Enzyme Activity Of Macrophages Research Articles

Methyl and Isopropyl N-Methylanthranilates Affect Primary Macrophage Function - An Insight into the Possible Immunomodulatory Mode of Action.

To complement the knowledge on the anti-inflammatory activity of methyl and isopropyl N-methylanthranilates, two natural products with panacea-like properties, we investigated their effects on thioglycolate-elicited macrophages by evaluating macrophage ability to metabolize MTT, macrophage membrane function, and macrophage myeloperoxidase and phagocytic activities. Moreover, two additional aspects of the inflammatory response of these compounds, their inhibitory activity on xanthine oxidase and catalase, were studied. It was found that these two compounds regulate elicited macrophage functions, most probably by interfering with the function of cell membranes and changing the reducing cellular capacity or enzyme activity of macrophages. Nonetheless, no significant inhibitory action either towards xanthine oxidase or catalase was found, suggesting that the inhibition of these enzymes is not involved in the anti-inflammatory mode of action of these two esters.

Abstract 10865: Elucidating the Variant-to-Function Relationship for LIPA, a Risk Locus of Coronary Artery Diseases

Introduction: Genome-wide association studies revealed a robust association between genetic variants at the LIPA (lysosomal acid lipase) locus and coronary artery diseases (CAD). eQTL studies support that the risk alleles of LIPA CAD variants are associated with higher LIPA mRNA and enzyme activity in human monocytes, but not other blood or vascular cells, suggesting that increased myeloid LIPA may confer CAD risk. Herein, we aim to establish the causality of the variant-to-function relationship for the LIPA locus and elucidate how increased myeloid LIPA impact atherosclerosis in vivo. Hypothesis: We hypothesized that causal variants in LIPA lead to increased LIPA expression and enzyme activity in macrophages and myeloid-specific overexpression of Lipa promotes atherosclerosis. Results: We first confirmed that in human monocyte-derived macrophages, LIPA mRNA, protein and enzyme activity are higher in the risk allele carriers of CAD variants. High-resolution HiC revealed an intronic enhancer region showing strong interaction with the LIPA promoter. Within the enhancer region, both rs1320496 and rs1412445 had independent association with CAD and their risk alleles led to increased enhancer activity measured by luciferase assay. Risk allele of rs1320496 also demonstrated increased binding for PU.1, a myeloid-specific transcription factor. To establish how increased myeloid LIPA impact atherosclerosis, we generated myeloid-specific Lipa overexpression mice ( Lipa Tg , Ldlr -/- ). Lipa Tg significantly increased atherosclerotic lesion size without affecting plasma cholesterol level. scRNA-seq analysis showed that Lipa Tg led to reduced lipid-enriched yet increased inflammatory macrophage subsets, and upregulation of genes in chemokine signaling. This is further confirmed by reduced neutral lipid accumulation in both plaque and peritoneal macrophages in Lipa Tg mice. Mechanistically, Lipa Tg led to reduced expression of modified-LDL receptors, increased expression of inflammatory chemokines, and increased circulating IL18 and CXCL2 that likely drive immune cells infiltration during atherosclerosis. Conclusions: We for the first time established that LIPA risks alleles drive increased myeloid LIPA and aggravate atherosclerosis.

A preliminary study on the proinflammatory mechanisms of Treponema pallidum outer membrane protein Tp92 in human macrophages and HMEC-1 cells

AimsTo determine proinflammatory mechanisms of Treponema pallidum outer membrane protein Tp92 in the early syphilis infection in human macrophages and HMEC-1 cells. MethodsRecombinant Tp92 protein was used to stimulate target human macrophages and HMEC-1 cells. PDTC (Pyrrolidinedithiocarbamic acid), SB202190 and Z-YVAD-FMK were used to block the MyD88/NF-κB, MAPKs/p38 and NLRP3/Caspase-1 pathway, respectively. TNF-α, IL-1β, IL-6, IL-8,NLRP3, casepase-1 were detected by ELISA or Western blot. Lactate dehydrogenase (LDH) activity was measured. ResultsTp92 protein could significantly induced the secretion of proinflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 in HMEC-1 cells, but not in macrophages except IL-8. When MyD88/NF-κB pathway was blocked, differences in the secretion of TNF-α, IL-6 and IL-1β levels and LDH enzyme activity between Tp92 group and Tp92 + PDTC group were not significant (P > 0.05) in HMEC-1 cells and macrophages except IL-8(P < 0.05). When MAPKs/p38 pathway was blocked, differences in the secretion of TNF-α, IL-1β, IL-6 and IL-8 and LDH enzyme activity both Tp92 group and Tp92 + SB2010190 group were not significant (P > 0.05) in HMEC-1 cells and macrophages. In contrast, when NLRP3/Caspase-1 pathway was blocked with Z-YVAD-FMK, TNF-α, IL-6 and IL-1β levels, LDH enzyme activity, and Caspase-1 and NLRP3 protein levels were significantly declined (P < 0.05) in HMEC-1 cells except IL-8(P > 0.05). The LDH enzyme activity in macrophages was decreased before and after Z-YVAD-FMK blocking (P < 0.05),however, differences in the secretion of TNF-α, IL-1β, IL-6 and IL-8 between Tp92 group and Tp92+Z-YVAD-FMK group in macrophages were not significant (P > 0.05). ConclusionsTp92 protein may promote proinflammatory cytokines TNF-α, IL-1β, IL-6 secretion of HMEC-1 cells, but not in macrophages, and increase the LDH enzyme activity of HMEC-1 cells and macrophages through NLRP3/Caspase-1 pathway. However, Tp92 protein may promote IL-8 secretion of HMEC-1 cells and macrophages through MyD88/NF-κB pathway.

Enzyme characterisation and gene expression profiling of Atlantic salmon chicken- and goose-type lysozymes

Lysozymes represent important innate immune components against bacteria. In this study, Atlantic salmon (Salmo salar) goose (g-) and chicken (c-) types of lysozyme were subjected to protein characterisations and tissue expression analyses. Specific bacterial protein inhibitors of g- and c-type lysozymes were employed to discriminate between respective enzyme activities. Blood, gills and liver contained activities exclusive for the g-type lysozyme. Only haematopoietic organs (head kidney and spleen) contained enzyme activities of both g- and c-lysozyme enzymes and c-type activity was not found outside these organs. Gene transcript levels proportional to enzyme activity levels were detected for the g-type lysozyme but not for the c-type. In vitro studies revealed significant induction of c-type gene expression and enzyme activity in macrophages after incubation with lipopolysaccharide (LPS) while expression of the g-type lysozyme gene was unaffected. The activity of purified native c-type enzyme was profoundly reduced by divalent cations and displayed low tolerance to monovalent cations, while the native g-type lysozyme was stimulated by monovalent cations and tolerated low concentrations of divalent cations. Activities of both enzymes increased with temperature elevations up to 60°C. The native g-type lysozyme responses to temperature in particular are in apparent conflict to the ones for the recombinant salmon g-lysozyme.Our results imply separate expression regulations and different functions of c- and g-type lysozymes in salmon. LPS-induced expression of c-type lysozyme and broad constitutive tissue distribution of g-type lysozyme in salmon is different from findings in other studied fish species.

The Role of Progestogens in Regulating Matrix Metalloproteinase Activity in Macrophages and Microglial Cells

Although the systemic effects of progestogens have been extensively studied, little is known in regards to the cellular effects of these compounds. Using a cellular model for vascular (macrophages) and brain (microglial) cells, we studied the effects of various progestogens, either alone or in combination with 17β-estradiol (E(2)) on the activity of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme involved in vascular remodeling and plaque destabilization in cardiovascular events, blood-brain barrier breakdown in stroke and brain regeneration and neurovascular remodeling during repair phases of brain injury. In the absence of E(2), medroxyprogesterone acetate (MPA), a synthetic progestogen and progesterone (PG) metabolites tended to increase MMP-9 enzyme activity in macrophages and microglial cells, whereas PG decreased such activity in macrophages; exceptions being that MPA and the PG metabolite, pregnanediol (Pdiol) had no effect on macrophage MMP-9 enzyme activity and PG had no effect on microglial cell MMP-9 enzyme activity. In the presence of E(2), an opposite affect was observed whereby MPA and the PG metabolites tended to decrease MMP-9 enzyme activity from macrophages and microglial cells, whereas PG had no effect; exceptions being that MPA and Pdiol had no effect on macrophage MMP-9 enzyme activity. In conclusion, these results demonstrate that the effects of PG, PG metabolites and MPA on MMP-9 enzyme activity differ across vascular and brain cells when administered alone or in combination with E(2) which could have important mechanistic implications for hormone therapy.

Neurotrophins in COPD

Background: Numerous studies have demonstrated evidence of oxidative stress in COPD, in both respiratory tract lining fluids and biopsies. Antioxidant response in airway inflammatory cells to a pro-oxidative environment is however poorly understood. We have previously demonstrated an enhanced antioxidant enzyme activity in macrophages from asthmatics. This study was performed to assess whether a similar adaptation in intra-cellular antioxidants occurred in subjects with COPD. The activities of enzymatic antioxidants were examined in alveolar mixed cell populations and compared to healthy age-matched controls (ACs) and young adults, to permit the relative contributions of disease state and natural ageing to be disentangled.

Antitumor Effects and Immunomodulating Activities of Phellinus linteus Extract in a CT-26 Cell-Injected Colon Cancer Mouse Model

The antitumor effects of Phellinus linteus extract (Keumsa Linteusan) were investigated in a CT-26 cell-injected colon cancer mouse model. When administered orally (250~1,000 mg/kg body weight), Keumsa Linteusan significantly inhibited the growth of solid colon cancer. The highest dose was highly effective, reducing tumor formation by 26% compared with the control group. The anticomplementary activity of Keumsa Linteusan increased in a dose-dependent manner. Lysosomal enzyme activity of macrophages was increased by 2-fold (100 μg/ml) compared with the control group. Keumsa Linteusan can be regarded as a potent enhancer of the innate immune response, and can be considered as a very promising candidate for antitumor action.

Polysaccharide isolated from Glycyrrhiza uralensis Fisch induces intracellular enzyme activity of macrophages

Polysaccharide isolated from Glycyrrhiza uralensis Fisch induces intracellular enzyme activity of macrophages

Polysaccharide isolated from Glycyrrhiza uralensis Fisch induces intracellular enzyme activity of macrophages

A purified fraction of water-soluble polysaccharides was isolated from Glycyrrhiza uralensis Fisch using ion exchange and size exclusion chromatography. The present study was undertaken to discuss the preliminary immunoregulation mechanism of glycyrrhiza polysaccharide (GP) by cytochemistry and quantitative analyses, and intracellular enzyme measurement of macrophages. Acid phosphatase (ACPase), adenosine triphosphatase (ATPase), acid α-naphthyl acetate esterase (ANAE) and succinate dehydrogenase (SDH) in macrophages were stained with different methods. The results indicate that GP increased the production of ACPase, ATPase, ANAE and SDH; the activities of lysozyme (LSZ) and superoxide dismutase (SOD) of macrophages were also induced by GP. Our data suggest that the beneficial therapeutic effects of GP may be attributed partly to its ability to modulate macrophage immune functions.

Regulation of antioxidant enzyme activities in male and female rat macrophages by sex steroids.

Human and animal immune functions present sex dimorphism that seems to be mainly regulated by sex hormones. In the present study, the activities of the antioxidant enzymes total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured in intraperitoneal resident macrophages from adult male and female rats. In addition to comparing males and females, we also examined the regulation of these enzyme activities in macrophages by sex steroids. GSH-Px activity did not differ between male and female macrophages. However, both total SOD and CAT activities were markedly higher in females than in males (83 and 180%). Removal of the gonads in both males and females (comparison between castrated groups) increased the difference in SOD activity from 83 to 138% and reduced the difference in CAT activity from 180 to 86%. Castration and testosterone administration did not significantly modify the activities of the antioxidant enzymes in male macrophages. Ovariectomy did not affect SOD or GSH-Px activity but markedly reduced (48%) CAT activity. This latter change was fully reversed by estrogen administration, whereas progesterone had a smaller effect. These results led us to conclude that differences in the SOD and CAT activities may partially explain some of the differences in immune function reported for males and females. Also, estrogen is a potent regulator of CAT in macrophages and therefore this enzyme activity in macrophages may vary considerably during the menstrual cycle.

The expression and regulation of inducible nitric oxide synthase gene differ in macrophages from chickens of different genetic background

It has been previously reported that lipopolysaccharide (LPS)-stimulated macrophages from Cornell K-Strain chickens (B 15B 15) and a transformed cell line, MQ-NCSU, (broiler origin) produced significantly higher levels of inducible nitric oxide synthase (iNOS) mRNA than macrophages isolated from GB1 (B 13B 13) and GB2 (B 6B 6) chickens. The purpose of this study was to determine the basis of such differential iNOS gene expression and to study the relationship of high or low expression of iNOS mRNA with iNOS enzyme activity in macrophages from GB2 (low iNOS mRNA expresser), K-strain and MQ-NCSU (high iNOS mRNA expressers). The enzyme activity in lysates from LPS-stimulated macrophages was lower in GB2 (range: 23 to 41 μM, P<0.05) as compared with the K-strain and MQ-NCSU macrophages that exhibited intermediate (range: 27 to 59 μM) and the highest (range: 144 to 217 μM) activity, respectively. Total RNA collected from LPS-treated macrophages at various time-points post-actinomycin D treatment revealed comparable iNOS mRNA levels in MQ-NCSU, GB2, and K-strain macrophages, suggesting that post-transcriptional regulation mechanism(s) do not account for the difference in iNOS mRNA expression. To determine if differences in the transcription rate are the basis of the differential iNOS gene expression, macrophages were stimulated with or without LPS and nuclei-isolated. Inducible NOS mRNA probes were generated and hybridized with immobilized iNOS cDNA (reverse Northern blot). The resulting lumigraph yielded enhanced transcriptional activity from K-strain and MQ-NCSU macrophages whereas this activity was lower in GB2 macrophages. Therefore, these studies suggest that the previously reported genetically-based difference in iNOS mRNA expression further translates into differences in iNOS enzyme activity, and that the iNOS gene in chickens is transcriptionally regulated.

Anti-Osteoporotic activity of metal complexes of amine carboxyboranes

Metal complexes of trimethylamine carboxyborane successfully suppressed calcium flux from both paired pup calvaria bones and rat UMR-106 osteosarcoma cultured cells over a 48 h period. These agents increased uptake of calcium into the cell cultures and accelerated [3H]proline incorporation into collagen. Copper and iron complexes of the trimethylamine carboxyborane were more potent compared with the cobalt and chromium complexes. The agents effectively reduced Iysosomal enzyme activity and also proteolytic enzyme activities of macrophages. Since macrophages invade the bone surface and assist in the demineralization and digestion of collagen, those agents may be potentially useful to retard diseases involving bone reconstruction. Influx of white blood cells and macrophages to sites of degradation most probably would be inhibited by the agents, based on sponge test observations in mice. Osteoporosis induced by ovariectomy was minimized by injections of tetrakis[u-(trimethylamine-boranecarboxylato)-bis(trimethylamine-carboxyborane)dicopper(II)] into rats at 3.5 mg kg−1 day−1 for 14 days. Bone volume, density, weight and calcium content returned to normal baseline control values. In addition, the copper complex returned serum calcium, serum parathyroid hormone (PTH) and vitamin D3 values to normal levels. One possible mode of action of these derivatives is the regulation of the production and release of chemical mediators initiating bone loss, e.g. tumor necrosis factor, TNF α and interleukins 11 or 11-2.

Effect of Vitamin E Deficiency on Oxidative Metabolism and Antioxidant Enzyme Activity of Macrophages

In AUG rats, deprived of vitamin E for 90 days, we noted a 3-fold increase of kinetic parameters of luminol-dependent chemiluminescence of macrophages, stimulated with opsonized zymosan, superoxide dismutase activity decrease and increment of plasma membrane lipid bilayer microviscosity, which was estimated by fluorescent probe pyrene eximerization method. Vitamin E deficiency did not affect glutathione peroxidase and glutathione reductase activities of macrophages.

Proteolytic enzyme activities of macrophages and lymphocytes in neurological diseases

Studies showed a significant decrease in the macrophage neutral protease and lymphocyte acid protease activities in patients with multiple sclerosis in remission, a significantly decreased neutral protease activity in macrophages in patients with myasthenia gravis and a significantly decreased acid protease activity in macrophages and lymphocytes in patients with polymyositis. No remarkable abnormalities were found in patients with myotonic dystrophy. These results suggest that multiple sclerosis, myasthenia gravis and polymyositis have an abnormality in immunological function.

Possible involvement of angiotensin I-converting enzyme in the inactivation of bradykinin by intact macrophages

As intact macrophages inactivated bradykinin, the subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig macrophages. The bradykinin-inactivating activity was found to be present in membrane and cytosol fractions but not in granular and nuclear fractions. The bradykinin-inactivating activity of the membrane fraction was inhibited by captopril, a specific inhibitor of angiotensin I-converting enzyme, whereas that of the cytosol fraction was hardly inhibited by various proteinase inhibitors used. Angiotensin I-converting enzyme activity was located predominantly in the membrane fraction and its activity was inhibited by captopril. Angiotensin I-converting enzyme activity measured with a synthetic substrate was competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for macrophage angiotensin I-converting enzyme. When macrophages were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent, both the bradykinin-inactivating activity and the angiotensin I-converting enzyme activity of macrophages decreased significantly without any inhibition of the cytosol bradykinin-inactivating activity. These findings seem to suggest that the angiotensin I-converting enzyme would be responsible for the inactivation of bradykinin in intact macrophages.

Lipolytic Enzyme Activity of Macrophages in Bovine Mammary Gland Secretions

The ability of macrophages isolated from the involuted bovine mammary gland and pooled raw milk to secrete lipolytic enzymes was investigated. Macrophages obtained from the involuted gland and maintained in cell culture secreted lipolytic enzymes into culture medium for up to 120h. Leukocytes in pooled raw milk were separated using Ficoll discontinuous density gradients. Macro-phages secreted lipolytic enzymes into the gradient while fractions containing polymorphonuclear leukocytes and lymphocytes did not possess lipolytic activity. Enzyme activity of macrophages from pooled raw milk averaged .1% of the total milk lipoprotein lipase activity present in the original milk samples. Fresh raw milk with a macrophage concentration increased to 2.5×106 cells/ml contained 11.6% higher milk lipoprotein lipase activity after storage for 48h at 4°C. These results indicate that macrophages isolated from bovine mammary secretions produce lipolytic enzymes that could influence milk lipoprotein lipase activity in raw milk over storage.

Immunoactive peptides, FK 156 and FK 565. IV. Activation of mouse macrophages.

We investigated the effects of the immunoactive peptides, FK 156 and FK 565, on functions of mouse macrophages. FK 156 and FK 565 given parenterally or orally to mice enhanced spreading of peritoneal macrophages, phagocytosis of latex particles and intracellular killing of bacteria by peritoneal macrophages. FK 156 and FK 565 also enhanced the production of superoxide anion and lysosomal enzyme activities of macrophages. The peptides also activated mouse spleen macrophages, and the kinetics of this activation differed from that of the peritoneal macrophages. In addition, both drugs directly enhanced the production of superoxide anion by mouse peritoneal macrophages treated in vitro and enhanced the functions of peritoneal macrophages of athymic nude mice. Both these phenomena suggest that direct activation might be one of the mechanisms of macrophage activation by the peptides.

Leishmania donovani: Action of excreted factor on hydrolytic enzyme activity of macrophages from mice with genetically different resistance to infection

The effect of purified excreted factor from promastigotes of Leishmania donovani upon the activity of four enzymes from lysed peritoneal exudate cells of mice (C3H and C57BL) was determined. There was no demonstrable effect on acid phosphatase (EC3.1.3.2), β-glucuronidase (EC3.2.1.21), and N-acetyl-β-glucosaminidase (EC3.2.1.29), but β-galactosidase (EC3.2.1.23) was inhibited up to 72% after 3 hr of incubation at 37 C. Inhibition of C57BL mouse enzymes was not significantly different from that of C3H mice. Protamine sulfate combined with the highly negatively charged excreted factor of L. donovani to migrate as a single positively charged band on immunoelectrophoresis. Protamine sulfate also reversed the β-galactosidase inhibition, though this was without direct effect on the enzyme. The excreted factor did not change or lose its charge or antigenicity with regard to precipitating antibody, when incubated with extracts of mouse peritoneal exudate cells, splenocytes, or liver homogenate—irregardless of whether the mice had been infected with leishmaniasis for 1 or 2 weeks or were uninfected.

Effect of irradiation on lysosomal enzyme activation in cultured macrophages.

The effect of gamma-rays on lysosomal enzyme activity of normal and immune macrophages of DBA/2 mice cultured in vitro has been studied. Quantitative cytochemical methods have been used for measuring lysosomal enzyme activity. A dose of 500 rad did not significantly affect lysosomal enzyme activity 3 hours after irradiation but caused the activity to increase to 1x4 times the control value 22x5 hours after irradiation. 22x5 hours after a dose of 3000 rad the enzyme activity increased to 2x5 times the control. Lysosomal enzyme activity of the macrophages was also markedly increased by immunization of the mice with D lymphoma cells, before culture in vitro, but irradiation of these cells with a dose of 500 rad caused a further increase in lysosomal enzyme activity. The results indicate that immunization and irradiation both cause stimulation of lysosomal enzyme activity in macrophages but that the mechanisms of activation are unlikely to be identical.

Comparison of Peritoneal Macrophages from Germfree and Conventional Mice

Morphology, lysosomal enzyme activities, and phagocytosis via immunological receptors were tested in peritoneal macrophages from germfree and conventional mice. Nonstimulated macrophages from germfree mice showed less spreading and were more easily detached when seeded on glass than conventional macrophages. The activities of the lysosomal acid phosphatase and cathepsin D were similar in the two cell groups, whereas beta-glucuronidase showed higher activity in macrophages from germfree mice. F(c) receptor-mediated phagocytosis of opsonized sheep erythrocytes was equally effective in germfree and conventional macrophages, and both cell types attached but did not internalize erythrocytes via the C(3)b receptor. Intraperitoneal injections of mineral oil caused a significantly higher influx of macrophages in conventional mice than in germfree mice, whereas the influx of polymorphonuclear cells was enhanced in both animals. Stimulation in vivo with oil or Escherichia coli endotoxin increased cell size, spreading ability, membrane ruffling, and lysosomal enzyme activities in macrophages from both conventional and germfree mice. The Fc-mediated phagocytosis was not influenced by stimulation, whereas the capacity to internalize via C(3)b receptor was triggered in macrophages from conventional mice, but not in corresponding cells from germfree mice. Similar results were obtained after stimulation with endotoxin in vitro. Culture in fetal calf serum for 72 h caused intracellular rises in all three enzyme activities tested in macrophages from conventional mice, whereas only the activity of acid phosphatase was increased in macrophages from germfree mice. Stimulation with zymosan in vitro caused selective release of lysosomal enzyme activity in macrophages from both animal groups. We conclude that peritoneal macrophages from germfree mice share several properties with cells from conventional mice, however, unstimulated beta-glucuronidase activity was increased, whereas spreading on glass, chemotactic response, in vitro induction of lysosomal enzymes, and the capacity to internalize via the C(3)b receptor after stimulation were reduced or absent.