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.     

Fc-receptor mediated protein phosphorylation in murine peritoneal macrophages

The effect of Fc receptor engagement on protein phosphorylation in murine peritoneal macrophages has been investigated. Treatment of macrophage cultures with insoluble immune complexes resulted in enhanced phosphorylation of six proteins at 73, 66, 53, 37, 31 and 25 kD. Comparison of the protein phosphorylation patterns induced by immune complexes with those induced by agents which mimic the actions of well known intracellular second messengers (i.e., A23187, dibutyryl cAMP, or phorbol myristate acetate) revealed substantial similarity between Fc receptor induced events and those induced in response to phorbol diesters. There were, however, two phosphorylated proteins which were only seen following stimulation with immune complexes. Thus, more than one kind of protein kinase activity appears to be involved in Fc receptor mediated stimulation of macrophage function.     

Peroxidase-induced enhancement of chemiluminescence by murine peritoneal macrophages

A number of substances have been shown to enhance the respiratory burst (RB) of macrophages. Many of these substances are not normally found in vivo. The present study suggests that a group of enzymes characterized as peroxidases have the ability to significantly enhance the RB and concomitant phagocytosis by murine peritoneal macrophages. Horseradish peroxidase (HRP), lactoperoxidase (LPO), and microperoxidase (MPO) can significantly augment these functions. Both resident and thioglycollate-induced macrophages exhibited enhanced chemiluminescence (CL) upon exposure to HRP, however, the effect was more pronounced with the latter. The increase in CL was correlated with an increase in production of superoxide, which was measured by reduction of cytochrome c. Horseradish peroxidase immobilized on an inert carrier, was capable of enhancing the RB suggesting that it does not have to enter the cell in order to function. Hemin, hematoheme and hematoporphyrin had little effect on macrophage stimulated CL. All of the peroxidases tested caused increased phagocytosis of opsonized zymosan. These studies indicate that peroxidases are capable of stimulating the RB, phagocytosis and possibly other macrophage functions.     

Inactivation of thrombin by murine peritoneal macrophages.

We recently showed that murine peritoneal macrophages cultured in vitro express potent prothrombinase activity (Lindahl, U., Pejler, G., B?gwald, J., and Seljelid, R. (1989) Arch. Biochem. Biophys. 273, 180-188). In the present report, we demonstrate that the macrophages also express anticoagulant activity by inactivating the thrombin that is formed due to the action of the prothrombinase. Addition of exogenous purified thrombin to the macrophage cultures resulted in inactivation of the enzyme at a maximum rate of approximately 5 micrograms/h/10(6) cells. The inactivation appeared to be specific for thrombin, since neither Factor Xa, chymotrypsin, nor trypsin, three serine proteases exhibiting homology with thrombin, were inactivated by the macrophages. Thrombin-inactivating activity was not secreted into the culture medium. Inhibitors of endocytosis did not decrease the rate by which thrombin was inactivated, suggesting that internalization of the coagulation factor was not required. In contrast, the thrombin-inactivating activity was strongly inhibited by the polycation Polybrene. Anion-exchange chromatography of extracts obtained after Triton X-100-solubilization of the macrophages demonstrated that the thrombin-inactivating activity exhibited a high negative charge. Incubation of the thrombin-inactivating activity recovered after anion-exchange chromatography with unlabeled thrombin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, showed that thrombin was proteolytically cleaved into defined fragments. Similar proteolytic fragments were obtained when 125I-labeled thrombin was added to macrophage cultures. Degradation of thrombin was blocked by phenylmethanesulfonic fluoride, an inhibitor of serine proteases, but not by inhibitors of other classes of proteases. Thrombin that had been chemically modified at its active site was degraded at the same rate by the macrophages as active thrombin. Taken together, these findings indicate that the murine macrophages express surface-bound serine protease activity that specifically inactivates thrombin by proteolytic cleavage. The significance of thrombin-inactivating activity in relation to the involvement of macrophage procoagulant activity in the immune response is discussed.     

Maleylated-BSA suppresses IFN-gamma-mediated Ia expression in murine peritoneal macrophages

Maleylated bovine serum albumin (maleyl-BSA) and other polyanionic polymers that are recognized by cell surface receptors on macrophages have been shown to induce chemotaxis, protease secretion, and tumoricidal function in this cell type. In this paper the effect of maleyl-BSA on Ia antigen expression has been evaluated. In a fashion similar to LPS, maleyl-BSA suppressed IFN-gamma-induced expression of Ia in a time- and dose-dependent manner. Also like LPS, maleyl-BSA stimulated the production and secretion of substantial amounts of PGE2 over a 24-hr period. This did not, however, appear to be the primary mechanism by which expression of Ia was suppressed, because co-treatment of the cells with indomethacin, which totally inhibited the production of PGE2, only minimally affected the suppressive activity. Surprisingly, the suppressive activity of both maleyl-BSA and LPS could be largely abrogated by co-treatment of the cells with cyclohexamide during the time period when Ia expression was sensitive to suppression. This effect was selective in that PGE2- or dibutyryl cyclic AMP-induced suppression of Ia expression was not affected by cyclohexamide treatment. The data support the concept that there are multiple molecular mechanisms involved in the negative regulation of IFN-gamma-induced Ia expression in macrophages. Such mechanisms may include, in addition to the synthesis of PGE2 and consequent elevation in intracellular levels of cyclic AMP, one or more proteins made early after treatment with either maleyl-BSA or LPS. Thus the function of some of these early gene products may be to regulate expression of functional genes such as that encoding Ia antigen.     

LPS regulation of specific protein synthesis in murine peritoneal macrophages

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) analysis of biosynthetically labeled proteins of murine peritoneal macrophages elicited by inflammatory and activating stimuli indicated that the accumulation of a small number of cell-associated proteins was altered after in vitro treatment with bacterial lipopolysaccharide (LPS). Both increases and decreases in the accumulation of specific proteins were observed after LPS stimulation. Proteins of approximately 87, 43, 37, 30, and 28 Kd were similarly regulated by LPS in proteose peptone-, P. acnes-, and M. bovis BCG-elicited macrophages. Thioglycollate-elicited and resident peritoneal macrophages showed very few changes in the pattern of proteins synthesized after LPS treatment. Many of the proteins whose accumulation was increased by LPS in the elicited macrophages (proteins of approximately 87, 52, 43, 37, and 28 Kd) were already synthesized at high levels in resident macrophages. LPS stimulation also altered the accumulation of many of the same proteins in bone marrow-derived macrophages, indicating the lack of T lymphocyte influence on the LPS-induced changes in macrophages. LPS stimulation of highly purified B cells caused changes in the accumulation of several proteins of 70 and 78 Kd, which were different from those regulated by LPS in peritoneal macrophages.     

Induction of metalloelastase mRNA in murine peritoneal macrophages by diethylmaleate

Macrophage-specific metalloelastase (MME) hydrolyzes elastin and other matrix proteins and plays an important physiological role in tissue remodeling and pathological tissue destruction. We have examined the effects of diethylmaleate (DEM), an electrophilic agent that reacts with sulfhydryls, on the expression of MME mRNA in mouse peritoneal macrophages. Quantification of MME mRNA by Northern blot analysis revealed that basal mRNA levels were quite low in freshly isolated cells, although mRNA levels increased markedly and reached a steady level within 12 h when cells were cultured in a serum-supplemented RPMI 1640 medium. When macrophages were challenged with DEM at 0.05-1.0 mM for 8 h the expression of the MME gene was enhanced further. In the presence of 0.1 mM DEM, the level of the MME mRNA increased 2-fold compared to the control levels after 6-9 h and decreased to control levels in 24 h. Other electrophilic agents, catechol and 1-chloro-2,4-dinitrobenzene, also enhanced MME gene expression. However, oxidative stress agents such as hydrogen peroxide, menadione, paraquat (an O-2 generator), sodium arsenite and cadmium chloride had no effect on MME gene expression. These results indicate that the electrophilic agents selectively enhance the expression of MME mRNA during primary culture of the macrophages.     

Structural characterization of the disialogangliosides of murine peritoneal macrophages

Sialoglycosphingolipids (gangliosides) have been increasinglyimplicated as regulators of membrane signaling events. Macrophageganglioside patterns dramatically increase in complexity whenmurine peritoneal macrophages are stimulated in vivo with theappearance of the sialidase-sensitive monosialoganglioside GMlb(cisGMl) as a major component Gangliosides from stimulated murineperitoneal macrophages were separated into monosialo and polysialofractions and the polysialo fraction structurally characterizedby enzymatic, chemical, and mass spectra methods. All detectablecomponents of the polysialo fraction were determined to be disialogangliosides.Treatment of the polysialo fraction with Clostridium perfringenssiali-dase produced mostly the sialidase-resistant monosialoganglioside,GMIa, and a minor amount of asiaJoGMI. Perio-date oxidationand mass spectrometry analyses demonstrated the lack of tandemdisialo moieties which indicated the absence of GD1b or GD1c(GDI) entities. The combined data showed the major disialogangliosidesconsisted of GDla entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer,IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, andIV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted ofGDl entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGcGgOse4Cer,and also positional iso-mer(s) of GDl(NeuAc, NeuGc). These isomericcomponents were identified by collision analysis and tandemmass spectrometry. Consistent with previous analyses, the cer-amideportion of all polysialo (disialo) gangliosides contained solelyC18 sphingosine with C16 and C24 fatty acid moieties. Theseresults, combined with the previous characterization of macrophagemonosialogangliosides, indicate normal murine macrophage gangliosidebiosynthesis proceeds along the "a" ganglioside pathway, e.g.,GM3GM2GMlaGDl, and the proposed asialogan-glioside or "" pathway,asialoGMlGMlbGDl. The presence of totally sialidase-sensitivegangliosides appears to be characteristic of functional murineperitoneal macrophages while they are reduced in geneticallyimpaired cells. ganglioside GDla ganglioside GDl murine macrophages tandem mass spectrometry collision induced dis-association electrospray ionization     

Interferon-gamma modulates protein kinase C activity in murine peritoneal macrophages

The content of Ca2+-, phospholipid-dependent protein kinase activity (protein kinase C) in murine peritoneal macrophages treated with recombinant interferon-gamma (IFN-gamma) has been investigated. Protein kinase C activity was solubilized by nonionic detergent extraction of sonicated cells and separated by high performance liquid chromatography on a TSK 4000 SW gel filtration column. The enzyme eluted from the column in a molecular weight range of 60-80 X 10(3) and was identified by virtue of Ca2+ and phospholipid requirements. Macrophages treated with recombinant IFN-gamma exhibited a substantial increase in total protein kinase activity which could be accounted for entirely by increased protein kinase C activity. This activity was enhanced as much as 5-fold over that seen in untreated macrophages and was specific for IFN-gamma in that other agents known to signal changes in macrophage function had no effect. The time required for the elevation of kinase activity was identical to that required for induction of other functions by IFN-gamma in macrophages. These observations suggest that protein kinase C may be a focus of regulatory action in IFN-gamma-mediated macrophage activation.     

Effects of endotoxic shock in several functions of murine peritoneal macrophages

Gram negative sepsis and septic shock continue to be a major medical problem, with a complex physiopathology and it is associated with high mortality. Although secretion of cytokines such as tumor necrosis factor-alpha by macrophages is the principal host mediator of septic shock, other characteristic functions of macrophages implicated in their phagocytic capacity have not been studied in the process of endotoxic shock. In the present study we have used an intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg) in order to obtain an endotoxic shock model in adult female BALB/c mice. Peritoneal cell suspensions were obtained at several times (2, 4, 12 or 24 h) after injection and the following functions were studied on the peritoneal macrophages: adherence to substrate, mobility (spontaneous and directed or chemotaxis), ingestion of particles and superoxide anion production. The results showed a stimulation of adherence, ingestion and superoxide production as well as a decrease of chemotaxis in the animals injected with LPS. These effects changed with time after LPS injection. Thus, the increase of adherence and the decrease of mobility were higher during the first hours, whereas the increase in ingestion and superoxide production turned larger with time.     

LPS induces altered phosphate labeling of proteins in murine peritoneal macrophages

Covalent modification of proteins via phosphorylation is a well-documented mechanism whereby intracellular events are controlled by external stimuli. Treatment of thioglycollate-elicited, C57Bl/6 murine peritoneal macrophages with nanogram quantities of bacterial lipopolysaccharide (LPS) consistently results in altered 32Pi labeling of a specific set of proteins (e.g., proteins of 67, 37, 33, and 28 kD), as measured by autoradiography after SDS-polyacrylamide gel electrophoresis. Induction of this pattern of phosphorylation is duplicated by the lipid A moiety of LPS. The LPS-stimulated changes in phosphate labeling are both dose- and time-dependent. Of various pharmacologic agents tested, the phosphorylation pattern induced in macrophages by the tumor promoter phorbol myristic acetate shows similarity to the pattern induced by LPS. Analysis of pp 28 and pp 37 from both LPS- and PMA-treated macrophages by limited proteolysis demonstrates that these phosphoproteins are structurally related and that the sites of phosphorylation are similar for both treatment conditions. Macrophages from the genetically LPS-unresponsive C3H/HeJ strain show no alteration in their pattern of phosphorylation after treatment with LPS. Control macrophages, from C3H/HeN mice, respond to LPS in a fashion identical to that seen in C57Bl/6 macrophages. Pretreatment of macrophages with IFN-gamma potentiates the effect of LPS (i.e., yields a level of altered phosphate labeling greater than that observed with LPS or PMA alone). Together, the data indicate that LPS causes altered phosphate labeling of a defined set of proteins, and that the circumstances of this response are consistent with a possible role in coupling LPS-initiated signals to the induction of functional competence in macrophages.     

Ultrastructural cytochemistry of atrial muscle cells

Summary Sections of atrial cardiocytes from young rats were subjected to radioautography after a single intravenous injection of L-leucine-4,5 ³H to identify the sites of synthesis and to follow the migration of newly-formed proteins. As early as 2 min after injection of L-leucine ³H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 5 min, most of the label had migrated from the RER to the Golgi complex. Some label was already present over specific granules by 2 min but the peak was reached at 1 h. By 4 h, the label over the specific granules had diminished, possibly indicating a release of newly-synthetized secretory material outside the cell. The label over myofilaments and Z-bands was relatively high at most time intervals, suggesting an early and important incorporation of leucine into the contractile and structural proteins of these organelles. The label over the cytosol was initially high and increased even further at 5 and 20 min but decreased to a very low level at 4 h. In contrast, the label over the cell surface rose continuously and peaked at 4 h. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the cytosol before reaching their destination.     

Ultrastructural cytochemistry of human spermatozoa]

Enzymatic activities and repartition of glycoproteins were studied with electron microscopy in human ejaculated spermatozoa. Enzymatic activities are localised in the head of spermatozoon: arylsulfatase in the acrosome, acid phosphatase in the periacrosomal cytoplasm. Phosphotungstic acid at low pH and collo?dal iron allow detection of glycoproteins and acid groups on the sperm cell surface. Glycoproteins are present in the acrosome. These results are slightly different to those obtained in other species.     

Ultrastructural carbohydrate cytochemistry of gastric epithelium

Synopsis On examination with ultrastructural methods for visualizing thevicinal glycols and acid groups of complex carbohydrates, the most superficial surface epithelium of the rat gastric corpus displayed biphasic mucous droplets consisting of a cortex of hexose-rich (i.e. periodate-reactive) neutral mucosubstance and an uncharacterized denser core plus monophasic droplets with the neutral mucosubstance. In many surface epithelial cells of the foveolae, the biphasic and monophasic droplets with the neutral mucosubstance intermingled in varying proportions with monophasic droplets showing uniform periodate reactivity, a variable degree of dialyzed ironbinding—demonstrative of acidic glycoconjugate, and high iron—diamine affinity—demonstrative of sulphomucin. Deep foveolar epithelium displayed only monophasic droplets, most of which contained acidic periodate-reactive complex carbohydrate. Underiying cells, designated isthmus cells, exhibited monophasic or occasional biphasic granules containing sulphated, hexose-rich mucosubstance. Nascent droplets or granules near the Golgi zone differed from the mature organelles in the distribution of the glycoconjugate. Mucous neck cells occupied a deeper stratum and displayed a uniform population of monophasic mucous droplets with a loose meshwork of neutral mucosubstance.Techniques for demonstrating hexoses ultrastructurally stained all Golgi cisternae in the mucigenic epithelium, showing increasing reactivity toward the maturing face. Distinctive cistemae with moderate reactivity in the Golgi complex of isthmus cells were interpreted as GERL. Acidic mucosubstances were visualized only in the inner, mature cisternae of the Golgi complex of cells storing acidic glycoconjugates, and not in cisternae interpretable as GERL.The apical plasmalemma of isthmus cells uniquely exhibited abundant sulphated glycoconjugate and that of parietal cells revealed a less prominent, periodic neutral mucosubstance. Lateral and basal plasmalemmae varied from unstained to slightly reactive; basement membranes showed moderate reactivity with methods for visualizing complex carbohydrates. Abundance of glycogen further characterized surface epithelial cells of the corpus and of some parietal cells