Correlation Between Modification of Membrane Phospholipids and Some Biological Activity of Lymphocytes, Neutrophils and Macrophages

Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. the results are as follows.

a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested.

b) the phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids.

c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs.

d) the cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at c16 cause a decrease in the formation of phagolisosomes in the macrophages tested.     

Changes in the incorporation of free fatty acids upon the stimulation of human polymorphonuclear leukocytes

We have investigated the incorporation of free fatty acids into the cellular lipids of human polymorphonuclear leukocytes (PMN). Resting PMN incorporated both saturated and unsaturated fatty acids into triacylglycerol with only small amounts incorporated into the phospholipids. In contrast, PMN stimulated with the calcium ionophore A23187 incorporated significantly higher amounts of fatty acids, predominantly those other than arachidonic acid, into phosphatidylcholine and phosphatidylinositol, with reduced incorporation into triacylglycerol. Stimulation of PMN with serum-treated zymosan or the chemotactic peptide f-met-leu-phe but not phorbol myristate acetate, also increased the incorporation of fatty acids into these phospholipids. This stimulation-induced incorporation of fatty acids into cellular phospholipids was directed exlusively into position 2 of the lipid and probably reflects the reacylation of lysophospholipids after the release of arachidonic acid by phospholipase A2.     

Effects of fatty acids on growth of Bordetella pertussis in defined medium.

The effects of saturated and unsaturated fatty acids on the growth of Bordetella pertussis strain 114 in defined medium were tested. Individual fatty acids were found to be either inhibitory or stimulatory to growth, depending on concentration. Myristic (C14), pentadecanoic (C15), and palmitic (C16) acids were the most inhibitory saturated fatty acids tested. B. pertussis 114 was extremely sensitive to the unsaturated fatty acids oleic (C18; cis-9), elaidic (C18; trans-9) and petroselinic (C18; cis-6).     

Dialysis fluids and local host resistance in patients on continuous ambulatory peritoneal dialysis

The ability of polymorphonuclear leukocytes, monocytes and peritoneal macrophages to mount a respiratory burst in continuous ambulatory peritoneal dialysis (CAPD) fluids was tested in a phorbolmyristate acetate stimulated chemiluminescence assay. Fresh CAPD fluids depressed the chemiluminescence response of all three types of phagocytes tested to less than 18% of their chemiluminescence response in control buffer. When tested in spent CAPD fluids the suppression of chemiluminescence was 30–32%. Oxygen consumption of polymorphonuclear leukocytes was depressed in fresh CAPD fluids to below 40%. Both phagocytosis ofEscherichia coli by and bactericidal capacity of polymorphonuclear leukocytes and monocytes were suppressed in fresh CAPD fluids but not in spent effluents. The influence of acidic pH and hyperosmolality on phagocytic functions were studied separately by modifying the acidity or the glucose content of the control buffer. pH values below 6.0 significantly inhibited chemiluminescence but not phagocytosis. Under hypertonic conditions, both phagocytosis and chemiluminescence were inhibited. We conclude that the currently available CAPD solutions are beyond the limits of acid and osmotic tolerance of human phagocytic cells, and may thus compromise the peritoneal defenses of CAPD patients.     

Lipid chemotaxins isolated from culture filtrates ofEscherichia coli and from oxidized lipids

Lipid extracts of sterile culture filtrates ofEscherichia coli were shown to contain approximately 75% of the chemotactic activity for human polymorphonuclear leukocytes and rabbit alveolar macrophages. Fractionation and purification of these lipids revealed the presence of many unknown lipids of widely different properties, but all were anionic and at very low concentrations, chemotactic. The only one of active molecules that could be identified was an unsaturated ultraviolet-absorbing hydroxy fatty acid, which, following catalytic reduction with hydrogen, was found to be hydroxyeicosanoic acid. This fatty acid's Chromatographic behavior was very similar to that of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), which is a potent chemotaxin for polymorphonuclear leukocytes and macrophages. Unknown chemotaxins could be generated by the oxidation of known unsaturated lipids. Prostaglandins A₂ and E₂ produced potent chemotaxins upon aerobic oxidation. Malonaldehyde, a peroxidation product of unsaturated lipids, when reacted with phosphatidylethanolamine in aerobic conditions, also produced strong chemotactic agents. The chemotactic activity of these products could be destroyed by catalytic reduction with hydrogen and by methylation with dry methanolic HCl. These data indicate that the nonenzymatic oxidation of unsaturated lipids generates some products that are potent chemotaxins for mammalian inflammatory cells.     

Motility of rabbit alveolar cells: role of unsaturated fatty acids.

A mechanism for the specific accumulation of macrophages in alveoli or other biologic cavities following injury is presented. The data herein indicate that unsaturated fatty acids, ie, linoleic and linolenic acids, which accumulate in rat pleura following injection of carrageenan or during incubation of rabbit alveolar macrophages (AMs), strongly activate migration in vitro of AMs but not of human polymorphonuclear leukocytes (PMNLs). Other anionic lipids, ie, phosphatidylglycerol, as well as various nonspecific proteins, such as gelatin, or albumin were also shown to be potent activators of migration of AMs and not of PMNLs. These observations suggest that the elaboration of unsaturated fatty acids, as well as of nonspecific proteins, is responsible for the specific accumulation of macrophages in injured body spaces, such as alveoli or pleura.     

Arachidonic acid metabolism in polymorphonuclear leukocytes from patients with chronic granulomatous disease.

The effect of the calcium ionophore A23187 on the release and metabolism of [3H]arachidonic acid was examined in normal polymorphonuclear leukocytes and those obtained from patients with chronic granulomatous disease. The ionophore A23187 which stimulates oxidative metabolism in normal polymorphonuclear leukocytes was ineffective in increasing oxidative metabolism (chemiluminescence) in polymorphonuclear leukocytes from patients with chronic granulomatous disease. However, the ionophore A23187 stimulated the release of [3H]arachidonic acid from chronic granulomatous disease neutrophil phospholipids and stimulated its metabolism into hydroxyeicosatetraenoic acids and leukotrienes.     

Macrophage fatty acid composition and phagocytosis: effect of unsaturation on cellular phagocytic activity.

In order to manipulate the physical properties of the macrophages membrane, methods were developed which potentiated the incorporation of exogenously supplied fatty acids into membrane lipids. Chromatograms of macrophages which were grown in the presence of a variety of fatty acids demonstrated that exogenously supplied unsaturated fatty acids (palmitoleic, oleic, elaidic, linoleic, linolenic and arachidonic acids) were readily incorporated into the cells and selectively altered the fatty acyl composition of macrophage phospholipids. Up to 38% of the total cellular phospholipids were found to be derived from the exogenously added fatty acid supplements. The incorporation of the different fatty acids into cellular phospholipids had striking effects on cellular phagocytic activity. These effects were found to correlate with the degree of unsaturation, and the cis- or trans-double bond configuration. Thus, macrophage phagocytic ingestion rates of 125I-labelled Shigella flexneri were found to alter by more than 2-fold after the cells were cultivated in the presence of cis unsaturated fatty acids.     

Chemiluminescence by human alveolar macrophages: stimulation with heat-killed bacteria or phorobol myristate acetate.

Chemiluminescence of human alveolar macrophages (AM) was evaluated in vitro. Unstimulated AM generated chemiluminescence that remained constant during incubation. Addition of heat-killed Staphylococcus aureus 502A (HKB) or a chemical agent, phorbol myristate acetate, produced high rates of chemiluminescence that were significantly (P less than 0.05) increased over unstimulated AM. Phorbol myristate acetate-and HKB-stimulated increases in AM chemiluminescence were completely blocked by the enzyme superoxide dismutase. In comparison with unstimulated polymorphonuclear leukocytes, unstimulated AM had significantly (P less than 0.005) greater levels of chemiluminescence. However, after stimulation by phorbol myristate acetate or HKB, AM showed less chemiluminescence than similarly treated polymorphonuclear leukocytes.     

Inhibition of interferon gamma-induced macrophage microbicidal activity against Leishmania major by liposomes: inhibition is dependent upon composition of phospholipid headgroups and fatty acids

Multilamellar liposomes of phosphatidylcholine and phosphatidylserine at a 7:3 molar ratio significantly inhibited activation of murine resident peritoneal macrophages by recombinant murine interferon-gamma for cytotoxicity against amastigotes of the protozoan parasite Leishmania major; other macrophage effector functions, such as particle phagocytosis or tumoricidal activity, were unaffected. This inhibition was not due to direct toxic effects of liposomes against parasite or macrophage, was fully reversible, and was directed at one or more early events in macrophage-LK interactions which ultimately induce microbicidal activity. Liposomes containing some natural phospholipids (phosphatidylserine, phosphatidylethanolamine, phosphatidic acid or diphosphatidyl glycerol), but not phosphatidylcholine, phosphatidylglycerol, or several synthetic saturated phospholipids, prevented the induction of macrophage microbicidal activity. Inhibition by liposomes of various composition was not related to the efficiency with which these vesicles were ingested by macrophages. Inhibitory activity was directly influenced by changes in the phospholipid head group, as well as by the number of unsaturated bonds in phospholipid fatty acids: for a given phospholipid in liposomes, inhibition was directly related to the number of unsaturated bonds among the fatty acids. These data support a role for phospholipids in postbinding regulation of macrophage activation and add to our understanding of how liposome delivery systems can be designed to avoid potential microbicidal suppressive effects.     

Striatal dopamine in the response of brain and liver unsaturated and saturated free fatty acids following administration of C75 in CD-1 mice

In order to clarify the mechanism of action of cerulenin analog, C75, known to suppress feeding behavior, food intake was measured in adult CD-1 male mice n = 5 per group, treated i.p. with 10 and 20 mg/kg of C75. Animals in both treatment groups had significantly lower 24 h food consumption rate relative to the control group injected with vehicle. Striatal monoamine neurotransmitters and striatal as well as liver long chain free fatty acids concentrations were subsequently evaluated in another group treated i.p. with 20 mg/kg C75. Acute exposure to C75 at 20 mg/kg led to approximately 50% increase in the striatal dopamine levels and a decrease in dopamine turnover for up to 24 h following the injection. The concentration of serotonin remained unchanged. Concentration of saturated fatty acids in the liver and striatum did not change, while striatal unsaturated myristoleic acid (cis-9-tetradecenoic acid) levels were significantly higher as early as 2 h post-injection and remained elevated at 24 h post-injection. These preliminary data suggest a central regulatory role of unsaturated fatty acids under dopaminergic control in the C75-induced anorexia. Pharmacological alterations in fatty acid metabolism may prove beneficial in the treatment of obesity.     

Enzymes of phospholipid metabolism in airway secretions of patients with asthma,cystic fibrosis,and alveolar proteinosis

Phospholipase A₂, lysolecithinase, and lysolecithin-lysolecithin acyltransferase are present in the airway secretions of patients with asthma, cystic fibrosis (CF), and alveolar proteinosis. Assays of these enzymes under the same conditions in extracts of human polymorphonuclear leukocytes, alveolar macrophages, and pig tracheal mucosa indicated that these extracellular airway enzymes were probably not derived from these cell types. Although the above three enzymes were present in secretions of all patients with these three diseases, large amounts of palmitoyl lysolecithin, free fatty acids, and dipalmitoyl lecithin were found only in patients having alveolar proteinosis and asthma. The amount of free fatty acids in these secretions was sufficient to inhibit both the lysolecithinase and the lysolecithin-lysolecithin acyltransferase, but not the phospholipase A₂. These findings suggest that the large amount of dipalmitoyl lecithin, free fatty acids, and lysolecithin found in these secretions results from extracellular remodelling by these enzymes. Because the phospholipase A₂ was present in large amounts, it was possible after delipidation to purify it to homogeneity. It is a stable enzyme with an apparent molecular weight of 75,000 as estimated by SDS-mercaptoethanol gel electrophoresis.     

Effects of dietary fish oil supplementation on polymorphonuclear leukocyte inflammatory potential

Polymorphonuclear leukocytes (PMNLs) are an important contributor to inflammation and are thus a part of the pathophysiology of many human diseases. We assessed the effect of fish oil on PMNL inflammatory potential by measuring chemiluminescence and Superoxide production before and after six weeks of daily cod liver oil ingestion by healthy volunteers. Phagocytosing PMNLs demonstrated a 27% decrease in chemiluminescence (P < 0.05) and a 64% decrease in Superoxide production (P < 0.01), following the cod liver oil supplementation. Analysis of PMNL and platelet fatty acids revealed the appearance of eicosapentaenoic acid and a significant decrease in arachidonic acid in both types of cells.     

Inhibitory Action of Fatty Acids on the Growth of Neisseria gonorrhoeae

Fatty acids of various chain lengths (C(1) to C(24)) were examined for their effects on growth, oxygen consumption, and in vitro reduced nicotinamide adenine dinucleotide oxidase activity of Neisseria gonorrhoeae CS-7. The growth inhibition caused by saturated fatty acids increased with increasing chain length to a maximum with palmitic acid (C(16)). Stearic acid (C(18)) and longer saturated fatty acids showed little inhibition of growth. However, unsaturated fatty acids of chain length C(16) to C(20) were inhibitory. Similar inhibition was observed with Bacillus subtilis and a deep rough mutant of Salmonella typhimurium. Wildtype S. typhimurium and Pseudomonas aeruginosa were more resistant to medium-chain (C(7) to C(10)) fatty acids and completely resistant to long-chain (C(12) to C(18)) fatty acids. Thus, sensitivity of N. gonorrhoeae to long-chain fatty acids appears to be related to the permeability of the outer membrane. Growth inhibition by short-chain (C(1) to C(6)) fatty acids was pH dependent; inhibition of growth increased with decreasing pH. Saturated fatty acids inhibited oxygen consumption by log-phase cells of N. gonorrhoeae. This inhibition increased with increasing chain length to a maximum observed with myristic acid (C(14)). Whereas stearic acid (C(18)) had little effect upon oxygen consumption, unsaturated C(18) fatty acids were inhibitory. An in vitro inhibition of reduced nicotinamide adenine dinucleotide oxidase activity by saturated (C(1) to C(12)) and unsaturated (C(16) to C(20)) fatty acids was also observed. Although the inhibitory concentrations were generally higher than those required to inhibit growth or oxygen consumption, an inhibition of electron transport may be partially responsible for the observed growth inhibition.     

Changes in phospholipid fatty acid composition and triacylglycerol content in mouse tissues after infection with bacille Calmette-Guérin.

Changes in the lipids of tissues from mice infected with bacille Calmette-Guérin (BCG) have been detected by gas-liquid chromatography. Infection with BCG resulted in (1) an increase in the polyunsaturated to saturated fatty acid ratio of phospholipids and (2) a decrease in the total triacylglycerol fatty acid content of spleen, liver and peritoneal macrophages. The alteration in fatty acid composition was significant in the phosphatidylethanolamine fraction of the phospholipids. The relation of these findings to an increased sensitivity to bacterial endotoxins is discussed.     

Production of luminol-reactive oxygen radicals during Plasmodium vinckei infection.

We tested the ability of whole blood and enriched fractions of peripheral blood polymorphonuclear leukocytes obtained from mice during the course of infection with Plasmodium vinckei to produce luminol-mediated chemiluminescence in response to phagocytic and nonphagocytic stimuli. The chemiluminescence response of whole blood to all stimuli increased dramatically and nonlinearly as the infection progressed, and there was a concomitant increase (80%) and decrease (70%) in the total numbers of leukocytes and erythrocytes, respectively. The proportion of polymorphonuclear leukocytes in the total leukocyte population increased threefold. On a per cell basis and at a constant hematocrit, the chemiluminescence response of peripheral leukocytes from infected animals to phorbol myristate acetate or opsonized zymosan was only slightly greater than that of cells from uninfected animals. Polymorphonuclear leukocytes isolated from the blood of infected animals also showed no large increase per cell in chemiluminescence responsiveness. Thus, although leukocyte numbers increase during a murine malarial infection, there appears to be no major change in the capacity of individual peripheral blood leukocytes to produce activated species of oxygen. However, the physiological reduction in the total concentration of hemoglobin at high parasitemia, due to hemolysis and hemoglobin digestion by the parasites, increases the possibility of oxygen radical-mediated damage to tissues and intraerythrocytic parasites as a result of decreased antioxidant protection.     

Role of 5-lipoxygenase-activating protein in the regulation of 5-lipoxygenase activity in human neutrophils

Using intact and fractionated human polymorphonuclear leukocytes (PMNL), we provide evidence that the enantioselective leukotriene synthesis inhibitor (LSI) BAY X 1005, (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid binds specifically to a high-affinity binding site, which is most likely identical to FLAP.BAY X 1005 blocks the translocation of 5-lipoxygenase (5-LOX) in PMNL stimulated by the calcium ionophore A23187 or chemotactic stimuli such as PAF, C5a or fMLP as does MK-886. In contrast to the direct 5-LOX inhibitors (LOI) A-64077 and AA-861, the degree of leukotriene synthesis inhibition declines with increasing duration of A23187-induced leukocyte activation in the presence of BAY X 1005 and MK-886. Kinetic studies performed with BAY X 1005 showed that this effect was not accompanied by a significant translocation of 5-LOX from the cytosol to the microsomal fraction. Because FLAP has been implicated in the transfer of arachidonic acid to 5-LOX and A23187 is a potent activator of leukocyte phospholipase A₂, we hypothesized that the observed loss of leukotriene synthesis inhibition may be due to competition of BAY X 1005 binding by endogenously released arachidonic acid. Accordingly, binding of BAY X 1005 to FLAP in intact and fractionated cells is dose-dependently inhibited by arachidonic acid and other unsaturated long-chain fatty acids, but not by saturated fatty acids. Therefore, we conclude that BAY X 1005 or MK-886 inhibit leukotriene biosynthesis by binding to FLAP, thereby preventing 5-LOX translocation and substrate transfer to the enzyme.     

Changes in phospholipid fatty acid composition and triacylglycerol content in mouse tissues after infection with bacille Calmette-Guérin

Changes in the lipids of tissues from mice infected with bacille Calmette-Guérin (BCG) have been detected by gas-liquid chromatography. Infection with BCG resulted in (1) an increase in the polyunsaturated to saturated fatty acid ratio of phospholipids and (2) a decrease in the total triacylglycerol fatty acid content of spleen, liver and peritoneal macrophages. The alteration in fatty acid composition was significant in the phosphatidylethanolamine fraction of the phospholipids. The relation of these findings to an increased sensitivity to bacterial endotoxins is discussed.     

Effect of amino acids on luminol-dependent chemiluminescence generated by peripheral blood polymorphonuclear leukocytes.

Individual amino acids and amino acid mixtures caused a dose-dependent increase in chemiluminescence generated by peripheral blood polymorphonuclear leukocytes. A visual assay for opsonophagocytosis, however, failed to identify any quantitative differences between leukocytes incubated with amino acids and those incubated in amino-acid-free solutions. The results of this study suggest that the presence of amino acids may interfere with the proper interpretation of luminol-dependent chemiluminescence curves.     

Fatty Acid Requirements of the Kazan 5 and Reiter Strains of Treponema pallidum

The fatty acid requirements of two avirulent treponemes were investigated by using a “lipid-poor” albumin-thioglycolate medium. The Kazan 5 and the Reiter stains of Treponema pallidum required a pair of fatty acids for growth. One member of the pair was saturated and the other was unsaturated. The saturated fatty acids could contain either an odd or even number of carbon atoms, but a chain length of at least 14 carbon atoms was necessary. Unsaturated fatty acids with one, two, or three double bonds were satisfactory if they contained 15 or more carbon atoms. The pair of fatty acids could be replaced with a single 18-carbon monounsaturated fatty acid if it was in the trans configuration rather than the naturally occurring cis form.