Estradiol inhibits differentiation of mouse macrophage into a pro-inflammatory phenotype by upregulating the IRE1α-XBP1 signaling axis / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway.@*METHODS@#Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR.@*RESULTS@#Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist.@*CONCLUSION@#Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.

Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis

BACKGROUND Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

Correction to: High-fat diet or low-protein diet changes peritoneal macrophages function in mice

The original version of this article [1], published on 28 June 2016, contains a mistake. The part labels in Fig. 1 are missing. The corrected version of Fig. 1 is given below.

Modulatory effects of α- linolenic acid on generation of reactive oxygen species in elaidic acid enriched peritoneal macrophages in rats.

Fatty acids are known to influence the ability of macrophages to generate reactive oxygen species (ROS). However the effect of elaidic acid (EA, 18:1 trans fatty acid) on ROS generation is not well studied. Rat peritoneal macrophages were enriched with elaidic acid by incubating the cells with 80 µM EA. The macrophages containing EA generated higher amounts of superoxide anion (O2·-), hydrogen peroxide (H2O2) and nitric oxide (NO˙) by 54, 123 and 237%, respectively as compared to control cells which did not contain EA. To study the competition of other C18 fatty acids with EA macrophages were incubated with EA along with stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and α- linolenic acid (ALA, 18:3). ALA significantly reduced the incorporation of EA into macrophage lipids. This also significantly reduced the generation of O2· -, H2O2, NO˙ by macrophages. Studies were also conducted by feeding rats with diet containing partially hydrogenated vegetable fat (PHVF) as a source for EA and linseed oil (LSO) as a source for ALA. The rats were fed AIN-93 diet containing PHVF with 17% EA and incremental amounts of linseed oil for 10 weeks. The peritoneal macrophages from rats fed partially hydrogenated vegetable fat generated higher levels of O2·-, H2O2, NO˙ by 46, 161 and 76% respectively, when compared to rats fed control diets containing ground nut oil. Macrophages from rats fed PHVF with incremental amounts of LSO produced significantly lower levels ROS in a dose dependent manner. Thus ALA reduces the higher levels of ROS generated by macrophages containing EA.

In vitro studies on inhibitory effect of proanthocyanidins in modulation of neutrophils and macrophages.

The role of proanthocyanidins (PC), a novel flavonoid extracted from grape seeds was studied in vitro in the modulation of neutrophil and macrophage function. We attempted to assess the levels of non-enzymatic and enzymatic mediators in the presence or absence of PC in 4-phorbol-12--myristate-13-acetate (PMA)-stimulated neutrophils isolated from humans and rats, E. coli endotoxin-stimulated macrophages and macrophages isolated from E. coli endotoxin-induced experimental periodontitis in rats. Addition of PC at a concentration of 50 µg/ml effectively blocked the release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and exhibited a marked inhibition of myeloperoxidase (MPO) and lysosomal enzymes (p<0.001), as compared to PMA-stimulated neutrophils (human and rats) and neutrophils isolated from experimental periodontitis in rats. The levels of ROS, RNS and lysosomal enzymes were found to be elevated (p<0.001) and addition of PC significantly (p<0.001) reduced these levels as compared to those from E. coli endotoxin-stimulatedmacrophages from rats and macrophages isolated from experimental periodontitis in rats (p<0.001). Thus, the study demonstrated that PC decreased the levels of ROS and RNS and also inhibited the MPO and lysosomal enzymes activities in experimental periodontitis in rats. In addition, this study clearly indicated that PC could be developed as an effective antiinflammatory agent.

Avaliação dos perfis de resposta de macrófagos peritoneais de balb/c infectados in vitro com Leishmania amazonensis ou Leishmania braziliensis / Evaluation of response profiles of peritoneal macrophages of Balb / c infected in vitro with Leishmania amazonensis and Leishmania braziliensis

Macrófagos são células do sistema imunológico que desempenham importante papel na defesa contra Leishmania. Neste trabalho, foram investigados alguns fatores e mecanismos que podem estar envolvidos na determinação dos perfis de resposta de Macrófagos peritoneais de BALB/c infectados in vitro com L. amazonensis ou L. braziliensis. Observou-se que IFN-y não foi capaz de reduzir a infecção causada por ambas as espécies de Leishmania. A sinergia entre IFN-y e TNF-a leva a uma diminuição do percentual de infecção dos macrófagos por La e Lb no tempo de 72h. Tanto o pré-tratamento com IFN-y quanto com IFN-y e TNF-a não alterou a viabilidade de L. amazonensis isoladas de Macrófagos em relação ao grupo que não sofreu pré-tratamento. No entanto, L. braziliensis se mostrou susceptível a ação dessas citocinas, já que o pré-tratamento dos Macrófagos com IFN-y foi capaz de diminuir o número de parasitos viáveis isolados. A sinergia entre IFN-y e TNF-a se mostrou bastante efetiva nesse grupo uma vez que o número de Lb isoladas a partir dos Macrófagos infectados mostrou-se bastante reduzido. Embora não tenha sido detectada produção de NO nos grupos de Macrófagos infectados com La e Lb, o bloqueio da produção dessa molécula, tanto in vitro quanto in vivo, é capaz de aumentar a infecção por Lb, mas não altera a infecção por La. A avaliação da produção de IL-12, revelou que Macrófagos infectados com Lb produzem maior quantidade dessa citocina e apresentam uma maior expressão de RNAm de TNF-a nas primeiras 6h após infecção que os infectados por La. Por outro lado, a infecção com La induz uma maior produção de TGF-J3, maior expressão de RNAm de IL-10 e menor expressão de RNAm de quimiocinas e receptores dessas moléculas em relação ao grupo infectado com Lb. A investigação da atividade de arginase de La e Lb demonstrou que promastigotas de La apresentam uma elevada atividade de arginase, apresentando-se aproximadamente 50 a 100 vezes maior que nos promastigotas de Lb. Esses dados correlacionam-se com a maior carga parasitária e maior sobrevivência desse parasito no interior dos Macrófagos, enquanto que a menor atividade dessa enzima em Lb pode estar relacionada com a baixa carga parasitária, alta susceptibilidade aos mecanismos microbicidas dos Macrófagos e uma conseqüente diminuição na sobrevivência dessa espécie de Leishmania. Esses dados demonstram que La e Lb induzem perfis diferentes de resposta à infecção, tanto in vitro quanto in vivo, e sugerem que fatores produzidos pelo parasito, a exemplo de arginase, podem estar envolvidos no desenvolvimento desses perfis diferenciados de resposta.

Multiplicación de Brucella abortus y producción de óxido nítrico en dos líneas celulares de macrófagos de distinto origen / Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin

Brucella abortus es una bacteria que causa abortos e infertilidad en el ganado y fiebre ondulante en el hombre. Se multiplica en el citoplasma celular evadiendo los mecanismos de muerte intracelular. El óxido nítrico (NO) es importante en la regulación de la respuesta inmune. En el presente trabajo estudiamos la habilidad de tres cepas de B. abortus para sobrevivir intracelularmente en dos líneas celulares de macrófagos. La multiplicación de bacterias en ambas líneas celulares fue determinada a distintos tiempos en número de UFC/ml, también fue observada al microscopio de campo claro y de fluorescencia utilizando Giemsa y naranja de acridina, respectivamente. La tinción de ambas líneas celulares inoculadas con B. abortus mostró un resultado concordante con el encontrado en la determinación del número de UFC. Fue confirmada la presencia de B. abortus por microscopía electrónica. Para medir la producción de NO se utilizó el reactivo de Griess. La multiplicación de la cepa rugosa RB51 disminuyó en ambas líneas celulares y los niveles de NO fueron mayores en células inoculadas con dicha cepa que cuando fueron inoculadas con las cepas lisas (S19 y 2308). Estos resultados sugieren que probablemente la ausencia de cadena O en el lipopolisacárido afecta el crecimiento intracelular de B. abortus.

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably has affects the intracellular growth of B. abortus.

Identification and characterization of lipids from endosomes purified by electromagnetic chromatography.

Traditional separation techniques do not yield endolysosomes of sufficient purity to permit detailed biochemical characterization of this important class of intracellular vesicles. Here, we have used a magnetic chromatography technique to isolate the endosomes from rat peritoneal macrophages and studied their lipid composition. Electromagnetic isolation works by retention of colloidal iron containing vesicles on magnetic column. The data suggested that both early and late endosomes were rich in cholesterol, whereas sphingomyelin (SM) and specific phospholipids like phosphatidylcholine. phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine are enriched in the late compartments. Our results also indicated that the purified fractions are enriched in raft lipids like SM, but not in cholesterol. The endosomal purification method described here yields pure endosomes with little or no contamination from mitochondria and hence could be used for further biochemical and marker analysis, giving insight into mechanisms of endocytic traffic.

Activation of inducible nitric oxide synthase by Kagamjuaguiem in peritoneal macrophages in mice.

BACKGROUND & OBJECTIVE: A Korean herbal formula Kagamjuaguiem (KJE) has been used for the purpose of the tumour therapy. However, its mechanism of action is not clear. Nitric oxide (NO) as a potent macrophage-derived effector molecule against a variety of tumours has received increasing attention. In this study, using mouse peritoneal macrophages, we have examined the mechanism by which KJE regulates NO production. METHODS: Peritoneal macrophages were cultured with recombinant interferon-gamma (gammaIFN-gamma) for 6 h. The cells were then stimulated with various concentrations of KJE. NO synthesis in cell cultures was measured by Griess method, and inducible NOS expression was measured by western blotting. The amount of tumour necrosis factor-alpha (TNF-alpha) secreted by the cell was measured by a modified enzymelinked immunosorbent assay. RESULTS: When KJE was used in combination with gammaIFN-gamma there was a marked co-operative induction of NO production. However, KJE had no effect on NO production by itself. The increased production of NO from rIFN-gamma plus KJE-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate, an inhibitor of nuclear factor kappa B (NF-kappaB). Further, treatment of peritoneal macrophages with rIFN-gamma plus KJE caused a significant increase in TNF-alpha production. INTERPRETATION & CONCLUSION: Our findings demonstrate that KJE increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of KJE.

Extract of gum resins of Boswellia serrata L. inhibits lipopolysaccharide induced nitric oxide production in rat macrophages along with hypolipidemic property.

Boswellia serrata, Linn F (Burseraceae) is commonly used in Indian system of medicine (Ayurvedic) as an anti-inflammatory, analgesic, anti-arthritic and anti-proliferative agent. This study was planned to investigate the water-soluble fraction of the oleoresin gum of Boswellia serrata (BS extract) on lipopolysaccharide (LPS) induced nitric oxide (NO) production by macrophages under in vivo and in vitro conditions. In the previous condition, rats were fed on atherogenic diet (2.5% cholesterol, 1% cholic acid, 15.7 % saturated fat) along with the BS extract for 90 days. Blood was collected for lipid profile and toxicological safety parameters. Peritoneal macrophages were isolated and cultured to see the LPS induced NO production. Under in vivo experiment, BS extract significantly reduced serum total cholesterol (38-48 %), increased serum high-density lipoprotein- cholesterol (HDL-cholesterol, 22-30%). Under in vitro experiments with thioglycolate activated macrophages, it inhibited LPS induced (NO) production with IC 50 value at 662 ng /ml. Further, this fraction, in the dose of 15 mg/100 g body wt for 90 days, did not show any increase in serum glutamate-pyruvate transaminase (SGPT) and blood urea, in normal control animals. However, it significantly reversed the raised SGPT and blood urea in the atherogenic diet-fed animals. Transverse section of liver and kidney also supported its protective effect. Thus it may be concluded that water extract of Boswellia serrata possesses strong hypocholesterolemic property along with increase in serum HDL. It inhibits the LPS induced NO production by the activated rat peritoneal macrophages and show hepato-protective and reno-protective property.

Receptor-mediated endocytosis of biofilm-forming Enterococcus faecalis by rat peritoneal macrophages.

BACKGROUND & OBJECTIVES: Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics. Studies were taken up to identify virulence factors and to characterise pathogenic mechanisms of such infections to evaluate potential targets for treatments alternative to antibiotic therapy. This study was carried out to evaluate the contribution of extracellular polysaccharide expressed by Enterococcus faecalis to resistance to phagocytosis and survival within rat peritoneal macrophages. METHODS: Six E. faecalis clinical isolates were tested for their ability to survive within rat peritoneal macrophages. Cytochalasin D, colchicine and monodansylcadaverine were used to investigate the route of enterococcal entry inside macrophages. RESULTS: Four of the isolates were able to produce extracellular polysaccharide and form biofilm after growth in glucose-supplemented medium, while no production could be detected in glucose deficient medium. Two isolates were polysaccharide-negative in both conditions. Isolates expressing extracellular polysaccharide were able to survive for more than 24 h compared to polysaccharide-negative bacterial cells of the same strain grown in glucose-deficient medium, which were readily cleared. Cytochalasin D virtually abolished the number of viable intracellular bacteria, after growth in either trypticase soy broth (TSB) or TSB supplemented with glucose; colchicine and monodansylcadaverine strongly affected survival of polysaccharide-positive bacteria, significantly more than that of polysaccharide-negative ones. INTERPRETATION & CONCLUSION: Biofilm-forming E. faecalis survived within rat peritoneal macrophages significantly better than polysaccharide-negative isolates. Perturbators of cytoskeleton and of surface receptors turnover, indicated receptors-mediated endocytosis as the most likely route for enterococcal entry into macrophages.

Oxidative and non-oxidative activation of murine peritoneal macrophages by histone H1.

The present study aimed at assessing the role of histone H1 in activating macrophages. Histone H1, injected intraperitoneally at a dose of 1 mg/kg body weight as multiple regimens weekly, significantly increased the number of peritoneal macrophages post 21 days of injection. The oxidative and non-oxidative activation of peritoneal macrophages by histone H1 was assessed. For the assessment of oxidative activation the levels of superoxide radical and nitric oxide radical were assessed. The oxidative activation was evident from release of significantly high levels of superoxide and nitric oxide radicals liberated by macrophages of animals treated with histone H1 (P < 0.001) than in untreated animals. In addition, the higher activities of superoxide dismutase indicated protective effect of histone H1, to keep away the macrophages from noxious effects of superoxide. The catalase activity was decreased significantly in macrophages of histone H1 treated animals. The levels of reduced glutathione were significantly (P < 0.001) lowered in treated animals, whereas the levels of lipid peroxides generated were non-significant. The non-oxidative activation was assessed from the activities of lysosomal enzymes released and also from cytolysis of NO-insensitive L929 cells. The activities of lysosomal enzymes-acid phosphatase and beta-glucuronidase released were significantly high in treated animals than in untreated animals (P < 0.001). Histone H1 stimulated the cytolysis of macrophages in L929 cells than in untreated animals. These results suggest that histone H1 stimulates macrophages by oxidative and non-oxidative mechanisms, which favor its future therapeutic prospects.

Goat serum: an alternative to fetal bovine serum in biomedical research.

Serum is frequently added to the defined basal medium as a source of certain nutritional and macromolecular growth factors essential for cell growth. Although a number of synthetic media have been prepared serum continues to be used in cell culture by many investigators. The best supplementation to a basal medium is fetal bovine serum (FBS) that is most frequently used for all types of cell cultures. During last four decades National Institute of Virology, Pune, has been working on isolation and identification of viruses from clinical specimens, employing tissue culture. Initially FBS was used for this purpose. However, due to its prohibitive cost and uncertain supply an alternative was sought. Commercially available sera from newborn calf, sheep, horse, human and serum obtained from goat blood (available from local abattoir) were tried. Goat serum (GS) was found to be suitable for most of the cell lines and primary cultures. Primary cultures from guinea pig embryo, monkey kidney, chick embryo, mouse peritoneal macrophages, and established cell lines were prepared and grown in growth media supplemented with GS. These cultures were studied for their morphology and growth in comparison with cultures grown in FBS containing media, and were used for mass cultivation of cells, quantitation and susceptibility of various virus strains, studies on effects of different nutrients and natural substances on cellular metabolism and virus replication, epitope analysis of various strains of Japanese encephalitis (JE) virus, strain differentiation studies, studies on antibody dependent plaque enhancement, assay of murine migration inhibition factor. Monoclonal antibodies against JE virus adapted to GS were characterised for their retention of functionalities. The results were comparable to those of cell cultures grown in FBS containing media. Similar results on chromosome studies were obtained from patient's whole blood cultures prepared in GS and FBS containing growth media. Organ cultures from mammalian, reptile and avian hosts; successfully grown in GS supplemented growth media, were used for different virological studies. Growth media supplemented with GS were used for in vitro cultivation of malarial parasites. Thus since the last three decades many scientists are using GS in place of FBS, in various fields of biomedical research. The present article reviews an account of the same.

In-vitro study of mouse peritoneal macrophage response to virulent and avirulent strains of mycobacteria.

An immunological study of pathogenesis of tuberculosis was carried out in BALB-c mice in-vitro. Peritoneal macrophages obtained from BALB-c mice were challenged with virulent (H37Rv) and avirulent (H37Ra, BCG, M. phlei) strains of mycobacteria. Activated peritoneal macrophages showed enlargement, presence of intracellular bacteria and vacuolation. These significant changes in macrophage morphology were clearly evidenced in cells infected with virulent strains of Mycobacterium tuberculosis i.e. H37Rv while being absent in cells infected with avirulent H37Ra, BCG and M. phlei. Virulent mycobacteria (H37Rv) survive the phagocytic action of macrophages by residing inside the vacuoles. The capacity of virulent and avirulent strain to stimulate TNF-alpha production from peritoneal macrophage of BALB-c mice was also examined at different time interval i.e. 1,2,4,6 and 8th day by measuring cytolytic activity of culture supernatant against murine fibroblast cell line. The pattern of highest TNF release was in case of H37Rv and least with M. phlei as measured in culture supernatant after 1,2,4,6 and 8th day.

Soluble factor from tumor cells induces heme oxygenase-1 by a nitric oxide-independent mechanism in murine peritoneal macrophages

Tumor target-derived soluble secretary factor has been known to influence macrophage activation to induce nitric oxide (NO) production. Since heme oxigenase-1 (HO-1) is induced by a variety of conditions associated with oxidative stress, we questioned whether soluble factor from tumor cells induces HO-1 through NO-dependent mechanism in macrophages. We designated this factor as a tumor-derived macrophage-activating factor (TMAF), because of its ability to activate macrophages to induce iNOS. Although TMAF alone showed modest activity, TMAF in combination with IFN-gamma significantly induced iNOS expression and NO synthesis. Simultaneously, TMAF induced HO-1 and this induction was slightly augmented by IFN-gamma. Surprisingly, however, induction of HO-1 by TMAF was not inhibited by the treatment with the highly selective iNOS inhibitor, 1400 W, indicating that TMAF induces the HO-1 enzyme by a NO-independent mechanism. While rIFN-gamma alone induced iNOS, it had no effect on HO-1 induction by itself. Collectively, the current study reveals that soluble factor from tumor target cells induces HO-1 enzyme in macrophages. However, overall biological significance of this phenomenon remains to be determined.

Influence of 7beta-hydroxycholesterol on glutathione status and nitric oxide production in macrophages: in vitro studies.

Incubation of murine peritoneal macrophages with 7beta-hydroxycholesterol (7beta-OH) for 24 hr led to dose-dependent reduction in cellular glutathione content as well as nitrite levels in the medium. Treatment with an inorganic form of selenium, sodium selenite which is a potent antioxidant, elevated the cellular glutathione levels and decreased nitrite levels. Our results suggest that 7beta-OH may exert its pro-atherogenic effect by inhibiting glutathione synthesis and nitric oxide production by macrophages present in the arterial wall and thus, impair the cellular antioxidant defense system.

Stimulation of macrophage function by interphotoreceptor retinoid-binding protein: production of nitric oxide

In this study, we investigated whether retinal soluble proteins, such as interphotoreceptor retinoid-binding protein(IRBP), play a role in the induction of nitric oxide by macrophages in vitro. Cells from the murine macrophage cell line RAW 264.7 and rat and rabbit peritoneal macrophages were incubated in the presence of retinal soluble protein. The nitrite level in the cultured supernatant was evaluated for nitric oxide production using the Griess reaction. IRBP induced significant, dose-dependent nitrite production in both RAW 264.7 and rat peritoneal macrophages. Induction of inducible nitric oxide synthase (iNOS) by retinal proteins was inhibited by the iNOS-specific inhibitor, aminoguanidine, and the tyrosine inhibitor, genistein. These results show that soluble retinal proteins significantly induce nitric acid production by macrophages. Increased production of reactive oxygen species by macrophages in the presence of this soluble retinal protein in vivo may accelerate photoreceptor degeneration in uveitis.

Internalization of dengue virus-induced suppressor cytokine during transmission of the suppressor signal via macrophage.

In dengue type 2 virus (DV)-induced suppressor T cell cascade TS1 cells secrete a suppressor cytokine (SF) which acts via syngeneic macrophages (M phi) to recruit TS2 cells. SF binds to both high and low affinity receptors (SF-R) on M phi. In the present study the fate of SF in M phi during transmission of suppressor signal is investigated. It was observed that SF bound to high affinity receptors internalized through receptor mediated endocytosis. This was inhibited by pretreatment of M phi with anti-SF-R-antiserum and didansylcadaverine, a potent inhibitor of endocytosis. Internalized SF was degraded by lysosomal activity as shown by inhibition of suppressor activity by pretreatment of M phi with monensin and NH4Cl. Degraded SF was transported to a site other than SF-R on M phi membrane for recruitment of TS2 cells. This was inhibited by anti-SF-antiserum. Transmission of suppressor signal is inhibited if M phi are treated first with H-2K-mAb and then with SF (shown earlier) but when M phi were treated first with SF and after 1 hr with H-2K-monoclonal antibody, the inhibition did not occur. As SF requires binding to H-2K and SF-R for mediation of suppression, the binding of H-2K occurred with degraded SF within the cell. Thus SF is internalized, degraded and binds to H-2K antigen before its recognition by native T cells.

Identification and characterization of receptor for dengue virus-induced macrophage cytotoxin.

The present study was undertaken to identify the receptor for dengue virus type 2 (DV) induced macrophage cytotoxin (CF2) on mouse peritoneal macrophages (MPhi). The binding of 125I-labelled CF2 to MPhi was saturable (15 nM), reversible, temperature, pH- and time-dependent. The saturation concentration was similar to that causing cell death. Scatchard analysis showed the presence of intermediate type of affinity receptor and the number of receptor sites was 1.1 x 10(6) per cell with dissociation constant of 14.28 nM.

Modulation of Fc and C3b receptor activity of mouse peritoneal macrophages elicited by preformed immune complexes.

A study was undertaken to reveal the role of Fc and C3b receptor of mouse peritoneal macrophages (MPM) in the uptake of radiolabelled immune complexes. Large latticed preformed complexes consisting of human serum albumin (HSA)-anti HSA at equivalence (IC-Eq) and with antibody excess (IC-Ab) were observed to be avidly taken up by resident macrophages unlike small size complexes with antigen excess (IC-Ag). Macrophages elicited by thioglycollate (Tg) showed higher IC-binding capacity while IC-elicited MPM showed reduction in the same when compared to the resident cells. However, complement coated complexes were significantly taken up by these IC-elicited macrophages. Uptake studies were further extended to determine the expression of Fc and C3b receptor activity in MPM when elicited with preformed IC. Tg-elicited MPM were observed to bind greater number of IgG-coated erythrocytes (E-IgG) than resident MPM whereas IC-elicited MPM bound E-IgG poorly. When Fc receptors were blocked by in vitro IC treatment, poor binding of complement coated E-IgG [E(IgG)C] was recorded in resident MPM. The present complement medicated rosetting data tends to show enhanced expression of C3b receptors on IC-elicited macrophages.