Different effects of concanavalin A and its succinylated derivative on superoxide release in peritoneal macrophages

The effects of tetravalent concanavalin A and its succinylated derivative on the intracellular production of superoxide anion (O₂^?) and its release into cell exterior of peritoneal macrophages were observed. Both tetravalent concanavalin A and its succinylated derivative induced marked enhancement of intracellular reduction of nitroblue tetrazolium, which could be inhibited by α-methyl-D-glucoside. The extent of activation of nitroblue tetrazolium reduction induced by both types of the lectin paralleled the activation ratio of oxygen consumption.There was littele difference in the extent of intracellular O₂^? production induced by two types of the lectin. Nitroblue tetrazolium reduction was not affected significantly by pretreatment with colchicine, rotenone or malonate, inhibitors of the cytoskeletal system and of the electron transport system. In contrast, tetravalent concanavalin A induced a higher rate of superoxide release than did succinylated divalent concanavalin A, which lacks the cross-linking activity of surface glycoproteins.These results indicate that superoxide production following oxygen consumption and superoxide release into cell exterior are controlled independently by a separate membrane mechanism and that superoxide production system is not essentially dependent on the involvement of the cytoskeletal system.     

Different effects of concanavalin A and its succinylated derivative on superoxide release in peritoneal macrophages.

The effects of tetravalent conconavalin A and its succinylated derivative on the intracellular production of superoxide anion (O-2) and its release into cell exterior of peritoneal macrophages were observed. Both tetravalent concanavalin A and its succinylated derivative induced marked enhancement of intracellular reduction of nitroblue tetrazolium, which could be inhibited by alpha-methyl-D-glucoside. The extent of activation of nitroblue tetrazolium reduction induced by both types of the lectin paralleled the activation ratio of oxygen consumption. There was little difference in the extent of intracellular O-2 production induced by two types of the lectin. Nitroblue tetrazolium reduction was not affected significantly by pretreatment with colchicine, rotenone or malonate, inhibitors of the cytoskeletal system and of the electron transport system. In contrast, tetravalent concanavalin A induced a higher rate of superoxide release than did succinylated divalent concanavalin A, which lacks the cross-linking activity of surface glycoproteins. These results indicate that superoxide production following oxygen consumption and superoxide release into cell exterior are controlled independently by a separate membrane mechanism and that superoxide production system is not essentially dependent on the involvement of the cytoskeletal system.     

Effect of C-reactive protein on peritoneal macrophages. I. Human C-reactive protein inhibits migration of guinea pig peritoneal macrophages

The effect of human C-reactive protein (CRP) isolated and purified from pooled patients' sera on macrophage function, especially on macrophage migration, was studied. Peritoneal exudate cells (PEC) from guinea pigs were used for macrophage migration inhibition (MMI) test of capillary method. Migration of either PEC or adherent purified macrophages exposed to CRP were inhibited dose-dependently. These findings indicate that CRP inhibits macrophage migration directly, not via activation of lymphocytes contained in PEC. As control, we examined the effect of normal human serum, anti C-polysaccharide antibodies isolated from patients' sera, and free endotoxin at the dose contaminated in CRP preparation on macrophage migration and found that none of them were effective. The effect of CRP on MMI of sensitized PEC exposed to antigen was also studied. Large amounts of CRP inhibited MMI induced by antigen, indicating the possibility that CRP may act on macrophages competitively with migration inhibitory factor (MIF) and may modulate MMI. CRP possesses MIF-like activity and may play a functional role at the site of tissue injury by causing the accumulation of macrophages.     

Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig

The majority of glycosaminoglycans synthezied in peritoneal macrophages from the guinea pig in vitro were secreted into culture medium. The secreted glycosaminoglycans were reduced in size with alkali treatment, indicating that the glycosaminoglycanas existed in the form of proteoglycans. After the glycosaminoglycans were digested with chondroitinase AC and ABC, the high voltage paper electrophoretic analysis and the descending paper chromatographic analysis indicated the presence of a considerable amount of unsaturated disulfated disaccharides. Based on the enzymatic assay with chondro-4- and 6-sulfatase, the positions of sulfation in the disulfated disaccharide have been identified as the 4- and 6-position of N-acetylgalactosamine, Moreover, the results of the ion-exchange chromatography and the chondroitinase AC and ABC digestion indicate that ΔDi-diS_E derived from dermatan sulfate. This suggests that peritoneal macrophages are capable of synthesizing oversulfated proteodermatan sulfate as main component. The proportion of synthesized oversulfated dermatan sulfate to the total glycosaminoglycans was independent of the incubation time, and the distribution of oversulfated dermatan sulfate in cell and incubation medium also did not change. After exposure of macrophages to Escherichia coli for 15 min, the incorporation of [³⁵S]sulfate and [³H]glucosamine into the glycosaminoglycans was increased by about 40% with no significant change in the proportion of synthesized oversulfated dermatan sulfate, but the relese of glycosaminoglycans into the culture medium remains essentially unchanged. The difference of the existence of oversulfated dermatan sulfate is not yet understood.     

Degradation of bradykinin by peritoneal and alveolar macrophages of the guinea pig

Peptidase inhibitors and identification of the peptide fragments were used for the characterization of the bradykinin metabolism by alveolar and peritoneal macrophages. Both cell types show differences in the rate of inactivation and in the quantity of the metabolites generated. BK(1-5), BK(1-8), and BK(1-7) are the predominant direct metabolites. Metalloendopeptidase 24.15, carboxypeptidase M, and an unidentified peptidase are responsible for their formation. Angiotensin-converting enzyme and neutral endopeptidase 24.11 do not play a crucial role in the degradation of bradykinin by macrophages. In the bronchoalveolar space, other cells than the macrophages are more important to the breakdown of this peptide.     

Activation of guinea pig peritoneal macrophages by platelet activating factor (PAF) and its agonists

The platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and its analogs were examined to determine their effects on guinea pig peritoneal macrophages. PAF activated macrophages, but its effect on macrophages was much weaker than that observed on platelets: the concentration required for 50% maximum activation was 8.5 X 10(-6) M for macrophages and 2.9 X 10(-10) M for platelets. Three PAF agonists, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-glycero-3-phosphocholine (Compound I), 1-O-octadecyl-2-acetamido-2-deoxy-glycero-3-phosphocholine (Compound II), and 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (Compound III), showed higher activity in stimulating macrophage function than PAF. The abilities of these non-metabolizable PAF agonists to activate macrophage paralleled their relative potency to induce platelet activation. The sn-3 enantiomers of PAF and Compound III exhibited activity, while the sn-1 did not. By comparing the activities of derivatives of Compound III, it was shown that the long-chain alkyl-ether group in the glycerol-1 position, a relatively small size of the substituent on the hydroxy group at the sn-2 position, and the choline moiety in the glycerol-3 position must play critical roles in the process of macrophage activation. A specific PAF antagonist, CV3988, which inhibits PAF-induced platelet activation and hypotension, inhibited the activation of macrophages caused by PAF and its agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.     

Effect of glycosylation inhibitors on binding properties of the Fc gamma receptors from guinea pig peritoneal macrophages.

Guinea pig peritoneal macrophages possess on their surfaces receptors for the Fc region of IgG immunoglobulins (Fc gamma R). The cells treated with the glycosylation inhibitors tunicamycin or monensin interacted with IgG with a higher affinity and showed a lower number of IgG-binding sites in comparison with control cells. This indicates that the interaction of IgG with the macrophage Fc gamma receptor depends on the degree of glycosylation of the cell surface glycoconjugates and that glycosylation of the macrophage Fc gamma receptor is important for the expression of the mature form of the receptor.     

Ca2+ release in the endoplasmic reticulum of guinea pig peritoneal macrophages

Passive permeability of the endoplasmic reticulum of saponin-treated macrophages to Ca2+ was studied by the filtration method using 45Ca. The Ca2+ release from the endoplasmic reticulum of macrophages was enhanced by the presence of submicromolar concentrations of Ca2+ in the medium. The Ca2+ release was enhanced by caffeine, and suppressed by MgCl2. These phenomena are similar to the Ca2+-induced Ca2+ release reported for the sarcoplasmic reticulum of skeletal muscle. On the other hand, adenine suppressed the Ca2+ release from the endoplasmic reticulum, while it reportedly enhanced the Ca2+-induced Ca2+ release of the skeletal muscle. The threshold concentration of Ca2+ for the Ca2+-induced Ca2+ release was approximately 10(-8) M in the presence of 0.95 mM MgCl2 in macrophages. The spontaneous spreading of macrophages and spontaneous migration of macrophages were inhibited by adenine, and also by caffeine in spite of the enhancement of the Ca2+-induced Ca2+ release.     

Functional and metabolic changes of guinea pig peritoneal macrophages induced by bacterial vaccines

Phagocytic activity of peritoneal macrophages of guinea pigs injected subcutaneously with bacterial vaccines was found to be increased. The phagocytic index remained unchanged or was decreased. In addition, a decreased candidacidal activity was observed. Metabolic activation of macrophages, measured by the INT test, was inhibited in unstimulated cells while cells stimulated with zymosan or opsonized zymosan exhibited higher values of the INT test as compared with control animals. After injection of vaccines the number of peritoneal macrophages was increased, but the spleen mass decreased. Translated by I. Miler     

Effect of nifedipine treatment on oxidative metabolism of peritoneal macrophages and neutrophils of Plasmodium berghei-infected mice.

The oxidative metabolism of peritoneal macrophages (PM) and neutrophils from nifedipine (calcium channel blocker)-treated, Plasmodium berghei (NK 65)-infected and normal infected Swiss Albino mice was studied. A significant fall in oxidative metabolism as evidenced by decreased chemiluminescence (CL) response (P less than 0.001) was recorded both in PM and neutrophils from nifedipine-treated mice compared to the control animals. When the oxidative metabolism of these phagocytes was studied after infection of the host, higher CL response was recorded from both PM and neutrophils isolated during the early course of infection (0-1 and 5-10% parasitaemia) when compared to uninfected mice (P less than 0.001). A similar pattern was observed in the case of nifedipine-treated and infected mice even though the CL response was much lower. The increasing parasite load not only resulted in subnormal CL response but also prolonged the time required for the phagocytes to exhibit peak oxidative activity both in normal infected and CCB-treated infected mice, but the time taken to show peak CL response was shortened following drug administration compared to controls. These observations revealed the profound in vivo effect of CCB on the functioning of phagocytic leucocytes and thereby questions the use of CCB in combination with chloroquine for reversal of drug resistance.     

Alteration of hormone-stimulated cyclic AMP synthesis in guinea pig peritoneal macrophages.

The decrease of PGE-stimulated cyclic AMP synthesis due to pretreatment of intact cells with PGE (hormone-specific desensitization) was shown to be a rapid process in macrophages. Desensitization was found to be extensive after 5-min treatment of macrophages with PGE₂ and almost complete after 20 min. Furthermore, incubation of intact macrophages with colchicine caused a two- to sixfold increase in the rate of PGE₁-stimulated cyclic AMP synthesis in intact macrophages. Colchicine alone did not alter cyclic AMP levels. The enhancing effect of colchicine is related to its ability to disrupt microtubules. Vinblastine, another microtubule-disrupting agent, caused similar enhancement of PGE-stimulated cyclic AMP synthesis; no enhancement was found when lumicolchicine was used. Hormonestimulated cyclic AMP synthesis by colchicine-treated macrophages was also measured after cell homogenization. The enhancement of hormone sensitivity by colchicine was found to be lost upon homogenization. These findings suggest that colchicine acts at the interior of the cell to reversibly affect adenylate cyclase.     

Complex study of the oxidative metabolism of peritoneal macrophages attached to glass

A method of obtaining a peritoneal macrophage enriched population on a slide in a special chamber has been worked out and tested. It has been shown that such cells keep specific morphological and functional peculiarities, including the ability to phagocytosis. They are in active state and can be utilized for studying the biological oxidation processes. A relative activity of oxidative metabolism of key enzymes has been evaluated. The possibility of studying the respiratory chain activity in these cells was shown by polarographic and fluorescent methods. Alternative oxygen-dependent systems were investigated. It was determined that NADPH-oxidase of peritoneal macrophages attached to a slide, responsible for "the respiratory burst" formation and defining their bactericidal properties and peroxide generating ability, is in the active state.