Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.     

Ultrastructural cytochemistry of non-specific esterase in murine peritoneal macrophages

Summary Non-specific esterase (NSE) activity was demonstrated in glutaraldehyde-fixed monolayers of murine peritoneal macrophages. Using 2-naphthylthiol acetate (NTA) as substrate and Fast Blue BB as coupling agent a strong osmiophilic reaction product was obtained. The reaction product was observed as electron-dense dots covering cisterns of the rough endoplasmic reticulum, Golgi saccules and vesicles, or as large aggregates in lysosomes. Using -naphthyl butyrate (ANB) as substrate and hexazotized pararosaniline as coupling agent the osmiophilic reaction product was observed extracellularly on the plasma membrane as an electron-dense continuous layer, whereas intracytoplasmic staining of lysosomes was rare. Substitution of the coupling agents in the respective media resulted in a slight reaction with the ANB medium whereas with the NTA medium reaction product was observed only in lysosomal structures. The substrate specificity of the different types of esterases was confirmed after isoelectric focusing on thin-layer polyacrylamide gels. The results indicate that in murine peritoneal macrophages different types on NSE are detected with NTA and ANB, having distinct ultrastructural localizations.     

Simultaneous azo-coupling method for steroid acetate hydrolyzing enzyme

A simultaneous azo-coupling method for histochemical localization of steroid acetate hydrolyzing enzyme is described. It is based on the observation that d-equilenin, a natural oestrogenic steroid hormone, forms deeply coloured insoluble reaction products with diazonium salts under reaction conditions suitable for histochemical purposes. An acetate at position 3 of d-equilenin is rapidly hydrolysed by tissue esterase and the liberated d-equilenin couples with a diazonium salt to form a coloured precipitate. Steroid acetate hydrolyzing enzyme activity was observed in various tissues of the rat; a comparison with nonspecific esterase activity using alpha-naphthyl acetate as substrate suggested that steroid acetate hydrolyzing enzyme activity represents the activity of one or several isozymes of classical nonspecific esterase. This conclusion has also been drawn previously from biochemical studies using esters of other steroids.     

The location of non-specific esterase in human lung macrophages

Summary The non-specific carboxyl (serine) esterase of the human pulmonary alveolar macrophage was localized ultrastructurally using -naphthyl acetate and hexazotized pararosanilin. The reaction product principally outlined the outer side of the plasma membrane. Consequently, this esterase is an ectoenzyme which may function as mediator of cell response to injurious agents from the outside.     

A cytochemical method for the demonstration of 5′-nucleotidase in mouse peritoneal macrophages,with cerium ions used as trapping agent

Summary A method was developed for the demonstration of 5-nucleotidase in murine peritoneal resident macrophages. The cells are incubated cytochemically without agitation and cerium chloride is used as a trapping agent. Under these conditions, the great majority of the macrophages in the unstimulated peritoneal cavity show enzyme activity in the plasma membrane. In the presence of AMP-S (an AMP analogue inhibiting 5-nucleotidase, as shown biochemically) there was a decrease in both the number of positive macrophages and the amount of reaction product on the plasma membranes. This indicates that the enzyme activity detected by our cytochemical procedure is attributable to 5-nucleotidase.     

Cathepsin B and D activity in stimulated peritoneal macrophages

Summary Highly sensitive and specific synthetic substrates were used to quantitate cathepsin B and D activity in peritoneal macrophages in response to stimulation in vivo with mineral oil and thioglycollate. After intraperitoneal instillation of mineral oil the activity of cathepsin B increased significantly (to 15 300 units/mg protein versus 7 340 in saline controls), reaching values approaching those found in alveolar macrophages (18 400 units/mg protein). Significantly greater stimulation of enzyme activity was obtained after intraperitoneal instillation of thioglycollate (23 600 units/mg protein). Cathepsin D activity also increased significantly after both mineral oil and thioglycollate. However, the increase was moderate (from 806 to about 1 200 units/mg protein), remaining still more than six times lower-than in alveolar macrophages. The data are the first to demonstrate that cathepsin B activity can be stimulated in vivo in peritoneal macrophages by instillation of agents that induce acute inflammation. They also point to a differential control of expression of cathepsin B and D activity in both peritoneal and alveolar macrophages in spite of the common lysosomal origin of the two enzymes.Abbreviations Cbz -N-benxyloxycarbonyl - 2NA 2-naphthylamine - EDTA ethylenediamine tetraacetate - DMSO dimethylsulfoxide - PBS phosphate-buffered saline - PM peritoneal macrophage - AM alveolar macrophage     

Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages.

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.     

Analytical isolation of plasma membranes of intestinal epithelial cells: Identification of Na,K-ATPase rich membranes and the distribution of enzyme activities

Summary A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and cole material. Sucrase specific activity in the purified brush border plasma membrane was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochromec reductase, nonspecific esterase, -glucoronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity.Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent³⁵S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein.Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils

We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.     

Pitfalls in ecto-5''-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages

Summary We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killedEscherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5-nucleotidase onto the adhering particle was fact or artifact, we localized 5-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.     

Specificity and induction of undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone

Undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone was strongly inhibited by organophosphates and other esterase inhibitors. Also, p-chloromercuribenzoate at 1 x 10(-4) M showed a 70% inhibition of esterase activity. The enzyme hydrolyzed both aliphatic and aromatic acetate esters at substrate concentrations of 0.25 M. Under these conditions the highest reaction rate was toward undecyl acetate. No lipase or proteolytic activity was demonstrated. Undecyl acetate esterase was classified as a carboxylesterase (B-esterase). Cell-free activity studies on the production of undecyl acetate esterase grown on different carbon sources plus zymogram studies demonstrated that the enzyme was inducible when 2-tridecanone, 2-tridecanol, undecyl acetate and, to a lesser extent, 1-undecanol were growth substrates. Induction of undecyl acetate esterase during oxidation of 2-tridecanone supports the view that undecyl acetate is an intermediate in the degradation of the methyl ketone.     

Ultrastructural demonstration of acetylcholinesterase activity of motor endplates via osmiophilic diazothioethers

Summary The ultrastructural chemical localization of acetylcholinesterase of motor endplates of rat intercostal muscle has been studied with three new esterase substrates. These substrates, although not specific for acetylcholinesterase, have differential affinities for various types of esterases; two of them (NTA and TAB) are hydrolyzed preferentially by acetyl esterase enzymes, and the third (TPB) is a propionic acid ester and is hydrolyzed preferentially by pseudocholinesterase and other esterases. The end-product of the enzymatic reaction is converted to a diazothioether (droplet form) and upon osmication this is converted to a coordination polymer of osmium which has ideal properties for electron microscopy.Although this study supports previous observations that enzymatic activity can be found primarily on the post-synaptic membranes of the motor endplate, no enzymatic activity was noted on the pre-synaptic membrane, within the synaptic cleft, or on the basement membrane unless incubation was prolonged, resulting in overstaining. Neither was enzyme activity seen on membrane-free ribosomes and the ribosome-studded sarcoplasmic reticulum. Axonal vesicles also failed to exhibit enzymatic activity which had been noted with the method using thiolacetic acid and lead. A correlation of esterase activity with ultrastructural localization, using the substrate TPB, suggests that a buffer zone of nonspecific esterase activity is present beneath the subneural apparatus which limits the aberrant, accidental, or abnormal distribution of acetylcholine within a clearly defined area of sarcoplasm in the vicinity of the motor endplate of the muscle fiber.This investigation was supported by research grants from the National Cancer Institute (CA-2078 and CA-02478) and National Institute of Neurological Diseases and Blindness (NB 04096). Acknowledgement for technical assistence is due Miss Julia Silhan.This investigation was carried out during the tenure of a Public Health Service research career program award NB 5820 from the N.I.N.D.B.     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD+ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyl groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.     

Polymorphism and antigenic specificity of monocytic acid esterase (EC 3.1.1.6)

With the aim to provide a biochemical or immunological marker for human blood monocytes, their differentiational derivatives, and neoplastic variants the polymorphism of acid α-naphthyl acetate esterase was investigated. Using an isoelectric focusing technique it could be shown that the five enzyme variants of normal human blood monocytes, which do not occur in other human blood cell types, were regularly detectable in human peritoneal and alveolar macrophages. Antisera, raised against isoenzymes of monocytic acid esterase in rabbits, reacted in all cases with every five isoenzymes of blood monocytes; no cross-reactivity was observed with the isoenzymes of acid esterase obtained from purified human granulocytes, peripheral lymphocytes, B lymphocytes, and thymocytes as shown by immunoprecipitation. The results indicate that monocytic acid esterase and its variants could be regarded as a biochemical as well as immunological marker for this cell line.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

The comparison of lysosomal enzymes activities in alveolar and peritoneal macrophages of rat

Studies were carried out on macrophages isolated from control and thioglycollate injected rats. Intraperitoneal injection of thioglycollate had no effect on the number of harvested alveolar macrophages but caused a marked increase in the number of peritoneal macrophages. The specific activities of all the investigated enzymes were significantly lower in peritoneal macrophages from control rats in comparison to alveolar macrophages. Thioglycollate stimulation of peritoneal macrophages caused increase in activities of all lysosomal hydrolases studied. Cathepsin B, N-acetylglucosaminidase and esterase showed the highest level of stimulation.     

Nonspecific esterase activity of human alveolar macrophages in routine cytology

Alpha-naphthyl acetate esterase (ANAE) activity in alveolar macrophages was demonstrated in air-dried smears from samples of 82 sputa, 47 bronchial secretions and 14 bronchial lavages from 113 patients. Enzyme activity was estimated by a semiquantitative scoring method. There was a 7.4% loss of activity after 24 hours of storing the unfixed material, increasing to 14.4% after three days, while storing the air-dried smears for up to four weeks did not change the ANAE activity. The mean cellular esterase activity was correlated to the clinical and cytologic findings. A stringent correlation could be found for patients smoking more than 30 cigarettes a day; they had a 17% increase in activity as compared to nonsmokers. In patients with bronchial asthma, the activity was 18% higher than the total mean. In three patients with pulmonary embolisms, the ANAE activity was also increased. Treatment with a combination of cytostatics and a corticoid caused a severe decrease. No correlation could be found to age, sex, inflammation or malignant or cardiac diseases. These findings indicate that the application of the ANAE reaction to routine cytologic specimens can contribute to the functional characterization of human alveolar macrophages.     

Nonspecific esterase activity expressed in Weibel-Palade bodies of cloned guinea pig aortic endothelial cells

We studied the localization of nonspecific esterase activities in cloned guinea pig aortic endothelial cells using ultrastructural cytochemistry. Weibel-Palade bodies (WPB), which are known to contain von Willebrand protein, were positive for esterase, defining a heretofore unrecognized activity of these organelles. Esterase activity was also found localized to the external surface of the plasma membrane, to cytoplasmic lipid bodies, and to the outer (cytoplasm-facing) surface of certain membrane-bound cytoplasmic vacuoles. Localization of esterase activity to these four discrete sites probably reflects the presence of a number of endothelial cell enzymes capable of hydrolyzing alpha-naphthyl acetate or butyrate. The physiological substrate and biological function of these enzyme activities are not presently understood.     

Poly(I):poly(C)-enhanced alveolar and peritoneal macrophage phagocytosis: quantification by a new method utilizing fluorescent beads

Phagocytosis is an important immune function to quantify. This immune response may be modulated by exposure to biological response modifiers or by exposure to pollutants. A new technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed that utilizes fluorescent polystyrene beads. When incorporated into inhalation studies, this technique can be used to determine whether the toxic effect of an inhaled pollutant is local (effect on alveolar macrophages), systemic (effect on peritoneal macrophages), or both local and systemic. This method results in a determination of both the level of phagocytosis (the percentage of phagocytic macrophages) and the macrophage specific activity (the number of beads phagocytized per macrophage). This method also allows a determination of adherence by quantifying the number of particles in contact with, but not phagocytized by, the macrophage. Macrophage preparations were incubated with fluorescent beads for 2 hr and cyto-centrifuged onto a glass slide. Fluorescent beads present on the slide or cell-associated but not ingested by phagocytosis were removed by immersing the slide containing the macrophage preparation in methylene chloride for 15-30 sec. Fluorescent beads ingested by phagocytosis were then easily quantified with a fluorescence microscope. This technique was used to assess the baseline levels of phagocytosis for rat alveolar and peritoneal macrophages from the same animal and the kinetics and level of enhanced phagocytosis for alveolar and peritoneal macrophages after injection with the interferon inducer polyinosinate-polycytidylate (poly(I):poly(C)). The kinetics of enhanced alveolar and peritoneal macrophage phagocytosis by poly(I):poly(C) were similar; however, stimulated phagocytic levels of peritoneal macrophages never reached the phagocytic activity observed for the resident, highly phagocytic alveolar macrophages. This elevated phagocytic activity is most likely due to interferon stimulated by particulate matter in the large volume of air processed by the lungs and is important for host defense against a number of different inhaled microorganisms.     

A comparative study of superoxide dismutase activity in polymorphonuclear leukocytes, monocytes, and alveolar macrophages of the guinea pig.

Superoxide dismutase, an enzyme which catalyzes the dismutation of superoxide radical formed during the univalent reduction of oxygen, was quantitated by observing the inhibition of cytochrome C reduction in three cell fractions in guinea pig peritoneal PMNs and monocytes and compared to alveolar macrophages. No differences were found in the 16,000 × g pellets containing mitochondria, membranes, and granules and representing 96% of total SOD activity in PMNs and monocytes but only 48% total SOD activity in alveolar macrophages. The 100,000 × g microsomal pellet of alveolar macrophages contained 8% of total SOD activity and two-five times more activity than the respective fractions from monocytes and PMNs. However, there was 70 times more SOD in the 100,000 × g supernatant from alveolar macrophages containing 44% of total enzyme activity than in the same fraction of PMNs and monocytes containing less than 2% total SOD activity. SOD activity is mainly located in the 16,000 × g particulate fraction of PMN and monocytes but more equally distributed between the particulate fractions and cytosol of alveolar macrophages.