Histochemical and immunological demonstration of purple acid phosphatase in human and bovine alveolar macrophages

Summary A tartrate-resistant purple acid phosphatase was localized in human and bovine alveolar macrophages by enzyme- and immuno-histochemistry using an antibody to bovine spleen purple phosphatase. The enzyme could be detected in human and bovine lung tissues as well as on cytospin preparations of alveolar macrophage suspensions from bronchoalveolar lavages. The immunological identity of human and bovine purple phosphatases from alveolar macrophages was demonstrated by Western blot analysis of material separated by polyacrylamide gel electrophoresis. A possible significance of the purple phosphatase as a marker enzyme of activated cells of the mononuclear phagocyte system is discussed.     

Immunohistochemical characterization of purple acid phosphatase-containing leucocytes in the human placenta

Summary A tartrate-resistant acid phosphatase activity was detected in the human placenta. This enzyme displayed immunological properties similar to those of the group of purple acid phosphatases that can be demonstrated with a rabbit polyclonal antibody against bovine spleen purple acid phosphatase. The placental enzyme was mainly localized immunohistochemically to neutrophil granulocytes of the maternal blood between the placental villi and within foetal capillaries using the bovine spleen antibody and the commercial monoclonal antibody M1 directed against an antigen found on mature granulocytes. A minor activity was detected in decidual cells and the syncytiotrophoblast. The presence of purple acid phosphatase in placental granulocytes may be related to special immunological conditions of pregnancy.     

Histochemical investigations on the localization of the purple acid phosphatase in the bovine spleen

Summary The localization of the purple tartrate-resistant, iron-containing acid phosphatase in the bovine spleen was studied by enzyme histochemistry at the light and electron microscopic levels as well as by immunohistochemistry. The purple phosphatase was localized only in lysosome-like organelles of cells belonging to the reticulo-phagocytic system. The same cells were identified as containing large iron(III)-deposits as ferritin in homogeneously granular accumulations and freely in the cytoplasm, or as hemosiderin in siderosomes. The phagocytosing cells containing purple phosphatase and ferritin often had close contact with clusters of aged and deformed erythrocytes.A possible catabolic role of the purple enzyme as a phosphatase degrading phosphoproteins of the erythrocyte membrane and the cytoskeleton was assumed.     

Histochemical investigations on the localization of the purple acid phosphatase in the bovine spleen

The localization of the purple tartrate-resistant, iron-containing acid phosphatase in the bovine spleen was studied by enzyme histochemistry at the light and electron microscopic levels as well as by immunohistochemistry. The purple phosphatase was localized only in lysosome-like-organelles of cells belonging to the reticulo-phagocytic system. The same cells were identified as containing large iron(III)-deposits as ferritin in homogeneously granular accumulations and freely in the cytoplasm, or as hemosiderin in siderosomes. The phagocytosing cells containing purple phosphatase and ferritin often had close contact with clusters of aged and deformed erythrocytes. A possible catabolic role of the purple enzyme as a phosphatase degrading phosphoproteins of the erythrocyte membrane and the cytoskeleton was assumed.     

Purification and characterization of purple acid phosphatase from developing rat bone

Tartrate-resistant acid phosphatase active on nucleoside di- and triphosphate substrates was isolated from developing rat bone and purified 2500-fold. The enzyme concentration had a purple coloration and activity that was sensitive to reducing agents. Mild reducing agents such as ferrous ion and ascorbic acid caused loss of purple color and increased activity toward substrates severalfold; however, a strong reductant such as dithionite caused loss of both color and activity which were partially restored by addition of ferrous ion and ascorbic acid. Enzyme activity was homogeneous with protein during the final gel permeation steps of chromatography and gave an apparent molecular size of about 40,000 Da. Determination of iron in the most pure preparation revealed the presence of 1.3 atoms of iron per molecule of the tartrate-resistant enzyme E2. Other properties of the purified enzyme include a pI of approximately 9.5 and sensitivity to inhibition by ions of copper, zinc, fluoride, and molybdate. Antibody prepared to the pre-concanavalin A (Con A)-Sepharose purified enzyme reacted with all protein from the Con A step, but it did not react with tartrate-sensitive acid phosphatase from rat bone or with potato acid phosphatase. Purple acid phosphatase from rat bone has many properties that parallel the iron-containing purple acid phosphatases from rat spleen, bovine spleen, and pig uterine secretions.     

Cellular acid phosphatase activity: Correlation of cytochemical and biochemical measurements

Summary Methods for comparing results of cellular acid phosphatase activities obtained by quantitative cytospectrophotometry with those obtained by biochemical analysis are needed to express the cytospectrophotometric data in biochemical units. Since naturally occurring cells have differing amounts of acid phosphatase, enzyme activity was measured cytochemically and biochemically in polymorphonuclear leukocytes and peritoneal and alveolar macrophages from male rats to determine if these measurements permitted construction of a line correlating the two parameters. Cellular acid phosphatase activity, as measured cytospectrophotometrically and biochemically, increased proportionately with polymorphonuclear leukocytes having the lowest activities and alveolar macrophages the highest. These values when subjected to linear regression analysis fixed a line with a correlation coefficient of 0.95 demonstrating that cytochemical and biochemical activities of acid phosphatase activity can be correlated using naturally occurring cells.     

The protective action of disodium cromoglycate on alveolar macrophages in phagocytosis of fibrogenic silica.

Treatment of fibrogenic silica (DQ-12) with Disodium Cromoglycate (DSCG) prior to its in-vitro and in-vivo phagocytosis by alveolar macrophages prevents the destruction of the cells and the fibrosis of lung tissue which are a consequence of phagocytosis. However, the treatment of alveolar macrophages with DSCG before phagocytosis of the silica had no, or a negligible, protective effect on the cells. Acid phosphatase activity which was significantly enhanced above the control in cells phagocytosing the silica was returned to the range found in phagocytosis of inert dust when silica treated with DSCG was phagocytized. The inhibitor of DNA- dependent RNA synthesis actinomycin D caused an increase of acid phosphatase activity. DSCG did not depress the phagocytic ability of alveolar macrophages. It appears that the catabolic enzyme process predominant in cells phagocytosing DQ-12 was under control in cells phagocytosing DQ-12 treated with DSCG and that DSCG probably acted as a regulator of the factors permitting catabolism. From the results it is suggested that the equilibrium of the enzyme reactions which accompany phagocytosis was such that the integrity of the phagocytes was preserved.     

Tartrate-resistant acid phosphatase of human lung: apparent identity with osteoclastic acid phosphatase

Extracts of human lung tissue contain appreciable activities of a tartrate-resistant acid phosphatase which is apparently identical with the analogous enzyme in bone extracts, with respect to electrophoretic mobility, apparent molecular weight (ca. 37,000), Michaelis constants and relative rates of hydrolysis of various substrates. The acid phosphatase appears to be a constituent of alveolar macrophages. Lung provides a convenient source for the preparation of tartrate-resistant acid phosphatase.     

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55A and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases.     

Crystal structure of a mammalian purple acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is a mammalian di-iron- containing enzyme that belongs to the family of purple acid phosphatases (PAP). It is highly expressed in a limited number of tissues, predominantly in bone-resorbing osteoclasts and in macrophages of spleen. We have determined the crystal structure of rat TRAP in complex with a phosphate ion to 2.7 A resolution. The fold resembles that of the catalytic domain of kidney bean purple acid phosphatase (KBPAP), although the sequence similarity is limited to the active site residues. A surface loop near the active site is absent due to proteolysis, leaving the active-site easily accessible from the surrounding solvent. This, we believe, gives a structural explanation for the observed proteolytic activation of TRAP. The current structure was determined at a relatively high pH and without any external reducing agents. It is likely that it represents an oxidized and therefore catalytically inactive form of the enzyme.     

Hyaluronic acid-degrading enzymes in rat alveolar macrophages and in alveolar fluid: stimulation of enzyme activity after oral treatment with the immunomodulator RU 41740

RU 41740 (Biostim) is an immunomodulator clinically used for the treatment of chronic bronchitis and recurrent pulmonary infections. In these diseases large amounts of mucus are produced which congest the bronchi. A major glycosaminoglycan constituent of this mucus is hyaluronic acid, one of the largest molecules in nature; its metabolic degradation is carried out by 3 acid hydrolases: hyaluronidase, beta-N-acetylglucosaminidase, and beta-glucuronidase. In the lung these enzymes are especially synthesized and active in alveolar macrophages. It was thus interesting to study the effect of RU 41740 administration on the hyaluronic acid-degrading activity of these cells. This compound was given by gastric gavage to rats and the activities of lung alveolar macrophage and alveolar fluid hyaluronidase, beta-N-acetylglucosaminidase, beta-glucuronidase, and acid phosphatase as a lysosomal marker were determined. The effect on macrophage proliferation was also examined. The results obtained showed that: (1) unstimulated alveolar macrophages display the remarkable property, compared with other cell types, that hyaluronidase activity is about equally distributed between the inside and the outside of the cell; (2) RU 41740 administration increases the total activity of the 4 enzymes studied in the alveolar macrophages without inducing any increase in the number of macrophages; (3) the intracellular activities of beta-N-acetylglucosaminidase and beta-glucuronidase are markedly increased, whereas intracellular hyaluronidase activity is not changed. However, in the extracellular fluid only hyaluronidase activity is highly increased; (4) even the lysosomal marker enzyme acid phosphatase has only its intracellular activity increased. This would suggest the possibility that other lysosomal enzymes may also be increased by this immunomodulator.(ABSTRACT TRUNCATED AT 250 WORDS)     

The active-site and amino-terminal amino acid sequence of bovine intestinal alkaline phosphatase

The active site of bovine intestinal alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was labeled with [32P]Pi, a radioactive CNBr peptide was isolated and the amino acid sequence was determined. The sequence of the active-site peptide has limited homology (26%) with the active-site sequence of Escherichia coli alkaline phosphatase except for the ten residues immediately flanking the active-site serine (70%). A possible amino acid sequence deduced from the amino acid composition of an active-site tryptic peptide from human placental alkaline phosphatase is very similar to the bovine intestinal active-site sequence. The amino-terminal sequence of bovine intestinal alkaline phosphatase is homologous (69%) with the human placental enzyme but not with the E. coli phosphatase.     

Localisation of High Acid Phosphotyrosine Phosphatase Activity in Afferent Arterioles and Glomeruli of Human Kidney

Summary Endothelial cells contain a variety of specific protein tyrosine phosphatases and an acid phosphatase differing from other known phosphatases. The highest activity of this acid phosphatase with artificial or unspecific substrates is present in the afferent arterioles and glomeruli of human kidney, and the activity is inhibited by nephrotoxic fluoride concentrations, suggesting that it plays a role in circulatory regulation. Here the activity was characterised with physiological substrates. An incubation mixture containing phosphotyrosine or phosphoserine was stable at pH 5 when phosphate-precipitating lead was chelated with tartrate. The activities were studied in frozen sections. Only phosphotyrosine was hydrolysed by some cells. High activity of tartrate-resistant phosphotyrosine phosphatase was present in lymphocytes, endothelial cells of afferent arterioles, and glomerular mesangial cells of kidney, decidual cells, and alveolar macrophages. In lymphocytes the activity was fluoride-resistant and vanadate-sensitive, in other cells fluoride- and vanadate-sensitive. In decidual cells and alveolar macrophages, the activity is due to specific osteoclastic/macrophagic tartrate-resistant acid phosphatase, in lymphocytes to specific protein tyrosine phosphatases, and in endothelial and mesangial cells to a protein tyrosine phosphatase-like acid phosphatase. The results suggest that in endothelial cells of the afferent arterioles, mesangial cells, and lymphocytes the cellular activities are regulated by high constitutive phosphotyrosine phosphatase activity and this may be related to the exceptional cyclosporin A sensitivity of these cells.     

Interaction of mycoplasmas and phagocytes

Aspects of the interaction of certain mycoplasmas with macrophages and neutrophils in vivo and in vitro have been studied using two systems, one involving M. pulmonis in mice and the other involving M. bovis with bovine leucocytes. Studies with M. pulmonis indicated that the disappearance of viable organisms from the peritoneal cavity was not enhanced in SPF mice in which a peritoneal exudate rich in neutrophils had been induced. However, viable M. pulmonis organisms disappeared more rapidly from the peritoneal cavities with exudates containing increased numbers of macrophages. Experiments in vitro studied the opsonic effect of bovine IgG isotypes for bovine neutrophils and alveolar macrophages. Both IgG1 and IgG2 promoted killing of M. bovis by alveolar macrophages but IgG2 was more effective than IgG1 at promoting mycoplasma killing by neutrophils. Further studies in vitro indicated that certain bovine mycoplasma could inhibit killing of Escherichia coli by bovine neutrophils.     

Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

Tartrate-resistant acid phosphatase in human alveolar macrophages

Tartrate-resistant acid phosphatase has been extracted from human alveolar macrophages, in which its specific activity is 10-fold that in whole lung. The apparent identity of the alveolar macrophage isoenzyme with that associated with osteoclasts suggests that both types of cell belong to the mononuclear phagocyte system. Within this system, expression of tartrate-resistant acid phosphatase appears to accompany certain kinds of differentiation.     

Presence of cytochrome b-558 in guinea-pig alveolar macrophages-subcellular localization and relationship with NADPH oxidase

The assignment of cytochrome b-558 as a component of the O2- (H2O2) -generating enzyme in guinea-pig alveolar macrophages was investigated. Guinea pig alveolar macrophages contained 76 pmol cytochrome b-558/mg protein, a value very similar to that of neutrophils. The rate of myristic acid-stimulated O2- generation by alveolar macrophages, calculated per cytochrome b-558, was only one-fourth that of neutrophils. An analysis of Percoll density gradient centrifugation profiles showed that the H2O2-generating activity of myristic acid-activated alveolar macrophages was concentrated in a single peak which was consistently associated with 5'-nucleotidase activity, a plasma membrane marker enzyme. A little H2O2-generating activity was seen with unactivated alveolar macrophages. Furthermore, the cytochrome b-558 of both myristic acid-activated and unactivated alveolar macrophages was also predominantly associated with 5'-nucleotidase activity and was found in trace amounts in a peak containing lysozyme activity, a marker of lysosome granules. Only about 6% of the cytochrome b-558 in plasma membranes from myristic acid-activated guinea-pig alveolar macrophages was anaerobically reduced by 0.5 mM NADPH, while under the same conditions about 30% of the heme protein of myristic acid-activated neutrophils was reduced. These results suggest two conclusions: firstly, that in both activated and unactivated alveolar macrophages, cytochrome b-558 is located in the plasma membrane, and the translocation of cytochrome b-558 does not occur during the activation of NADPH oxidase; and secondly, that a smaller part of cytochrome b-558 is associated with the activated NADPH oxidase of guinea pig alveolar macrophages compared with neutrophils.     

In vitro cytotoxicity of chrysotile asbestos to human pulmonary alveolar macrophages is decreased by organosilane coating and surfactant

Human pulmonary alveolar macrophages were used to quantitate the cytotoxic effect of surface-altered chrysotile asbestos. Little difference was observed in mortality between chrysotile asbestos that was surface-treated to a 42% extent by a hydrophobic organosilane or untreated chrysotile. Little or no effect on mortality was observed when human pulmonary alveolar macrophages were cultured with untreated chrysotile or acid-leached asbestos in the presence of 10 mM dipalmitoyl lecithin. However, when human pulmonary alveolar macrophages were cultured with a hydrophobically-treated (to a 42% or 95% extent) chrysotile asbestos in the presence of 10 mM dipalmitoyl lecithin, a statistically significant decrease in mortality was observed compared to untreated chrysotile. No mutagenic activity was observed when V79 cells were cultured with acid-leached, or 42% hydrophobically-treated chrysotile asbestos, even when human pulmonary alveolar macrophages were included as an activation source. The 95% hydrophobically-treated and acid-leached chrysotile also exhibited decreased binding of benzo[a]pyrene compared to untreated chrysotile asbestos.Abbreviations AHH aryl hydrocarbon hydroxylase - B(a)P benzo[a]pyrene - CA chrysotile asbestos - CHO Chinese hamster ovary - DL dipalmitoyl lecithin - DMEM Dulbecco's Modified Eagle's Medium - FBS fetal bovine serum - O^r resistance to ouabain - PAH polycyclic aromatic hydrocarbon - PAM pulmonary alveolar macrophage - SCE sister chromatid exchange Deceased.     

Characterization of a calmodulin-dependent protein phosphatase from human platelets

A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.     

Type 5 acid phosphatase. Sequence, expression and chromosomal localization of a differentiation-associated protein of the human macrophage

The purple acid phosphatases and uteroferrin belong to a diverse multifunctional class of binuclear iron-containing proteins that includes haemerythrin and ribonucleotide reductase. In the pig, uteroferrin has been implicated in the delivery of iron to the foetus, but the role of the related human type 5 acid phosphatase that is principally found in resident tissue macrophages is not yet clear. To define further the function of this metalloenzyme, we have isolated and sequenced a cDNA clone for type 5 acid phosphatase and investigated expression of its gene in human tissues. The phosphatase clone contains an open reading frame of 975 bp and encodes a protein of 325 amino acids, including a signal peptide of 19 residues and two potential sites for N-glycosylation. The type 5 acid phosphatase gene mapped to the short arm of human chromosome 19 and was found to have a restriction fragment length polymorphism on digestion with XbaI. Expression of phosphatase mRNA was restricted to mononuclear phagocytes and the enzyme was induced greater than 20-fold on transformation of normal human monocytes to macrophages by culture in serum-supplemented medium. Type 5 acid phosphatase thus represents a tightly regulated system for the study of molecular events in the differentiation programme of the normal macrophage.