Macrophage Activation by Tetrahymena pyriformis II. Active Protein Fractions from Tetrahymena

ABSTRACT. Water-soluble and 0.6 M KCl-soluble protein fractions prepared from Tetrahymena pyriformis , when inoculated into mice, could effectively induce activated macrophages having the ability to kill Toxoplasma gondii in vitro. This effect was not induced by other proteins tested, such as bovine serum albumin, pepsin from porcine stomach mucosa and chicken egg-white lysozyme, nor by muramyl dipeptide (MDP), a potent immunoadjuvant. Five fractions obtained by DEAE-Sephadex chromatography of the water-soluble protein fraction were compared with regard to induction of toxoplasmacidal activity in macrophages. The first peak was most effective for activation of macrophages. Five fractions obtained by chromatography of the 0.6 M KCl-soluble protein fraction were also examined and it was found that the first peak had the activity. No marked difference in activity was observed between the active fractions of water-soluble and 0.6 M KCl-soluble protein fractions. For practical use, we focused on the water-soluble active fraction. The minimum effective dose of the active fraction was 100 μg and the fraction could activate macrophages directly in vitro. Four fractions obtained by gel filtration of the active fraction on Sephadex G-200 were compared and the first peak had the activity. The first peak contained a single protein, revealed by SDS-polyacrylamide gel electrophoresis; its apparent molecular weight was 64,000.     

Effect of in vivo administration of Tetrahymena pyriformis on the in vitro toxoplasmacidal activity of mouse peritoneal macrophages

Tetrahymena pyriformis is a free-living ciliate protozoan in the freshwater system. Experiments were carried out to determine whether intraperitoneal administration of T. pyriformis (GL strain) to mice activates macrophages to be able to kill Toxoplasma gondii tachyzoites in vitro. Mice were also injected intraperitoneally with several synthetic activators; dimethyldioctadecylammonium bromide (DDA), dextran sulfate, complete Freund's adjuvant (CFA) as well as Toxoplasma and Tetrahymena lysates in order to activate mouse peritoneal macrophages. One week after the administration of activators, peritoneal cells were harvested and the adherent macrophages were challenged with Toxoplasma tachyzoites. Macrophage monolayers were then fixed with absolute methanol after washing, and stained with Giemsa solution. The percentage of the adherent cells infected and total number of organisms per 100 macrophages were calculated to make toxoplasmacidal activity of macrophages according to the cultivation time. Peritoneal macrophages from mice administered with Tetrahymena exhibited significant protection against target parasites as compared with those treated with synthetic activators. Among non-biological synthetic activators, DDA was evaluated as an excellent activator.     

Screening of a functional polysaccharide from Zizyphus Jujuba cv. Jinsixiaozao and its property

Chinese jujube has been used as a kind of Chinese medicinal herb for a long time. In this paper, the polysaccharides from Zizyphus Jujuba cv. Jinsixiaozao (ZSP), one of the major Chinese jujube varieties, were extracted and purified sequentially so that its water-soluble immunobiological fractions were screened. The crude ZSP was found to dramatically increase thymus and spleen indices in mice, enhance the proliferation of splenocytes and peritoneal macrophages, and have potential anti-complementary activity. Immunobiological tests indicated that two fractions of ZSP, coded ZSP3c and ZSP4b, were the main active components. ZSP3c was rich in pectin with a degree of esterification (DE) of 49%, which may be related to its stronger immunological activity.     

Detection of IgG antibody against Neospora caninum in cattle in Korea

A total of 492 cattle sera was screened by IgG-ELISA against Neospora caninum (Nc-1 strain and a Korean isolate, KBA-2) and Toxoplasma gondii. Out of 492, 113 sera (23.0%) reacted positively to either Nc-1 or KBA-2 strains of N. caninum. Among the 113 positive sera, 92 sera (81.4%) reacted with antigens of both strains, but 6 sera (5.3%) with Nc-1 and 15 sera (13.3%) with KBA-2 strain only. And with T. gondii antigen, 6 sera (1.2%) were positive but all reacted with N. caninum antigen also. Western blot revealed typical binding pattern according to ELISA values, such that high OD group reacted specifically to the major surface proteins including 43 kDa protein. Seroprevalence of 23.0% indicates that neosporosis seemed to be one of major causes of abortion in cattle. It is suggested here to establish more epidemiological researches nationwide systematically.     

Laboratory passage and characterization of an isolate of Toxoplasma gondii from an ocular patient in Korea

Toxoplasma gondii tachyzoites were isolated from the blood of an ocular patient, and have been successfully passaged in the laboratory, for over a year, by peritoneal inoculation in mice. The isolated parasite was designated the Korean Isolate-1 (KI-1) and its characteristics were compared with those of the RH strain, a wellknown virulent strain originating from a child who suffered from encephalitis. The morphology, pathogenicity, infectivity and cell culture characteristics of the KI-1 were similar to those of the RH strain. Both RH and KI-1 antigens were detected by an anti-T. gondii monoclonal antibody (mAb), Tg563, against the major surface protein SAG1 (30 kDa), whereas no reaction was observed against an anti-Neospora caninum mAb, 12B4. The KI-1 was confirmed as an isolate of T. gondii. A long-term laboratory maintenance and characterization of a local T. gondii isolate is reported for the first time in the Republic of Korea.     

Toxoplasma gondii partially inhibits nitric oxide production of activated murine macrophages

Activated macrophages produce nitric oxide (NO) and as such are able to control the multiplication of Toxoplasma gondii. Until now, no reports have described a possible modulation of NO production of macrophages after T. gondii infection. To investigate this possibility, murine blood monocyte-derived and peritoneal macrophages were activated in vitro with interferon-gamma and lipopolysaccharide and infected with T. gondii and Trypanosoma cruzi, and NO production was evaluated. NO was produced by monocyte-derived macrophages only if cultured in the presence of macrophage-colony-stimulating factor. Monocyte-derived or peritoneal macrophages infected with T. gondii presented a significant reduction in NO production. NO production inhibition was not detected after T. cruzi infection. Macrophages infected with higher T. gondii/macrophage ratios presented lower NO production. Furthermore, only viable T. gondii could cause partial inhibition of NO production. In macrophages activated 24 h before the interaction, partial inhibition was detected after 3 h of infection and continued for 48 h. In macrophages activated immediately after the interaction, partial inhibition was not detected at 12 h, but was observed at 24 h. T. gondii-infected macrophages present lower inducible nitric oxide synthase expression as assayed by immunofluorescence. T. gondii did not develop in monocyte-derived macrophages producing NO, but were not totally eliminated. These results demonstrate that T. gondii infection partially inhibits NO production by murine macrophages, suggesting that a deactivating macrophage mechanism may be used for better survival into phagocytic cells.     

Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif

Two bradyrhizobial strains, CTAW71(T) and CTAW69, previously isolated from root nodules of Cytisus villosus, have been analysed using a polyphasic approach. These strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium cytisi, whose type strain CTAW11(T) presented 99.8% identity with respect to strain CTAW71(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII harboured by strain CTAW71(T) were divergent to those from B. cytisi CTAW11(T), with identity values of 93%, 95% and 97%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 37% relatedness between strain CTAW71(T) and B. cytisi CTAW11(T). Phenotypic characteristics were identical for strains CTAW71(T) and CTAW69, but differed from those of the described species from genus Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose that strains CTAW71(T) and CTAW69 should be classified into a new species for which the name Bradyrhizobium rifense sp. nov. is proposed (type strain CTAW71(T)=LMG 26781(T)=CECT 8066(T)).     

Binding of calmodulin to the microfilament network correlates with induction of a macrophage tumoricidal response

Induction of mouse peritoneal macrophage cytotoxicity against SV3T3, a line of virally transformed mouse cells correlated with the distribution of cytoplasmic calmodulin in the macrophages. The organization of the cytoskeleton was examined by fluorescent microscopy and by transmission electron microscopy, using immunogold tagging after Triton-X-100 (TX-100) extraction of the macrophages. Macrophages that had been activated to a tumoricidal state in vivo by vaccinia virus or in vitro by lymphokine stimulation displayed cytoskeletal networks that were more extended and weblike than did resident macrophages. The organization of microfilaments and microtubules in the cytoskeleton was displayed by using either anti-actin or anti-tubulin. Immunogold labeling of tumoricidal macrophage cytoskeletons with anti-calmodulin revealed strong binding to the microfilament network and no binding to microtubules. Anti-calmodulin reacted weakly with the cytoskeletal network of resident macrophages, and this was not demonstrably greater than the reaction with normal sheep serum. However, resident macrophages displayed a high density of calmodulin (CAM) associated with unidentifiable structures in the perinuclear region when reacted with anti-calmodulin. These characteristic distributions of CAM in resident and activated macrophages was confirmed by immunofluorescence. The total and cytoskeletal-associated amounts of calmodulin per unit of protein were determined by radioimmune assay and 125I labeling followed by SDS-PAGE. No statistically significant differences were detected between resident and activated macrophages in either the total cell or cytoskeleton fractions. In summary, our results suggest that induction of tumoricidal activity of mouse peritoneal macrophages correlates with the translocation of calmodulin to the microfilament network of the cytoskeleton.     

Molecular and phenotypic characterisation of Bacillus thuringiensis isolated during epizootics in Cydia pomonella L

Twelve Bacillus thuringiensis strains were isolated from intestinal tracts of Cydia pomonella larvae during epizootics in different laboratory insect culture lines. Phenotypic and genetic similarity of these isolates, together with two cultured from Leucoma salicis larvae and 14 reference B. thuringiensis strains were determined. The epizootic bacteria did not form a single group based on numerical analysis of biochemical properties. Simple RAPD method with only one primer does not allow estimating the genetic similarity of B. thuringiensis strains. We propose a novel strategy based on combining several DNA patterns obtained by RAPD technique with different primers for B. thuringiensis typing. Majority of infections in the C. pomonella culture lines were caused by bacteria with different genotypes. However, two isolates cultured from infected insects at different time (one strain was isolated in 1990 and the other in 1992) had identical DNA fingerprint that suggested a possibility of these bacteria to survive in the laboratory and to cause infections in different years. The results of SDS-PAGE of whole-cell proteins revealed a possibility to apply protein profile analysis in epidemiological investigations of infections caused by B. thuringiensis. Strains with identical DNA patterns had very similar whole-cell protein profiles.     

Macrophage activation by Tetrahymena pyriformis. I. Active subcellular fractions of Tetrahymena

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H2O2) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H2O2 release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.     

A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb

The immunopotentiating effect of the roots of Astragalus membranaceus, a medicinal herb, has been associated with its polysaccharide fractions (Astragalus polysaccharides, APS). We herein demonstrate that APS activates mouse B cells and macrophages, but not T cells, in terms of proliferation or cytokine production. Fluorescence-labeled APS (fl-APS) was able to selectively stain murine B cells, macrophages and a also human tumor cell line, THP-1, as determined in flow cytometric analysis and confocal laser scanning microscopy. The specific binding of APS to B cells and macrophages was competitively inhibited by bacterial lipopolysaccharides. Rabbit-anti-mouse immunoglobulin (Ig) antibody was able to inhibit APS-induced proliferation of, and APS binding to, mouse B cells. Additionally, APS effectively stimulated the proliferation of splenic B cells from C3H/HeJ mice that have a mutated TLR4 molecule incapable of signal transduction. These results indicate that APS activates B cells via membrane Ig in a TLR4-independent manner. Interestingly, macrophages from C3H/HeJ mice were unable to respond to APS stimulation, suggesting a positive involvement of the TLR4 molecule in APS-mediated macrophage activation. Monoclonal Ab against mouse TLR4 partially inhibited APS binding with macrophages, implying direct interaction between APS and TLR4 on cell surface. These results may have important implications for our understanding on the molecular mechanisms of immunopotentiating polysaccharides from medicinal herbs.     

Glycosaminoglycan profile of peritoneal and bone marrow-derived macrophages. Changes associated with macrophage activation.

1. We have analysed the glycosaminoglycan patterns of peritoneal and bone marrow-derived macrophages obtained from four different mouse strains which are resistant (A/J) or susceptible (BALB/c, DBA and C-57) to murine hepatitis virus type 3 (MHV3) infection. The glycosaminoglycans were biosynthetically labelled by exposing the macrophages to 35S-sulphate. The medium and cell fractions were collected and the 35S-glycosaminoglycans formed were identified by a combination of agarose gel electrophoresis and enzymatic degradation with bacterial mucopolysaccharidases. 2. Both peritoneal and bone marrow-derived macrophages synthesize and secrete a mixture of dermatan sulphate, heparan sulphate and chondroitin sulphate. Dermatan sulphate is the main glycosaminoglycan and most of the synthesized glycosaminoglycans are released to the culture medium. 3. The glycosaminoglycan patterns vary depending on the macrophage source. Bone marrow-derived cells synthesize glycosaminoglycans at lower rates, release a lower glycosaminoglycan percentage to the culture medium and express higher amounts of heparan sulphate in comparison with their peritoneal counterparts. Furthermore, LPS-induced activation leads to an increased glycosaminoglycan expression in bone marrow-derived macrophages and to a decrease in 35S-glycosaminoglycans of peritoneal macrophages from BALB/c, A/J and C-57 mice. 4. We have not established any correlation between macrophage glycosaminoglycans and resistance to MHV3 infection, since the glycosaminoglycan patterns of resistant (A/J) and susceptible (BALB/c, DBA and C-57) mouse macrophages are similar. Furthermore, the in vitro infection of both control and LPS-activated peritoneal macrophages with MHV3 did not cause any changes in the expression of glycosaminoglycans.     

Purification and characterization of the 65-kDa protein phosphorylated in murine macrophages by stimulation with bacterial lipopolysaccharide

Modification of cellular proteins via phosphorylation is known to be a major regulatory mechanism whereby external stimuli control intracellular events. We demonstrated that bacterial LPS induced a distinct set of phosphorylated protein (pp) in murine peritoneal macrophages, and that the LPS-induced pp were specifically located in cytosol and/or membrane fractions. One of the most heavily phosphorylated substrate proteins with a molecular mass of 65 kDa (pp65) was purified to homogeneity via SDS-PAGE analysis and autoradiography by sequential chromatography on Sephacryl S-200, HPLC anion exchange, and hydroxyapatite HPLC. Our pp65 is apparently the first purified LPS-induced pp, and is thought to be a novel protein. Serine residues on pp65 were found to be exclusively phosphorylated, indicating a contribution by LPS-inducible serine kinase. Interestingly, LPS-induced phosphorylation of pp65 was not observed in macrophages from a LPS-nonresponsive C3H/HeJ strain of mice, although their macrophages had about the same amounts of unphosphorylated p65 as normal macrophages when detected under Western blot analysis by using polyclonal anti-pp65 antibodies. This suggests that the functional defect of C3H/HeJ macrophages exists somewhere in the process before the pp65 phosphorylation. Moreover, the degree of the pp65 phosphorylation in macrophages stimulated with LPS or lipd A correlated well to that of cellular responses such as IL-1 production in the same macrophages. Considering these observations, the pp65 seems to play a crucial role in macrophage activation, and the studies on the structure and function of the pp65 should lead to progress in our understanding of the mechanisms of macrophage activation by LPS.     

Trichinella spiralis: macrophage activity and antibody response in chronic murine infection

The role of macrophages, their products, and the specific antibody response were examined during chronic Trichinella spiralis infection in BALB/c mice. Adult T. spiralis in intestines were detected from 5 to 20 dpi. Muscle larvae numbers peaked at 45 dpi and thereafter a reduction was noted. The highest numbers of macrophages in the peritoneal cavity of infected mice were obtained up to 30 dpi. The production of NO by macrophages in infected mice was suppressed at 5 dpi, and then NO release increased until 45 dpi. The levels of NO in plasma and urine were lower in infected mice during the entire experiment in comparison to control. The production of O(2)(-) in peritoneal macrophages was inhibited during the first two weeks after infection and then increased until 90 dpi. Circulating T. spiralis antigens in plasma and urine were detected from 5 to 30 dpi. Specific IgM and IgA in serum increased until 20 dpi. IgG, IgG(1), and IgG(2) levels in serum increased until 60 dpi.     

Phenylpropanoid glycosides from Scrophularia scorodonia: in vitro anti-inflammatory activity

Five phenylpropanoid glycosides isolated from Scrophularia scorodonia L. (Scrophulariaceae), namely angoroside A (1), angoroside C (2), angoroside D (3), acteoside (4) and isoacteoside (5), had been evaluated as potential inhibitors of some macrophage functions involved in the inflammatory process. These compounds have been tested in two experimental systems: ionophore-stimulated mouse peritoneal macrophages and human platelets serve as source of COX-1 and 5-LOX, and mouse peritoneal macrophages stimulated with E. coli LPS are the means of testing for COX-2, NO and TNF-alpha activity. None of compounds assayed had a significant effect on LTC(4)-release from calcium ionophore-stimulated mouse peritoneal macrophages. However, the release of PGE(2) by mouse peritoneal macrophages stimulated with calcium ionophore was inhibited by most of these compounds. In the TXB(2)-release assay, acteoside (4), angoroside A (1) and angoroside C (2) showed a significant effect. These five compounds, except angoroside C (2) significantly inhibited LPS-induced PGE(2), NO and TNF-alpha in a concentration-dependent manner. In LPS-stimulated macrophages, the phenylpropanoid glycoside angoroside C (2) only had activity on NO. These results indicate that the pharmacology of these compounds may participate in the anti-inflammatory effect of Scrophularia scorodonia.     

Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

Abstract The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

Trypanosoma cruzi: effects of adrenalectomy during the acute phase of experimental infection

Glucocorticoid hormones have been implicated as an important modulator of Trypanosoma cruzi pathogenesis. Since adrenal steroid hormones play a fundamental role in modulating the immune response, we hypothesized that adrenalectomy affect the course of the experimental T. cruzi infection. This study was undertaken to determine the effects of adrenalectomy during the acute phase of T. cruzi infection. Blood and tissue parasitism, macrophages, nitric oxide (NO) production and IFN-γ were evaluated in male Wistar rats infected with the Y strain of T. cruzi. Our results show that adrenalectomized rats displayed increased number of blood and heart parasites accompanied by decreases in the total number of peritoneal macrophages and IFN-γ when compared to controls. Adrenalectomy also reduced the levels of NO released from peritoneal macrophages of infected animals. These results suggest that adrenal corticosteroid insufficiency due to adrenalectomy could be considered an important factor during development of acute phases of experimental Chagas’ disease, enhancing pathogenesis through disturbance of the host’s immune system.     

Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.     

Comparative analysis of proteinase activities of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae)

Proteinase activities were compared in soluble and membrane fractions of guts obtained from larvae of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis. Overall, serine proteinases from soluble fractions of the susceptible strain were more active than those of the resistant strain. The soluble trypsin-like proteinase activity of the resistant strain was approximately half that of the susceptible strain. The number and relative molecular masses of soluble and membrane serine proteinases were different. However, there were no significant differences in the activities of serine proteinases and aminopeptidases extracted from midgut membranes of the two strains. Cry1Ab protoxin hydrolysis by soluble proteinase extracts of the resistant strain was reduced approximately 20-30% relative to that of the susceptible strain. Reduced protoxin processing due to decreased activities of Bt protoxin activation proteinases may be associated with resistance to Bt toxin in this resistant strain of O. nubilalis.