Influence of whole peptidoglycan of bifidobacterium on cytotoxic effectors produced by mouse peritoneal macrophages

INTRODUCTIONBifidobacteria are physiologically beneficial bacteria which are perdominant in human intestine ,and possess the most important functions .They play an important role in maintaining microbial balance of the intestine .Furthermore , their presence is thought to be an important indication of health of the body [1-4].Whole peptidoglycan ( WPG) is the major component in the cell wall of bifidobacterium ,which is also a biological responsemodifier with nontoxic side dffcets.

THE EFFECTS OF RADIX SALVIAE MILTIORRHIZAE ON LIPID ACCUMULATION OF PEROXIDIZED LOW DENSITY LIPOPROTEIN IN MOUSE PERITONEAL MACROPHAGES ?LIPID ANALYSIS AND MORPHOLOGICAL STUDIES

Mouse peritoneal macrophages were incubated in DMEM with pox-LDL and Rradlx Salviae Miltiorrhizae (RSM) to investigate the effects of RSM on the internalization of peroxidized low density lipoprotein (pox-LDL) by using lipid analysis and electron microscopy. Lipid peroxide (LPO) concentrations were increased slightly in the medium after incubation of macrophages with normal LDL (n-LDL), while decreased significantly in the media after incubation of macrophages with pox-LDL. In the three groups with pox-LDL, it could be found that there was a dose-dependent decrease of concentrations of LPO and total cholesterol (TCH) in the two RSM groups, and the decrease in the two RSM groups was much greater than in the group without RSM. RSM accelerated a more decrease of LPO than cholesterol contents in the media containing pox-LDL. The ultrastructural studies also showed that RSM induced the accumulation of lipid droplets in the cytoplasm of mouse peritoneal macrophages. The results suggested that RSM could accelerate the phagocytosis and degradation of pox-LDL by macrophages.

Abdominal paracentesis drainage ameliorates severe acute pancreatitis in rats by regulating the polarization of peritoneal macrophages

AIM To investigate the role of peritoneal macrophage(PM) polarization in the therapeutic effect of abdominal paracentesis drainage(APD) on severe acute pancreatitis(SAP).METHODS SAP was induced by 5% Na-taurocholate retrograde injection in Sprague-Dawley rats. APD was performed by inserting a drainage tube with a vacuum ball into the lower right abdomen of the rats immediately after the induction of SAP. To verify the effect of APD on macrophages, PMs were isolated and cultured in an environment, with the peritoneal inflammatory environment simulated by the addition of peritoneal lavage in complete RPMI 1640 medium. Hematoxylin and eosin staining was performed. The levels of pancreatitis biomarkers amylase and lipase as well as the levels of inflammatory mediators in the blood and peritoneal lavage were determined. The polarization phenotypes of the PMs were identified by detecting the marker expression of M1/M2 macrophages via flow cytometry, qPCR and immunohistochemical staining. The protein expression in macrophages that had infiltrated the pancreas was determined by Western blot.RESULTS APD treatment significantly reduced the histopathological scores and levels of amylase, lipase, tumor necrosis factor-α and interleukin(IL)-1β, indicating that APD ameliorates the severity of SAP. Importantly, we found that APD treatment polarized PMs towards the M2 phenotype, as evidenced by the reduced number of M1 macrophages and the reduced levels of proinflammatory mediators, such as IL-1β and L-selectin, as well as the increased number of M2 macrophages and increased levels of anti-inflammatory mediators, such as IL-4 and IL-10. Furthermore, in an in vitro study wherein peritoneal lavage from the APD group was added to the cultured PMs to simulate the peritoneal inflammatory environment, PMs also exhibited a dominant M2 phenotype, resulting in a significantly lower level of inflammation. Finally, APD treatment increased the proportion of M2 macrophages and upregulated the expression of the anti-inflammatory protein Arg-1 in the pancreas of SAP model rats.CONCLUSION These findings suggest that APD treatment exerts antiinflammatory effects by regulating the M2 polarization of PMs, providing novel insights into the mechanism underlying its therapeutic effect.

Hsp70 confines tumor progression of rat histiocytoma and impedes the cytotoxicity induced by natural killer cells and peritoneal macrophages

Objective:To study the role of inducible form of heat shock protein 70(Hsp70) in the host tumor regression of rat tumor model.Methods:We examined the role of Hsp70 in host tumorigenicity and in vitro cellular cytotoxicity using a rat histocytoma.The differential tumor growth and regression kinetics were studied and correlated with the expression of Hsp70,activation of macrophages and natural killer(NK) cells,and circulating or tumor infiltrating immune molecules in the host system.Results:The sub cuteaneous(s.c.) tumor regression was correlated with increased serum cytokines such as IL-12,TNF P,IFNγand Hsp70.Despite of similar increase of Hsp70 in intraperitoneal(i.p.) tumor implanted animals,animals succumb to tumor growth,further,evidently,no immune molecule activation was observed.The viral promoter driven Hsp70 over expression in these tumor cells restrained solid tumor growth,however,failed to inhibit ascites growth.The NK cells from s.c.immunized animals induces cytotoxicity in the presence of anti-tumor antibody,which necessitated CD40-L expression,conversely,NK cells from i.p.immunized animals failed to induce cytotoxicity.The NK cells from s.c.or i.p.implanted animals with Hsp70 positive tumor cells failed to induce such cytotoxicity.The peritoneal macrophages isolated from s.c.tumor implanted animals when co-cultured with parental BC-8 cells lyses tumor cells,nevertheless entail macrophage specific TNFαexpression.On the contrary,Hsp70 expressing BC-8 tumor cells were resistant to peritoneal macrophage induced cytolysis.Conclusions:This study brings out that Hsp70 possibly involved in regulating the host tumor response and cellular cytotoxicity.

Aqueous extract of Ocimum gratissimum Linn and ascorbic acid ameliorate nicotine-induced cellular damage in murine peritoneal macrophage

Objective:To test the in vitro protective role of aqueous extract of Ocimum gratissimum Linn. (0.gratissimum) and ascorbic acid against nicotine-induced murine peritoneal macrophage. Methods:Peritoneal macrophages from mice were treated with nicotine(10 mM),nicotine (10 mM) with aqueous extract of O.gratissimum(1 to 25μg/mL),and nicotine(10 mM) with ascorbic acid(0.01 mM) for 12 h in cell culture media,while the control group was treated with culture media.Levels of free radical generation,lipid peroxidation,protein carbonyls,oxidized glutathione levels and DNA damage were observed and compared.Results:Phytochemical analysis of aqueous extract has shown high amount of phenolics and flavonoids compound present in it.The significantly increased free radical generation,lipid peroxidation,protein carbonyls,oxidized glutathione levels and DNA damage were observed in nicotine-treated group as compared to the control group:those were significantly reduced in aqueous extract of O. gratissimum and ascorbic acid supplemented groups.Moreover,significantly reduced antioxidant status in nicotine exposed murine peritoneal macrophage was effectively ameliorated by these two products.Among the different concentration of aqueous extract of O.gratissimum,the maximum protective effect was observed at 10μg/mL which does not produce any significant change in the normal cell.Conclusions:These findings suggest the potential use and beneficial role of O.gratissimum as a modulator of nicotine-induced cellular damage in murine peritoneal macrophage.

Recent insights into the characteristics and role of peritoneal macrophages from ascites of cirrhotic patients

Macrophages are a diverse myeloid cell population involved in innate and adaptive immune responses,embryonic development,wound repair,and regulation of tissue homeostasis.These cells link the innate and adaptive immunities and are crucial in the development and sustainment of various inflammatory diseases.Macrophages are tissue-resident cells in steady-state conditions;however,they are also recruited from blood monocytes after local pathogen invasion or tissue injury.Peritoneal macrophages vary based on their cell complexity,phenotype,and functional capabilities.These cells regulate inflammation and control bacterial infections in the ascites of decompensated cirrhotic patients.Our recent work reported several phenotypic and functional characteristics of these cells under both healthy and pathological conditions.A direct association between cell size,CD14/CD16 expression,intracellular level of GATA-6,and expression of CD206 and HLA-DR activation/maturation markers,indicate that the large peritoneal macrophage CD14^(high)CD16^(high)subset constitutes the mature phenotype of human resident peritoneal macrophages during homeostasis.Moreover,elevated expression of CD14/CD16 is related to the phagocytic capacity.The novel large CD14^(high)CD16^(high)peritoneal subpopulation is increased in the ascites of cirrhotic patients and is highly sensitive to lipopolysaccharide(LPS)-induced activation,thereby exhibiting features of inflammatory priming.Thus,phosphorylation of ERK1/2,PKB/Akt,and c-Jun is remarkably increased in response to LPS in vitro,whereas that of p38 MAPK is reduced compared with the monocyte-derived macrophages from the blood of healthy controls.Furthermore,in vitro activated monocyte-derived macrophages from ascites of cirrhotic patients secreted significantly higher levels of IL-6,IL-10,and TNF-αand lower amounts of IL-1βand IL-12 than the corresponding cells from healthy donor’s blood.Based on these results,other authors have recently reported that the surface expression level of CD206 can be used to identify mature,resident,inflammatory peritoneal macrophages in patients with cirrhosis.Soluble CD206 is released from activated large peritoneal macrophages,and increased concentrations in patients with cirrhosis and spontaneous bacterial peritonitis(SBP)indicate reduced odds of survival for 90 d.Hence,the level of soluble CD206 in ascites might be used to identify patients with SBP at risk of death.In conclusion,peritoneal macrophages present in ascites of cirrhotic patients display multiple phenotypic modifications characterized by reduced ratio of cells expressing several membrane markers,together with an increase in the ratios of complex and intermediate subpopulations and a decrease in the classiclike subset.These modifications may lead to the identification of novel pharmaceutical targets for prevention and treatment of hepatic damage.

Effect of triptolide on secretion of inflammatory cellular factors TNF-α and IL-8 in peritoneal macrophages of mice activated by lipopolysaccharide

Research has been carried out to look for safe and effective anti-inflammation drugs from traditional Chinese herbal medicine. As a powerful research technology of life science, molecular biology has entered many areas of traditional Chinese medicine.This study aimed to investigate the effect of triptolide on tumor necrosis factor-α （TNF-α） and interleukin-8 （IL-8） of peritoneal macrophages activated by lipopolysaccharide （LPS） in mice. Peritoneal elicited macrophages were separated, purified and activated by LPS in mice, then cultured in vitro with triptolide at different concentrations. The activity of TNF-a and the level of IL-8 of cellular supernatants were determined by MTT colorimetric assay and ELISA, respectively. The activity of TNF-a in macrophages was significantly inhibited （P〈0.01） by triptolide （10^-1-10^1μg/ml） during 4-24 hours in a time- and dose-dependent manner. The level of IL-8 in macrophages was significantly inhibited （P〈0.01） by triptolide （10^-1-10^1g/ml） in 12 hours in a dose- dependent manner. Triptolide could inhibit the activity of TNF-a and the level of IL-8 in macrophages activated by LPS.

Tumoricidal activation of murine resident peritoneal macrophages on pancreatic carcinoma by interleukin-2 and monoclonal antibodies

INTRODUCTIONMacrophages play an important role in tumor lysisand growth inhibition.They can be activated to atumoricidal state by a variety of agents such asIFNr,TNFα or IL2.The killing machanisms ofactivated macrophages have been extensivelyinvestigated.Recently,it has been proved thatantibody dependent cellular cytotoxicity (ADCC) isone of the potent arms to lyse tumor cells

The relationship between endocytosis of peritoneal macrophages induced by concanavalin A and intracellular free calcium in mouse

In this experiment the morphological changes of mouse peritoneal macrophages in the course of their conjugation with colloidal gold-labelled concanavalin A(ConA-Au) i by the surface receptor and then the endocytosis and transport of the ConA were observed

Effects of hyperbaric air exposure on the functions of mouse peritoneal macrophages

Objective: To study the effects of hyperbaric air exposure on the functions of peritoneal macrophages of mice. Methods: Forty-eight mice were equally randomized to 6 groups: (1) normal air group (NA); (2) hyperbaric air group 1 (HA1); (3) hyperbaric air group 2 (HA2); (4) hyperbaric air group 3 (HA3); (5) hyperbaric oxygen group (HO);(6) hyperbaric nitrogen group (HN). Every group was exposed to corresponding pressure for 60 min, twice a day for 3 d. Peritoneal macrophages were obtained at the corresponding time to observe the changes of phagocytosis, acid phos-phatase, antigen presentation function and the produce of NO and TNF-α. Results: Compared with those in NA group, the activity of phagocytosis, acid phosphatase, antigen presentation function and the produce of NO and TNF-a were markedly inhibited in hyperbaric oxygen group and hyperbaric air group 1 ( P < 0.05, P < 0.01) and they changed little in HN group. These changes could disappear in 3 - 5 d. Conclusion: The functions of mice peritoneal macrophages were obviously inhibited in simulated air diving environment and hyperoxia may play an important role in it.

Studies on the Electron Microscopic Localization of Con A and WGA Receptors in Human Peritoneal Macrophages

The electron microscopic topology of Con A and WGA receptors was investi-gated in human peritoneal macrophagcs by utilizing avidin-gold colloids.Our observa-tions confirmed that both the receptors were distributed on the cell membranes,yet thereceptor expression showed variations among the individual cells.Some cells had abun-dant receptors,while others had only few receptors.The data obtained in the presentstudy indicate that human peritoneal macrophages may be a functionally andbiochemically heterogenous population,and the study may also serve as a theorcticalreference for future clinical exploitation of human peritoneal macrophages.

THE EFFECT OF DIALYSIS ON THE RELEASE OF TNF-α FROM PERITONEAL MACROPHAGE IN VIVO

Objective To investigate the consistent inhibitory effect olperitoneal dialysate on the capacity ofperitoneal macrophage release of TNF- α and the inhibition associated with different glucose concentration invivo. Methods We compared the peritoneal macrophage activity obtained from the irrigation solution of theuremia patients (n=2) immediately after inserting the catheter and with those from the long - dwell time dialysate(≥10h) of peritoneal dialysis patients (n= 7). The capacity of peritoneal macrophage to release TNF - x was assayedby L929 cytotoxicity. At the same time, we compared TNF- 7 activity among different glucose concentration (1.5%,2.5% and 4.25% dextrose) of dialysate. Results The levels of TNF- a in CAPD and control group were 170 and517 pg/106 respectively (P<0.01). The patients who were using dialysate containing higher glucose concentrationhad lower TNF- x level (P<0.05, P<0.01). Conclusion It is suggested that the inhibitory effect of peritonealdialysate on peritoneal macrophages might continue at least 10h in vivo. The higher glucose concentration ofdialysate may have worse effect on peritoneal macrophage activity.

Effects of quercetin on hepatocyte stimulating factor production by mouse peritoneal macrophages

Objective: To study the effects of quercetin on hepatocyte stimulating factor production from mouse peritoneal macrophages. Methods: Hepatocyte stimulating factor was evaluated by the amount of fibrinogen synthesized in Hep3B cells. Interleukin-6 activity was measured by B9 cell proliferation methyl thiazolyl tetrazolium colorimetric method. Hep3B cell supernatant fibrinogen was quantitated with ELISA. Results: LPS induced the synthesis of hepatocyte stimulating factor in mouse peritoneal macrophages, and hepatocyte stimulating factor promotes the synthesis of fibrinogen from Hep3B cells. Quercetin(5 to 40μmol/ L) inhibited the synthesis of hepatocyte stimulating factor stimulated by LPS. Quercetin(5 to 20μmol/ L) inhibited release of interleukin-6 from mouse peritoneal macrophages induced by 0. 5 g/ L fibrin fibrinogen degradation products. Conclusion: Quercetin inhibits the synthesis of hepatocyte stimulating factor in macrophages.

Cytoprotective and anti-inflammatory effects of kernel extract from Adenanthera pavonina on lipopolysaccharide-stimulated rat peritoneal macrophages

Objective:To investigate mechanism of anti-inflammatory activity of Adenanthera pavonina(A.pavonina) extracts.Methods:Rat peritoneal macrophages were treated with different concentrations of lipopolysaccharide and H_2O_2 in the presence and absence of kernel extract from A.pavonina.Nitric oxide,superoxide anion generation,cell viability and nuclear fragmentation were investigated.Results:The pre-treatment of kernel extract from A.pavonina suppressed nitric oxide,superoxide anion,cell death,nuclear fragmentation in lipopolysaccharide and H_2O_2stimulated or induced macrophages,respectively.Conclusions:These results suggest that A.pavonina extract suppresses the intra cellular peroxide production.

Effects of Several Doses of ^(60)Co Gamma Irradiation on Mouse Peritoneal Macrophages

Effects of several doses of60Co gamma irradiation on mouse macrophages were in-vestigated by means of quantitative cytochemistry.It was found that the activities of lysosomalenzymes（acid phosphatase（AcPase）and a-naphthyl acetate esterose（ANAE））,adenosinetriphosphatase（ATPase）and positive substances of periodic acid Schiff’s reaction（PAS）inperitoneal macrophages were increased after total-body irradiation （1,3Gy）,and decreasedwith the increase of doses of total-body irradiation（5,8Gy）.Meanwhile,the phagocytic ca-pacity and the size of peritoneal macrophages of irradiated mice also changed as compared withthe control group.

ACUPUNCTURE EFFECTS ON INDUCIBLE NITRIC OXIDE SYNTHASE mRNA, iNOS PRODUCT AND HEAT SHOCK PROTEIN IN PERITONEAL MACROPHAGES OF THE MOUSE

To study the acupuncture effects on the inducible nitric oxide synthase (iNOS) mRNA,iNOS product and heat shock protein(hsp) 70 in the mouse macrophages, after peritoneal stimulation with steriled paraffin oil for 48 h, 24 Kunming mice were randomly divided into 3 groups: a, electroacupuncture (EA) groupl treated with EA; b, control 1 (C 1) group, peritoneal macrophages treated with culture; and c, control 2 (C 2) group, treated with neither EA nor culture. The macrophages of mice in 3 groups collected from respective peritoneal cavities were prepared into two kinds of specimens, including slide and nitrocellulose membrane (NCM). The iNOS mRNA, iNOS and hsp 70 were detected respectively with in situ hybridization, cytochemistry, immunohistochemistry and RNA and protein dot blots. The results showed that the signals of iNOS mRNA and iNOS product were localized in the macrophage cytoplasm; the immunoreactivity(IR) of hsp 70 localized in both cytoplasm and nucleus. The dot blot signal scanning value of those in 3 groups in comparison with each other showed as follows: EA group>C 2 group>C 1 group, (P<0.01).

Effect of polydatin on TNFα and NAG release from endotoxin-stimulated peritoneal macrophages in mice

Macrophages (M) are the major target cells in endotoxin (bacterial lipopolysaccharide, LPS) stimulation. Endotoxin triggers the inflammatory cells to release mediators, including tumor necrosis factor (TNF). This is one possible reason for endotoxin shock and organ damage. Polydatin (PD) is a component of the rhizoma of Polygonum cuspidatum,a Chinese herb medicine. our previous studies showed that it attenuated TNF and NAG elevations in experimental intestinal ischemia-reperfusion injury and endotoxin shock. The present study is designed in vitro to demonstrate whether the attenuation was due to the effect of PD. Mice peritoneal M were isolated and incubated in 5% CO2in air at 37℃. Different doses of polydatin were added respectively into the wells before and after LPS stimulation. Concentrations of TNF and N-acetyl-β-D-glucosaminidase (NAG, a lysosomal enzyme) in the supernatants were determined. The results showed that PD decreased the levels of TNF and NAG in the supernatant of LPS- stimulated M with statistically significant differences. In 0. 50, 0. 82 and 1. 95 mmol/L PD -pretreated groups, LPS caused lower TNF production, 1. 26±0. 24, 0. 71 ±0. 34, and 0. 78± 0. 32 U/ml,respectively, vs 2. 17± 0. 24 U/ml of the untreated control. The NAG released was also much less, 3. 52± 0. 13, 2. 03± 0.30 and 2. 28± 0. 40 U/L,respectively,vs 4. 58±0. 18 U/L of untreated control. In the supernatants of M stimulated by LPS for 1 h, followed by addition of PD for 1 h, TNF produced was also less in PD- pretreated groups: 1.51±0. 47, 1. 43±0. 43 vs 2. 17± 0. 23 U/ml; while NAG levels decreased to 2. 89±0. 23, 2. 68±0. 16., and 2. 83± 0. 13 U/L vs 4. 58±0. 18 of control (P < 0. 001). It is concluded that PD inhibits TNF and NAG release from endotoxin-stimulated peritoneal macrophages in mice.

Relationship between peritoneal macrophages and inflammatory reaction in a rat model of severe acute pancreatitis

Objective To investigate the relationship between peritoneal macrophages(PMAs)and inflammatory reaction in a rat model of severe acute pancreatitis(SAP).Methods Sprague-Dawley rats were randomly divided into control group and SAP group.To induce SAP in rats,40 g/L sodium taurocholate(0.1 mL/100 g)was injected into the pancreatic duct through retrograde exposure of pancreatic bile duct in hepatic porta.One-third of rats were sacrificed at 3,6 or 12 h after modeling.PMAs were extracted,and incubated for 24 h in a humidified 5% carbon dioxide incubator.The expressions of tumor necrosis factor alpha(TNF-α)and interleukin-1β(IL-1β)mRNA in PMAs were measured by semi-quantitative RT-PCR.The levels of TNF-α and IL-1β in culture medium and serum were evaluated.The histological changes of pancreas were examined.Results The expressions of TNF-α mRNA and IL-1β mRNA in PMAs were significantly higher in SAP group than in control group at each time point(P<0.01).The concentrations of TNF-α and IL-1β in culture medium and serum were significantly elevated in SAP group compared with control group(P<0.01).The histological analysis of pancreas indicated that the damage was more severe in SAP group than in control group(P<0.01).Conclusion PMAs secrete cytokines into pancreatitis-associated ascitic fluid,and this study demonstrates a correlation between SAP and the activation of PMAs.

Regulation of ApoE Gene Expression in Mouse Peritoneal Macrophages by VLDL

Mouse peritoneal macrophages (MPM) were incubated with ApoEpoor VLDL or ApoE-rich VLDL at same concentrations for 24 h. The ApoE mR NA content increased in both groups than that in control and the highest ApoEmRNA content was seen in MPM incubated with ApoE-poor VLDL. The results suggest that VLDL could stimuIate ApoE gene expression in MPM and the ApoE poor VLDL has more pronounced effect. We think that the ApoE secreted byMPM may be incorporated into VLDL, especially the ApoE-poor VLDL, and thereby enhance the uptake of those lipoproteins by MPM or other local cells via ApoE-mediated receptor pathways.

Influence of whole peptidoglycan of Bifidobacterium bifidum on cytokines production by peritoneal macrophages of nude mice

Objective: To explore the functions of whole peptidoglycan (WPG ) of bifidobacterium in regulating immune reactions. Methods: IL- 1 activity produced by peritoneal macrophages was investigated by murine thymocyte proliferating method; The content of IL-6 and IL-12 was detected by ELISA. Results: IL--1 activity and the contents of IL--6 and IL--12 secreted by peritoneal macrophages of nude mice in the WPG injection group were significantly higher than those in the control group (P < 0. 01 ). Conclusion: WPG of bifidobacteria can activate peritoneal macrophages to secret relatively large amount of IL- 1. IL- 6 and IL- 12.