Generation of a chemotactic lipid from arachidonic acid by exposure to a superoxide-generating system

Certain products of arachidonic acid have been demonstrated recently to possess chemotactic activity for human polymorphonuclear leukocytes (PMN). Enzymatic (lipoxygenase, cyclooxygenase) generation of these lipid chemotaxins proceeds through the formation of intermediate lipid peroxides. Since lipid peroxidation can be mediated by oxygen-derived free radicals, we have examined whether chemotactically active products of arachidonic acid could be produced by exposing this unsaturated fatty acid to a superoxidegenerating system. A lipid with potent chemotactic activity for human PMN was produced by incubating arachidonic acid with xanthine oxidase and acetaldehyde. Generation of chemotactic activity was time-dependent and could be inhibited to the greatest extent by scavengers of singlet oxygen (i.e., histidine, uric acid, and 2,5-dimethylfuran). Inhibition was also observed with scavengers of superoxide anion radicals (i.e., superoxide dismutase), hydrogen peroxide (i.e., catalase), and hydroxyl radicals (i.e., mannitol). Silica gel thin-layer radiochromatography demonstrated a single peak with chemotactic activity (R_f = 0.33–0.38) distinct from unaltered arachidonic acid. The product of arachidonic acid was chemotactic at a concentration of 3.0 ng/ml and chemokinetic at concentrations of 0.75–1.5 ng/ml. Since PMN produce oxygen-derived free radicals and singlet oxygen upon stimulation of their plasma membrane, and since arachidonic acid is widely distributed in human tissues, free radical-mediated generations of chemotactic activity from arachidonic acid may play an important role in amplifying inflammatory responses.Supported by grants from the National Institutes of Health (AM-18531, AM-11949, AM-25374, AM-11949, HL-19721, and HL-18828) and the Kroc Foundation.Dr. Perez is the recipient of a Clinical Investigator Award (AM-00463) from the National Institutes of Health.Dr. Goldstein is the recipient of a Career Scientist Award from the Irma T. Hirschl Trust.     

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.     

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.     

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

Abstract

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.     

Proteoglycans, proteases, chemotaxis, and aggregation of inflammatory, cells.

A trypsin-like protease activity present in rabbit alveolar macrophages was shown to be activated by hyaluronic acid (HA) at 10(-9) M, but not by other proteoglycans. This activation of proteases by HA was accompanied by aggregation of alveolar macrophages and by the release of chemotactic activity. This chemotactic activity was destroyed by proteases and was less active for polymorphonuclear leukocytes than for alveolar macrophages. These effects of HA appear to be sepcific for alveolar macrophages, because no similar effects of HA on human polymorphonuclear leukocytes or lymphocytes could be observed. These observations suggest that HA, which is secreted into airways, plays a specific role in the macrophage-mediated chronic inflammatory processes in the distal lung.     

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.     

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.     

New aspects of chemotaxis. Specific target-cell attraction by lipid and lipoprotein fractions of Escherichia coli chemotactic factor.

A chemotactic factor extracted from sterile filtrates of Escherichia coli cultures was strongly chemotactic for polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (RAM). Electrophoresis of the cytotactic material yielded five lipid fractions: one that was protein-free and active toward both PMN and RAM, and four lipid-protein complexes that were strongly chemotactic only for RAM. Thin-layer chromatography of the lipid-protein complexes resulted in an unmasking of PMN activity in a peptide-free lipid extract, while the isolated peptidic components were essentially noncytotactic. The original RAM activity was retained in the unmasked lipid, which possessed chemical and chromatographic properties similar to those of a previously reported cytotaxin synthesized from arachidonic acid. These data indicate that a class of lipids derived from bacterial and cellular sources is intrinsically cytotactic for PMN and RAM. When peptide moieties are associated with cytotactic lipids, the resultant lipid-peptide complex may exhibit cellular specificity not evident in the free lipid.     

Macrophage-produced oxygen radical generating activities for polymorphonuclear leukocytes

Guinea pig peritoneal macrophages were stimulated in vitro by bacterial lipopolysaccharide. After incubation, the supernatants of macrophage cultures were collected and tested for O ₂ ^– production on guinea pig peritoneal polymorphonuclear leukocytes. The supernatants of macrophage cultures stimulated by lipopolysaccharide had significantly higher levels of O ₂ ^– -generating activities in polymorphonuclear leukocytes, and these activities appeared in the macrophage cultures within 2 h after stimulation by lipopolysaccharide However, the supernatants obtained from the nonstimulated cultures could not produce these activities. These activities disappeared with heating or trypsin and were not produced in macrophage cultures by incubation with cycloheximide.     

Inactivation of chemotactic activity of C5a by the serratial 56-kilodalton protease.

The effects of the 56-kilodalton protease (56K protease) from Serratia marcescens on complement-derived chemotactic activity were examined. Fresh human serum was incubated with zymosan to produce C5a. This activated serum was then incubated with various concentrations of 56K protease, and the chemotactic activity of mouse peritoneal exudate polymorphonuclear leukocytes (PMN) and macrophages was evaluated. A significant dose-dependent decrease of chemotactic activity was observed after protease treatment. Furthermore, treatment of human recombinant C5a with 56K protease at a dose of 1.0 microgram/ml resulted in a complete loss of chemotactic activity. When the living bacteria of the virulent strain, which produced about 10 times more protease than did the less virulent strain, were injected intraperitoneally into mice, the magnitude of infiltration of polymorphonuclear leukocytes into the peritoneal cavity was much lower than that caused by the less virulent strain. Because complement-dependent chemotactic activity is an initial response to bacterial infection, these results suggest indirect pathogenic functions of serratial proteases that suppress chemotactic activity.     

Chemotactic peptide-induced arachidonic acid mobilization in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes prelabeled with tritiated arachidonic acid liberated radiolabeled products when exposed to the chemotactic peptide fMet-Leu-Phe. The effect was enhanced in the presence of phorbol-12-myristate 13-acetate or 1-oleoyl-2-acetyl glycerol; these agents activate phospholipid- and Ca2+-dependent protein kinase C. In contrast, arachidonic acid mobilization was suppressed by two compounds known to inhibit protein kinase C activity: polymyxin B and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. These results suggest that protein kinase C was involved in arachidonic acid mobilization in leukocytes stimulated with chemotactic peptide.     

Chemotactic Factors Released in Culture by Intact Developing and Healing Skin Lesions Produced in Rabbits by the Irritant Sulfur Mustard

Developing, peak and healing lesions were induced in the skin of rabbits by topical applications (on different days) of the chemical irritant sulfur mustard (SM). Immediately after the rabbits were euthanized, the intact lesions were excised and organ-cultured for 17 to 20 hours. The culture fluids from early, peak and healing SM lesions all showed high chemoctactic activity for both PMN and MN. This finding suggests that the PMN and MN, seen microscopically in tissue sections of the lesions, were entering continuously, even during the healing process. The chemotaxins identified were the eicosanoid LTB₄, the chemokine IL-8, and proteases producing the complement fragment C5a. Other studies from our laboratory showed that the number of cells containing IL-1, IL-8, MCP-1, and GRO mRNAs was increased in SM lesions. Chemotactic activity was released by both live and dead (frozen and thawed) cell suspensions of PMN, MN, and fibroblasts, suggesting that these cells were major sources of the chemotaxins produced by the SM lesion explants. Explants of normal skin produced considerable chemotactic activity for MN, but not for PMN. Chemotactic activity for PMN, and the release of LTB₄, IL-8 and proteases cleaving C5 to C5a, occurred only in explants infiltrated by leukocytes.     

Polymorphonuclear leukocyte chemotaxis by mixed anaerobic and aerobic bacteria

The induction of chemotactic activity of polymorphonuclear leukocytes (PMNL) by anaerobic and aerobic bacteria alone or in combination was evaluated. Washed cells as well as the supernate of Proteus mirabilis were chemotactic for leukocytes. The supernate of cultures of two strains of Bacteroides fragilis contained small amounts of chemotactic factors. No chemotactic factors were released from the non-fragilis Bacteroides strains. The supernates of cultures of anaerobic bacteria were capable of inhibiting chemotaxis of leukocytes to the chemotactic factors of P. mirabilis. P. mirabilis and two strains of B. fragilis generated chemotactic factors in serum but none of the other Bacteroides spp. tested were able to induce serum chemotactic factors.     

Function of h(2)o(2), myeloperoxidase, and hexose monophosphate shunt enzymes in phagocytizing cells from different species

The effect of phagocytosis on the H₂O₂ production and myeloperoxidase (MPO) activities of leukocytes from various species was investigated. The intracellular distribution of MPO, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase (6PGDH) of resting and phagocytizing guinea pig polymorphonuclear leukocytes has also been studied. Phagocytizing cells produce more H₂O₂ than the corresponding resting cells. This has been found to be true for human peripheral polymorphonuclear leukocytes, mouse peritoneal macrophage, and guinea pig and rat peritoneal polymorphonuclear leukocytes. All of these cells, except rabbit alveolar macrophages, have significant MPO activity. Generally an increased activity is noted with phagocytizing cells. Homogenization and differential centrifugation of guinea pig peritoneal polymorphonuclear leukocytes indicate that the whole homogenate and its fractions from phagocytizing cells have significantly higher MPO and NADPH oxidase activities, when compared to the corresponding fractions from the resting cells. The 27,000 × g supernatant fluid from phagocytizing cells has 6-fold more MPO and 2.5-fold more NADPH oxidase activity than similar supernatant fractions from resting cells. The enzyme 6PGDH was unaffected by phagocytosis. The relationship of these stimulated activities to the intracellular bactericidal function of the phagocytes has been discussed.     

Total Antioxidant Capacity of Blood Plasma from Healthy Donors Receiving Vitamin and Mineral Complex

Using an original automated analyzer of double bonds we determined the rate constants for oxidation of saturated and unsaturated mono- and dienoic fatty acids (in vivo substrates for -oxidation in the mitochondria) by the ozone titration method. The rate constant for O₃ oxidation is maximum for oleic monoenoic acid, lower for dienoic linoleic, and very low for saturated palmitic acid. The rate constant for oxidation of oleic fatty acid, which by one order of magnitude surpasses that for oxidation of essential arachidonic acid, indicates that oleic acid is a leading in vivo acceptor of active O₂ species. By the rate of trapping active oxygen species and in the quantitative aspect, endogenously produced oleic acid can be regarded as the main biological antioxidant.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 517–519, November, 2004     

Peroxidase activity of alveolar macrophages.

Peroxidase activity was studied in alveolar macrophages and compared to the peroxidase activity in polymorphonuclear leukocytes using cytochemical techniques. A dense reaction product for peroxidase was observed in the primary lysosomes of polymorphonuclear leukocytes, but no significant peroxidase or peroxidative enzymes could be detected in rabbit alveolar macrophages. Furthermore, following vigorous phagocytosis of zymosan particles by alveolar macrophages in vitro, no peroxidase could be detected in association with the phagocytic vacuole. Exogenous horseradish peroxidase was ingested readily by alveolar macrophages so that abundant reaction product was demonstrated in pinocytotic vesicles and phagocytic vacuoles. The uptake of exogenous peroxidase by pinocytosis appeared to be more vigorous in alveolar macrophages than in polymorphonuclear leukocytes. These studies demonstrate that alveolar macrophages do not contain significant quantities of peroxidase and suggest that, it contrast to polymorphonuclear leukocytes, peroxidative metabolism does not contribute in a major way to microbial killing by alveolar macrophages.     

Quantitation of polymorphonuclear leukocyte migration under agarose by enzyme assay

Migration of polymorphonuclear leukocytes under agarose is widely used to assay the mobility of these cells. We modified this assay so that directed and random migration of polymorphonuclear leukocytes might be quantitated rapidly and objectively by an enzyme method. The optimal method used gelatin-agarose (0.25% gelatin; 0.875% agarose) gel layered on glass coverslips. At the end of the migration period, the leukocytes which had not migrated under the agarose were removed by aspiration and washing. The remaining leukocytes were solubilized with Triton X-100 and the myeloperoxidase activity on the coverslips determined. With appropriate standards, this activity could be used to estimate the number of polymorphonuclear leukocytes which had migrated in a random or in a directed (chemotactic) manner. Excellent correlation was observed between the myeloperoxidase method and direct microscopic counting of the number of cells that had migrated. The sensitivity of the assay could be enhanced by inducing bidirectional migration under 2 simultaneous gradients of chemotactic factor situated 180 degrees apart. This assay is objective, reproducible and combines the simplicity of assessing migration by measuring distance of the 'leading cell front' with the accuracy of individual cell counting.     

Anaerobic phagocytosis, killing, and degradation of Streptococcus pneumoniae by human peripheral blood leukocytes.

Encapsulated Streptococcus pneumoniae of serotypes 2, 9N, 14, 21, and 23F and an unencapsulated variant of type 2 pneumococci were efficiently phagocytosed by both aerobically and anaerobically incubated human leukocytes. In the presence of O2, the pneumococci rapidly lost their viability, whereas during anaerobiosis, killing was considerably delayed. Type 14 pneumococci radiolabeled with [14C]choline or [14C]ethanolamine for cell wall teichoic acid, [14C]uracil for nucleic acids, or [14C]arachidonic acid for unsaturated cytoplasmic membrane lipids were used in studies of the fate of bacterial macromolecules after phagocytosis. The degradation of teichoic acid, RNA, and DNA during anaerobiosis approached that recorded in air at 60 min of incubation (45 to 70% and 55 to 75%, respectively). In contrast, the marked loss of [14C]arachidonic acid from pneumococcal membrane lipids observed in aerobic leukocytes did not occur during anaerobic incubation. Hence, lipid peroxidation could be involved in the rapid aerobic leukocyte killing of pneumococci, whereas a different leukocyte function of as yet unknown nature appears to be responsible for the killing seen in anaerobiosis. Autolysis-resistant type 14 pneumococci were obtained by substituting ethanolamine for choline in a defined culture medium. Differences between such bacteria and normal (autolytic) pneumococci in their killing and degradation by leukocytes were not detected in either the presence or the absence of O2. The aerobic and anaerobic handling of phagocytosed pneumococci by human blood leukocytes thus proceeded independently of the bacterial autolytic system.     

Chemotactic activity for polymorphonuclear leukocytes: meconium versus meconium-stained amniotic fluid

PROBLEM: Our previous study indicated that meconium-stained amniotic fluid (turbid AF) possessed a potent chemotactic activity for leukocytes, which may be dependent on interleukin-8. It is not known, however, whether meconium itself possesses this chemotactic activity. METHOD OF STUDY: Meconium samples were collected from mature neonates with and without turbid AF. A 5% meconium suspension in phosphate buffered saline was prepared and measured for its chemotactic activity for leukocytes using the blind well chamber technique. Concentrations of IL-8, TNFalpha and IL-1beta were also measured with ELISA. RESULTS: The number of leukocytes that migrated to the meconium suspension (35 +/- 27) was comparable with that of the clear AF (31 +/- 37), but was significantly lower than that of the turbid AF (184 +/- 62, P < 0.0001). The meconium suspension contained much lower levels of IL-8, TNFalpha and IL-1beta than the turbid AF. CONCLUSIONS: Meconium itself exhibits a lower chemotactic activity for polymorphonuclear leukocytes than turbid AF in vitro. The leukocyte chemotactic activity of turbid AF does not originate from meconium itself.     

Inhibition of the accumulation of macrophages and the generation of macrophage chemotactic activity by dexamethasone in concanavalin A-induced peritonitis of mice

A delayed-type inflammatory response was evoked in mice using concanavalin A (Con A) as a stimulus, and the effect of various anti-inflammatory agents on the inflammations was examined. The intraperitoneal injection of Con A in the mouse resulted in the marked accumulation of leukocytes, especially macrophages, in the peritoneal cavity between 16 and 48 hr after the injection. Prior to the accumulation of macrophages, the chemotactic activity for macrophages appeared in the peritoneal fluid, and was associated with protein(s) in the molecular weight range from 10000 to 100000 daltons. When the effect of various agents on Con A-induced peritonitis was examined, neither anticomplementary agents (FUT-175 and K-76 COONa), bromophenacyl bromide, nordihydroguaiaretic acid nor indomethacin affected the generation of chemotactic activity and the accumulation of macrophages, suggesting that C5a, prostaglandins and leukotriene B₄ are hardly involved in the Con A-induced macrophage accumulation. On the other hand, dexamethasone suppressed both the generation of chemotactic activity and the accumulation of macrophages. Taking into consideration the observation that the synthesis of macrophage chemotactic factors by mitogen-stimulated lymphocytes is inhibited by glucocorticoids these results suggest that the macrophage chemotactic lymphokines might be involved in the accumulation of macrophages in Con A-induced peritonitis.