, suggesting that the spin-labeled fatty acids bind to hydrophobic regions of calmodulin, and consequently inhibit calmodulin-dependent phosphodiesterase activity. These binding characteristics to calmodulin were different from those to bovine serum albumin. Moreover, the ESR spectra of two spin-labeled derivatives of lysophospholipid having a spin-labeled acyl group or a spin-labeled polar head group showed that it is the acyl chain of lysophospholipid that interacts with the hydrophobic region of calmodulin. The interactions of fatty acids and lysophospholipids with calmodulin seem to be quite different from those of acidic phospholipids, described previously [Suzuki, T., Katoh, H., & Uchida, M.K. (1986) Biochim. Biophys. Acta, 873, 379-386]. Thus, from the results of ESR study, we can obtain information on the function of fatty acids and lysophospholipids on calmodulin. Instead of enzyme assay, ESR spectroscopy is a useful means to examine lipid-protein interaction.     

Equilibrium binding of spin-labeled fatty acids to bovine serum albumin: suitability as surrogate ligands for natural fatty acids

Electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies were used to characterize the binding of spin-labeled fatty acid (SLFA) to bovine serum albumin (BSA). Association constants of three stearic acid derivatives labeled with a nitroxyl radical at C-5, C-12, or C-16 were estimated by EPR spectroscopy as the ratio of SLFA to BSA was increased from about 0 to 9. The values were compared to those for unmodified stearate. With all three SLFA, it was apparent that the nitroxyl residue modified the binding pattern. For SLFA:BSA ratios up to 1, which probably involves the site(s) on BSA most specific for long-chain FA, the C-16 derivative bound with an affinity similar to that of the natural FA. At higher ratios, the association constants for this SLFA were lower than those for stearate. The C-12 and C-5 derivatives showed only low-affinity binding relative to stearate. The spectral parameter, W, was constant for SLFA:BSA ratios between 0 and 1 in the case of C-16 compound, indicating physical homogeneity of the high-affinity binding site. At higher ratios, the spectra changed progressively, indicating inhomogeneity of the lower affinity binding sites although parallel changes in association constants were not observed. Changes in W due to Heisenberg spin exchange were ruled out. By examining the mobility profile of the bound SLFA by both EPR and ST-EPR techniques, it was shown that the nitroxyl group was maximally immobilized when attached near the center of the carbon chain of the bound SLFA.     

Metabolism of glucose, glutamine, long-chain fatty acids and ketone bodies by murine macrophages.

Maximum activities of some key enzymes of metabolism were studied in elicited (inflammatory) macrophages of the mouse and lymph-node lymphocytes of the rat. The activity of hexokinase in the macrophage is very high, as high as that in any other major tissue of the body, and higher than that of phosphorylase or 6-phosphofructokinase, suggesting that glucose is a more important fuel than glycogen and that the pentose phosphate pathway is also important in these cells. The latter suggestion is supported by the high activities of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. However, the rate of glucose utilization by 'resting' macrophages incubated in vitro is less than the 10% of the activity of 6-phosphofructokinase: this suggests that the rate of glycolysis is increased dramatically during phagocytosis or increased secretory activity. The macrophages possess higher activities of citrate synthase and oxoglutarate dehydrogenase than do lymphocytes, suggesting that the tricarboxylic acid cycle may be important in energy generation in these cells. The activity of 3-oxoacid CoA-transferase is higher in the macrophage, but that of 3-hydroxybutyrate dehydrogenase is very much lower than those in the lymphocytes. The activity of carnitine palmitoyltransferase is higher in macrophages, suggesting that fatty acids as well as acetoacetate could provide acetyl-CoA as substrate for the tricarboxylic acid cycle. No detectable rate of acetoacetate or 3-hydroxybutyrate utilization was observed during incubation of resting macrophages, but that of oleate was 1.0 nmol/h per mg of protein or about 2.2% of the activity of palmitoyltransferase. The activity of glutaminase is about 4-fold higher in macrophages than in lymphocytes, which suggests that the rate of glutamine utilization could be very high. The rate of utilization of glutamine by resting incubated macrophages was similar to that reported for rat lymphocytes, but was considerably lower than the activity of glutaminase.     

Incorporation of spin-labeled fatty acids into bovine brain clathrin coated vesicles

Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. An abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.     

Interaction of crotoxin and its isolated subunits with spin-labeled fatty acids

We have investigated the interaction of crotoxin (component A-component B complex) and of its isolated phospholipase subunit (component B) with hydrophobic compounds by ESR, using spin-labeled fatty acids as probes. The phospholipase subunit alone (component B) binds more than three labeled fatty acid molecules/molecule with different affinities, the highest corresponding to a Kd of 10 microM in the case of 5-doxyl palmitic acid. In contrast, the noncatalytic subunit (component A) and the crotoxin complex do not bind fatty acids. ESR studies of the component B-fatty acid complex reveal a strong immobilization of the whole length of the fatty acid chain, strong spin-spin interactions between bound fatty acids, and nonaccessibility of the bound paramagnetic probe to Ni2+ ions. This suggests that the phospholipase component B possesses a hydrophobic cleft which may contain one or two fatty acids. This hydrophobic cleft is not accessible to spin-labeled fatty acids in the crotoxin complex. An overall rotational correlation time of about 200 ns of the phospholipase component B was determined by saturation transfer ESR. This high value is incompatible with the diffusion of a polypeptide of 14,500 molecular weight. The hydrodynamic analysis of the fatty acid-component B complex led us to estimate an apparent molecular weight of 95,000 which reveals that fatty acids induce the formation of polymers (most probably octamers) of component B. We propose a model in which the phospholipase component B exists in two conformational states which differ by their hydrophobicity.     

Modulating effect of cystamine and unsaturated fatty acids on gamma-glutamyl transpeptidase: Relation to cellular oxidative status

Summary Cell surface gamma-glutamyl transpeptidese activity in cultured neoplastic astrocytes was significantly increased upon treatment of the cells with the hepatoprotective disulfide, cystamine. The cystamine effect was sensitive to cycloheximide and could be significantly depressed by exogenous glutathione. Surface gamma-glutamyl transpeptidase activity was also modulated by the presence in the culture medium of the unsaturated fatty acids, linoleic acid and arachidonic acid. Metabolism of the fatty acids via the cyclooxygenase pathway was not a prerequisite for their modulation of the glycoprotein ectoenzyme. Lipoxygenase, however, was found to potentiate the unsaturated fatty acid effect in neoplastic astrocytes. Lipoxygenase is reported to catalyze the conversion of unsaturated fatty acids to their corresponding peroxides. The data indicate an oxidative influence on the control of gamma-glutamyl transpeptidase activity.     

Lipidomics analysis of essential fatty acids in macrophages

The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.     

Ultrastructure and phagocytic activity of rat peritoneal macrophages exposed to low temperatures in vitro

Hypothermia affects various components of the immune system, leading to impaired immune resistance. To examine the in vitro effect of low temperature on the ultrastructure and phagocytic function of rat peritoneal macrophages, cells were incubated at 4, 10, 24, and 37 degrees C for 60 min. Subsequently, their ultrastructure and capacity to engulf latex particles and generate superoxide anions were evaluated. The results showed a close inverse relationship between incubation temperature and ultrastructural changes, i.e., the lower the temperature, the higher the number of altered cells. In addition, at lower temperatures the number of cells capable of phagocytosis was reduced; the cells engulfed fewer particles per cell and generated less superoxide anions. These findings may be relevant for explaining the increased susceptibility to bacterial infections under hypothermic conditions.     

Activation state alters the effect of dietary fatty acids on pro-inflammatory mediator production by murine macrophages

Studies investigating the effect of dietary fats on pro-inflammatory cytokine production by macrophages (M phis) have yielded conflicting results. We hypothesised that this may be due to the different capacities of the M phis studied commonly (resident, thioglycollate-elicited) to produce prostaglandin E(2)(PGE(2)) and leukotriene B(4)(LTB(4)) which inhibit and stimulate, respectively, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) production. To investigate this, male C57Bl6 mice were fed for 6 weeks on a low fat (LF) diet or on high fat diets which contained coconut oil (CO), olive oil (OO), safflower oil (SO) or fish oil (FO) as the main fat source. Production of TNF-alpha, IL-1 beta, PGE(2)and LTB(4)by lipopolysaccharide-stimulated resident and thioglycollate-elicited (i.e. inflammatory) peritoneal M phis was measured. PGE(2)production by both inflammatory and resident M phis was significantly decreased by FO feeding. FO also decreased LTB(4)production by resident M phis compared with LF feeding. Production of both cytokines by inflammatory M phis decreased with increasing unsaturation of the high fat diets, such that cells from FO-fed mice showed significantly decreased production of TNF-alpha and IL-1 beta compared to those from mice fed on each of the other diets. In contrast, resident M phis from mice fed FO showed increased TNF-alpha production compared to those from CO-fed mice. Thus, FO feeding decreases production of TNF-alpha and IL-1 beta by inflammatory M phis and increases production of TNF-alpha by resident M phis, at least in comparison to some other dietary fats. These results indicate the mechanisms by which dietary fats exert their effects upon pro-inflammatory cytokine production are most likely different for resident and inflammatory M phis.     

Methyl palmitate inhibits lipopolysaccharide-stimulated phagocytic activity of rat peritoneal macrophages

Macrophages, in general, are critical effectors of body's immune system. Chemical inhibition of phagocytic activity of such macrophages as Kupffer cells has been extensively studied. We have earlier shown that methyl palmitate (MP) inhibits the activation of Kupffer cells. To evaluate the potential of MP to inhibit the activation of other macrophages, we treated rat peritoneal macrophages with varying concentrations of MP. Its treatment led to a dose-dependent inhibition of phagocytic activity, which was found to be 34%, 47%, and 66% at 0.25, 0.50, and 1.0 mM MP, respectively, as measured by latex bead uptake. When MP-treated peritoneal macrophages were stimulated with lipopolysaccharide (LPS), the nitric oxide (.NO) release was inhibited at 6 h, while cyclooxygenase-2 expression decreased after 24 h. The treatment with MP increased the release of interleukin (IL)-10 in the LPS-treated cells at 6 h, while IL-6 and tumor necrosis factor-alpha were significantly increased both at 6 and 24 h. Our data suggest that MP inhibits phagocytic activity and .NO production similar to that observed in isolated Kupffer cells. Therefore, inhibition of phagocytosis by MP may be a general phenomenon, and it could be used as an inhibitor of macrophage function.     

Cytotoxic activity of unsaturated fatty acids to lymphocytes

Cytotoxic activities of unsaturated fatty acids against lymphocytes were studied by demonstrating the inhibition of RNA synthetic activity of incubated lymphocytes. In general, the cytotoxic activity of fatty acids increases with decreasing number of carbon atoms and increasing number of double bonds. An empirical formula predicting the degree of impairment of lymphocytes by various fatty acids was proposed. Furthermore, protective effects of serum on cytotoxic actions of unsaturated fatty acids to lymphocytes were studied and the significance of these findings was discussed.     

Stimulation of the phagocytic function in guinea pig peritoneal macrophages by physical activity stress.

A study was made of all the different stages of the phagocytic function in peritoneal macrophages from male guinea pigs [3 (SD 1) months old] before, immediately after, and 24 h after being subjected to stress from physical activity (swimming until exhaustion). The early (10 min) and late (40 min) adherence to tissue substrates, chemotaxis, attachment and phagocytosis of Candida albicans, ingestion of inert particles (latex beads), and basal oxidative metabolism [measured by nitroblue tetrazolium (NBT) reduction] were significantly stimulated by the physical activity. After 24 h, late adherence, attachment capacities, and basal oxidative metabolism returned to basal values, whereas early adherence, chemotaxis, phagocytosis of cells and inert particles, and microbicidal capacity (production of superoxide anion measured by NBT reduction in presence of ingested material) remained significantly increased. The stress produced by physical activity, reflected in increased serum corticosterone values, led to a global stimulation of the phagocytic function.     

Composition of cellular fatty acids in unidentified non-pathogenic corynebacteria

The composition of fatty acids in lipids of selected strains of non-pathogenic unidentified corynebacteria was determined with the aim to employ such determinations for their characterization. Four strains were characterized by the presence of branched iso- and anteisoacids. One strain corresponded according to its composition to mycobacteria and nocardiae.     

Characteristics of the membranes and functional activity of phagocytic alveolar macrophages

Alveolar macrophages (AM) accumulate products of lipid peroxidation (PLP) in the time of phagocytosis of zymosan particles (ZP) during 4 hours, that lead to increase of lipid viscosity and decrease surface membrane area, which were studied by fluorescent probes pyrene and HSPH-14. Preliminary stimulation of AM by i/v ZP-injection of A(100 mg/kg before 5 days) lead to a decrease of lipid membrane viscosity and intensification of AM functional activity. During phagocytosis of ZP experimental cells accumulate much less PLP, than control cells, and promote support of viscosity on a more low level, and functional activity (phagocytic, adhesion properties)--on more high level, than in control cells.     

Serum-induced inhibition of the phagocytic activity of cultured macrophages IC-21

Fetal bovine serum has been shown to considerably inhibit phagocytosis of non-opsonized 2-μm fluorescent latex beads by cultured macrophages IC-21. Phagocytic activity was assessed using fluorescent microscopy and specially devised ImageJ plugins. Phagocytosis percent, PP (percentage of the bead-containing cells in the cell population under study), and phagocytosis index, PI (mean number of beads per cell in the bead-containing population), were about 2 times lower in the cells incubated in the presence of 10% serum as compared to the respective parameters for the cells incubated in serum-free medium (55 ± 5% vs. 92 ± 1% and 2.0 ± 0.2 vs. 4.3 ± 0.2 beads/cell). The effect of serum was dose-dependent. Albumin (10 mg/ml) did not mimic the effect of serum, suggesting that fatty acid extraction was not the cause of the serum-induced inhibition. Serum is a source of exogenous cholesterol, therefore we checked if cholesterol removal could stimulate phagocytosis. Cholesterol-sequestering agent methyl-β-cyclodextrin (mβCD) in concentrations of 5–7 mM indeed caused an increase of the phagocytic activity but at 10 mM exerted an inhibitory effect in serum-free medium. Connexin channel blocker carbenoxolone (CBX, 250–500μM) in most cases inhibited phagocytosis; the presence of serum or mβCD modulated the CBX effects. The data indicate an important role of serum in regulation of the macrophage phagocytic activity. Stimulating effect produced by serum removal may partly be accounted for by a decrease of cholesterol concentration, which in turn may alter the functioning of integral proteins involved in the mechanisms of phagocytosis.