Recently it is known that nitric oxide(NO) generated by an activatedmacrophage plays an important role in tumoricidal or bactericidal activity. Thisstudy was done to know the effects of NO produced by the activated macrophages onthe growth of the murine bladder tumor cell line (MBT-2). For the activation ofmacrophages, RAW 264.7 cell line ( macrophage-derived cell line) and peritonealmacrophages from Balb/c mouse were treated with interferon-r (INF-r),lipopolysaccharide ( LPS) or INF-r plus LPS and for the evaluation of growthinhibition of MBT -2 cell line, tritiated thymidine (3[H] -thymidine)incorporation was measured after coculturing of MBT-2 cell line with macrophageswhich had been activated to produce NO. The results are as follows : l.Peritoneal macrophages from Balb/c mouse could be activated to produce NO onlywhen they are stimulated by the combination of INF-r and LPS (35+/-1.0uM/L). Theycould produce a slight increase amount of NO when they had been stimulated byINF-r (11+/-2.5uM/ L) or LPS (14+/-3.5uM/L) compared to control macrophages(8+/-2.5uM/L). The induction of NO production by INF-r plus LPS could be abrogatedby the use of NG-monomethyl-L- arginine (NGMMA), a competitive inhibitor of NOsynthase (5+/-1.5 M/L). 2. RAW 264.7 cell lines could be activated to produce NOby the treatment of INF-r, LPS, and INF-r plus LPS (40+/-2.5uM/L, 37+/-3.0uM/Land 51+/-2.6uM/L respectively). When they are treated with NGMMA, they produced nomore NO even though they had been stimulated with INF-r plus LPS (14+/-4.0uM/L),and they could produce small .amount of NO(19+/-1.0uM/L) in the absence of thestimulation. 3. The incorporation of 3[H] -thymidine of the MBT-2 and peritonealmacrophages was reduced when the cells had been treated with INF-r plus LPScompared to the control (14,519+/-1,087cpm, 20,716+/-1,474cpm respectively).There was no reduction in the incorporation of 3[H] -thymidine when the cellshad been treated with INF-r or LPS alone. The incorporation of 3[H]-thymidineincreased when the cells had been treated with INF-r plus LPS in the presence ofNGMMA(19,622+/-1,341cpm). 4. The incorporation of 3[H] -thymidine of the MBT-2 and RAW 264.7 cell lines were reduced when the cells had been treated with INF-r, LPS and INF-r plus LPS (19.068+/-144cpm, 15,070+/-122cpm and 7,543+/-85cpm respectively) compared to control( 20,708+/-142cpm). When the cells had been pretreated with NGMMA, the incorporation of 3[H]- thymidine recovered to same degree (12,605+/-108cpm) compared to the cells stimulated with INF-r plus LPS. The above correlation of the NO production from macrophages which had been stimulated by INF-r and/or LPS and the inhibition of growth of the tumor cells suggests that NO produced by stimulated macrophages might be the responsible molecule in the defense system of the body against tumors.
Animals ; Arginine ; Cell Line* ; Macrophages* ; Macrophages, Peritoneal ; Metabolism* ; Mice ; Nitric Oxide ; Nitrogen* ; Thymidine ; Urinary Bladder Neoplasms

In order to investigate the mechanism of elevated vascular endothelial growth factor (VEGF) in peritoneal fluids from patients with endometriosis, macrophages were recovered from peritoneal fluids obtained at the time of diagnostic laparoscopy from infertile women with endometriosis (EMT group, n=20) and without endometriosis (control group, n=20). Macrophages were cultured in vitro. The VEGF levels of peritoneal fluid and the supernatant of macrophages culture were determined by enzyme linked immunoassay (ELISA). Meanwhile, the eutopic (n=20) and ectopic endometrium (n=20) from endometriosis patients, and normal edometrium (n=20) from non-endometriosis patients were obtained for the analysis of VEGF expression by labeled Streptavidin Biotin (LSAB). It was found that VEGF levels in peritoneal fluid and macrophages culture supernatant were significantly higher in EMT group than in control group (P<0.01). In normal endometrium, VEGF showed a cyclic changes and similar in eutopic and ectopic endometrium from patients with endometriosis. There was no difference in the intensity of VEGF in endometrium between two groups within each menstrual phase. It is suggested that altered VEGF production by peritoneal macrophages and ectopic endometrium secretion may contribute to the elevated VEGF levels in the peritoneal fluid of patients with endometriosis.
Ascitic Fluid/*metabolism ; Cells, Cultured ; Endometriosis/*metabolism ; Macrophages, Peritoneal/pathology ; Vascular Endothelial Growth Factor A/*biosynthesis

The effect of treatment of mercury chloride on the nitrite and nitrate synthesis was observed in peritoneal macrophages from Balb/c mice and EMT-6 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with cytokines. Amounts of nitrite and nitrate in the culture media after 24 and 36 hours of culture were about 2-fold, and 3-fold of those measured after 12 hours respectively. There were very close associations between the amounts of nitrite and nitrate measured in the culture media, according to culture time. The survival rate of peritoneal macrophages was significantly decreased by mercury chloride added into the media in dose-dependent manner, however the survivals of EMT-6 cells were not influenced by mercury chloride concentration in media. Nitrite and nitrate syntheses were dose-dependently decreased by mercury chloride added in culture media. These results reported here suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seems to be caused by the inhibition of metabolism of cells.
Animals ; Culture Media ; Cytokines ; Immunity, Cellular ; Macrophages, Peritoneal* ; Metabolism ; Mice* ; Nitric Oxide ; Survival Rate

The effect of treatment of mercury chloride on the nitrite and nitrate synthesis was observed in peritoneal macrophages from Balb/c mice and EMT-6 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with cytokines. Amounts of nitrite and nitrate in the culture media after 24 and 36 hours of culture were about 2-fold, and 3-fold of those measured after 12 hours respectively. There were very close associations between the amounts of nitrite and nitrate measured in the culture media, according to culture time. The survival rate of peritoneal macrophages was significantly decreased by mercury chloride added into the media in dose-dependent manner, however the survivals of EMT-6 cells were not influenced by mercury chloride concentration in media. Nitrite and nitrate syntheses were dose-dependently decreased by mercury chloride added in culture media. These results reported here suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seems to be caused by the inhibition of metabolism of cells.
Animals ; Culture Media ; Cytokines ; Immunity, Cellular ; Macrophages, Peritoneal* ; Metabolism ; Mice* ; Nitric Oxide ; Survival Rate

In order to investigate the mechanism of elevated vascular endothelial growth factor (VEGF) in peritoneal fluids from patients with endometriosis, macrophages were recovered from peritoneal fluids obtained at the time of diagnostic laparoscopy from infertile women with endometriosis (EMT group, n=20) and without endometriosis (control group, n=20). Macrophages were cultured in vitro. The VEGF levels of peritoneal fluid and the supernatant of macrophages culture were determined by enzyme linked immunoassay (ELISA). Meanwhile, the eutopic (n=20) and ectopic endometrium (n=20) from endometriosis patients, and normal edometrium (n=20) from non-endometriosis patients were obtained for the analysis of VEGF expression by labeled Streptavidin Biotin (LSAB). It was found that VEGF levels in peritoneal fluid and macrophages culture supernatant were significantly higher in EMT group than in control group (P<0.01). In normal endometrium, VEGF showed a cyclic changes and similar in eutopic and ectopic endometrium from patients with endometriosis. There was no difference in the intensity of VEGF in endometrium between two groups within each menstrual phase. It is suggested that altered VEGF production by peritoneal macrophages and ectopic endometrium secretion may contribute to the elevated VEGF levels in the peritoneal fluid of patients with endometriosis.
Adult ; Ascitic Fluid ; metabolism ; Cells, Cultured ; Endometriosis ; metabolism ; Female ; Humans ; Macrophages, Peritoneal ; pathology ; Vascular Endothelial Growth Factor A ; biosynthesis

OBJECTIVETo study the effect of Mycobacterium smegmatis vaccine on the level of nitric oxide (NO) produced by peritoneal macrophages in immunized mice.

METHODSBalb/c mice were randomized into low-dose, middle-dose, and high-dose groups (injected with different doses of Mycobacterium smegmatis vaccine) and a control group (injected with normal saline). Then the peritoneal macrophages were cultured with lipopolysaccharide in vitro. The supernatants were collected and the concentrations of NO were analyzed through the reaction with Griess reagents.

RESULTSThe levels of NO produced by the peritoneal macrophages in the control group, low-dose group, middle-dose group, and high-dose group were (3.50 +/- 3.11), (16.63 +/- 6.47), (13.97 +/- 6.20), and (7.55 +/- 2.26) ng/ml, respectively. The levels of NO in all dosing groups were significantly different from that in control group (P < 0.01).

CONCLUSIONMycobacterium smegmatis vaccine can promote the peritoneal macrophages to produce NO in mice.

Animals ; Bacterial Vaccines ; therapeutic use ; Lipopolysaccharides ; Macrophages, Peritoneal ; metabolism ; Mice ; Mice, Inbred BALB C ; Mycobacterium smegmatis ; Nitric Oxide ; metabolism

The role of very low density lipoprotein receptor (LVLDR) in the process of foam cell formation was investigated. After the primary cultured mouse peritoneal macrophages were incubated with VLDL, beta-VLDL or low density lipoprotein (LDL), respectively for 24 h and 48 h, foam cells formation was identified by oil red O staining and cellular contents of triglyceride (TG) and total cholesterol (TC) were determined. The mRNA levels of LDLR, LDLR related protein (ILRP) and VLDLR were detected by semi-quantitative RT-PCR. The results demonstrated that VLDL, beta-VLDL and LDL could increase the contents of TG and TC in macrophages. Cells treated with VLDL or beta-VLDL showed markedly increased expression of VLDLR and decreased expression of LDLR, whereas LRP was up-regulated slightly. For identifying the effect of VLDL receptor on cellular lipid accumulation, ldl-A7-VR cells, which expresses VLDLR and trace amount of LRP without functional LDLR, was used to incubate with lipoproteins for further examination. The results elucidated that the uptake of triglyceride-rich lipoprotein mediated by VLDLR plays an important role in accumulation of lipid and the formation of foam cells.
Animals ; Arteriosclerosis ; metabolism ; pathology ; Cells, Cultured ; Cholesterol, LDL ; metabolism ; pharmacology ; Female ; Foam Cells ; cytology ; metabolism ; Lipoproteins, VLDL ; pharmacology ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Receptors, LDL ; metabolism ; Triglycerides ; metabolism

This study was aimed at exploring the expression pattern of P2X family receptors (P2XR) in peritoneal macrophages and their relationship with the activation states of macrophages in Notch1-induced mouse T-ALL model. After establishment of the leukemia model, F4/80(+) peritoneal macrophages, F4/80(+)CD206(+) M2-like and F4/80(+)CD206(-) M1-like peritoneal macrophages were sorted by flow cytometry based on F4/80 and CD206 surface markers. The expression of P2XR in each cell population was detected by real time RT-PCR. The results showed that macrophages,M1-like and M2-like macrophages moderately expressed P2XR except for P2X5R. The expression of P2XR varied with the development of leukemia. The expression of P2X1R and P2X7R in peritoneal macrophages increased steadily; the expression of P2X2R and P2X3R decreased at late stage of leukemia;the expression of P2X4R slightly decreased at intermediate stage;the expression of P2X6R kept unchanged. At intermediate stage of leukemia, the expression of P2XR in M1-like and M2-like peritoneal macrophages varied. M1-like macrophages expressed higher level of P2X1R than M2-like macrophages, whereas M2-like macrophages expressed higher level of P2X7R than M1-like macrophages, which suggested that the expression of P2XR were related to the activation states. It is concluded that the expression of P2XR in peritoneal macrophages from leukemia mice is related to the progression of leukemia and the activation states of macrophages, which lay a foundation for further studying the role of macrophages in the development of leukemia.
Animals ; Cytokines ; metabolism ; Disease Models, Animal ; Macrophages, Peritoneal ; metabolism ; Mice ; Mice, Inbred C57BL ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Receptor, Notch1 ; metabolism ; Receptors, Purinergic P2X ; metabolism ; Signal Transduction

BACKGROUNDThe immunomodulatory effects of glucocorticoids (GCs) have been described as bimodal. High concentration of GCs exerts immunosuppressive effects and low levels of GCs are immunopermissive. While the immunosuppressive mechanisms of GCs have been investigated intensely, the immunopermissive effects of GCs remain unclear. A lot of studies showed GCs could exert rapid non-genomic actions. We herein studied the rapid immunopromoting effects of GCs.

METHODSWe observed the rapid (within 30 minutes) effects of corticosterone on respiratory burst of mouse peritoneal macrophages and studied their mechanisms. The superoxide anions were measured by cytochrome C reduction assay. Protein kinase C phosphorylation was measured by Western blotting and membrane fluidity was evaluated by fluorescence polarization measurement.

RESULTSThe 10(-8) mol/L and 10(-7) mol/L corticosterone rapidly increased the superoxide anions production by macrophages, which were insensitive to GC-receptor antagonist, mifepristone, and protein-synthesis inhibitor, cycloheximide. Corticosterone coupled to bovine serum albumin was able to mimic the effects of corticosterone. The effects were independent of protein kinase C pathway and the change in membrane fluidity.

CONCLUSIONSThe results indicate that corticosterone rapidly promote the superoxide anions production by mouse peritoneal macrophages may through non-genomic mechanisms. This study may contribute to understanding the effects of GCs under stress condition and the physiological significance of nongenomic effects of GCs.

Animals ; Corticosterone ; pharmacology ; Macrophages, Peritoneal ; drug effects ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Respiratory Burst ; drug effects ; Superoxides ; metabolism

OBJECTIVETo investigate the effect of high mobility group box-1 protein (HMGB1) on apoptosis of macrophages in mice.

METHODSThe peritoneal macrophages were obtained from female BALB/c mice. After exposure to different doses of HMGB1 (10, 100, 1000, and 10000 ng/ml) for 24 hours, or exposure to 10 000 ng/ml for different time (6, 12, 24, and 48 hours), cells were stained with Annexin V-PE and 7-amino-actinomycin-D (7-AAD), then determined by flow cytometry and laser scanning confocal microscopy, respectively.

RESULTSExposed to HMGB1 for 24 hours resulted in a dose-dependent induction of apoptosis, with increased levels of apoptosis observed at 100, 1 000, and 10 000 ng/ml compared with controls [(12.91 +/- 3.89)%, (18.76 +/- 3.41)%, and (39.84 +/- 5.98)%, respectively, vs. (4.49 +/- 1.72)%]. Exposed to HMGB1 at 10000 ng/ml for 6, 12, 24, or 48 hours showed a time-dependent increase in apoptosis rate [(18.33 +/- 4.60)%, (25.02 +/- 5.27)%, (38.30 +/- 6.95)%, and (21.81 +/- 7.96)%]. The percentage of apoptotic cells markedly increased with a peak value at 24 hours after HMGB1 stimulation (P<0.01).

CONCLUSIONHMGB1 has a proapoptotic effect on murine peritoneal macrophages.

Animals ; Apoptosis ; Female ; HMGB1 Protein ; physiology ; In Vitro Techniques ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Mice, Inbred BALB C