Potentiation of tumoricidal properties of murine macrophages by Nocardia rubra cell wall skeleton (N-CWS)

Intraperitoneal injection of squalene-treated cell wall skeleton of Nocardia rubra (N-CWS) caused increase in number of peritoneal exudate cells (PEC). Adherent macrophages obtained from N-CWS-treated PEC suppressed growth of methylcholanthrene-induced fibrosarcoma (Meth-A), when injected intradermally with the tumor cells into BALB/c mice. The macrophages showed strong cytotoxicity against Meth-A cells in vitro. When treated with 10 micrograms/ml of N-CWS in vitro, proteose peptone-induced macrophages acquired tumoricidal property but resident macrophages showed no cytotoxicity after the treatment. In the supernatant of spleen cells cultured for 72 hours in the presence of N-CWS (10 micrograms/ml), the presence of (a) factor(s) with macrophage activating effect was observed. This factor, shown to be identical to macrophage activating factor (MAF) in molecular weight, showed synergy with N-CWS in potentiating macrophage cytotoxicity against tumor cells.     

Alteration of macrophage anti-tumor activity and transferrin receptor expression by exposure to dimethylnitrosamine in vivo

Dimethylnitrosamine (DMN), a potent immunomodulatory agent, produces its effects on cell-mediated immune reactions through alterations in macrophage production and/or macrophage maturation/differentiation from bone marrow stem cells. Macrophages obtained by the in vitro culture of bone marrow from animals exposed to DMN in vivo (bone marrow-derived macrophages, BMDM) demonstrated enhanced cytotoxicity against L929 target cells. The enhanced cytotoxicity was also present in concanavalin A- and Corynebacterium parvum-elicited peritoneal exudate cells (PEC) obtained from DMN-exposed animals while thioglycollate-elicited PEC from DMN-exposed animals displayed no increase in their cytotoxic activity as compared to vehicle-exposed animals. However, treatment of thioglycollate-elicited PEC with interferon-gamma induced cytotoxic activity in PEC obtained from DMN-exposed animals but not in PEC obtained from control animals. BMDM obtained from DMN-exposed mice also demonstrated an alteration in the kinetics of the expression of the membrane-associated transferrin-binding receptor (mTFR), a marker of the activational status of macrophages. BMDM from animals exposed to DMN in vivo exhibited maximal expression of mTFR on day 7 of culture in vitro as compared to day 5 for BMDM from vehicle-exposed animals. C. parvum- and concanavalin A-elicited PEC from DMN-exposed animals showed dose-related decreases in their expression of mTFR which were associated with their expected enhanced cytotoxicity. Likewise, thioglycollate-elicited PEC from DMN-exposed mice had dose-related decreases in mTFR expression and total transferrin-binding activity, suggesting a change in their state of activation. No alterations in mTFR expression were observed in splenic macrophages. BMDM cultured with T cell-derived lymphokines known to affect mTFR expression demonstrated enhanced expression of mTFR independent of changes in the cell cycle profiles. Furthermore, while lymphokines enhanced mTFR expression, there was no alteration in the kinetics of mTFR expression by BMDM obtained from DMN- or vehicle-exposed animals. These results support the hypothesis that DMN-induced alterations in macrophage hematopoietic differentiation/maturation are manifested in changes in macrophage function.     

OF4949, new inhibitors of aminopeptidase B. V. Effect on the murine immune system

OF4949-I inhibited the growth of the solid form of IMC carcinoma and protected against pulmonary metastases of Lewis lung carcinoma. It also augmented the cytostatic activity of mouse peritoneal macrophages, the natural killer activity, and the antibody-dependent cell-mediated cytotoxicity of mouse spleen cells. This substance was not cytotoxic to cultured tumor or normal cells even at high concentrations. These results suggest that a cell-mediated immune response stimulated by this compound might account for its antitumor activity.     

Modulation of cell-mediated resistance to listeriosis in mice given T-2 toxin

The modulating effect of preinoculation and postinoculation treatment with a single oral 4.0 mg/kg dosage of T-2 toxin on cell-mediated resistance was studied in mice inoculated with Listeria monocytogenes. Toxin treatment caused significant decreases in thymus and spleen weights, bone marrow cellularity, and in the total number of circulating leukocytes, lymphocytes, and neutrophils. Enhancement or suppression of resistance to listeriosis was dependent on the time of toxin administration relative to the time of Listeria challenge. Preinoculation treatment on Day 2 or 4 prior to Listeria challenge significantly enhanced resistance and decreased mortality due to listeriosis by as much as 50%. In contrast, resistance was suppressed and mortality was increased by 50% in mice that were treated with toxin after Listeria challenge. Enhanced resistance to listeriosis was accompanied by a significant increase in the influx of macrophages into Listeria-elicited peritoneal exudates. In addition, in vivo phagocytosis of sheep red blood cells by peritoneal macrophages was significantly increased in toxin-treated mice that were sensitized with sheep erythrocytes. The data indicate that T-2 toxin has a modulating effect on cell-mediated resistance and that enhancement of resistance to listeriosis in mice pretreated with T-2 toxin is associated with increased migration/activation of macrophage effector cells.     

Macrophage involvement in the antitumor activity of a synthetic acyltripeptide (FK-565) against experimental lung carcinoma metastases

Resident peritoneal macrophages can be activated to develop cytotoxicity against P815 mastocytoma target cells following incubation in vitro with either D-lactoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(L)-gl ycine (FK-156), heptanoyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alani ne (FK-565), or bacterial lipopolysaccharide (LPS) at a minimum concentration of 10 micrograms/ml. Subthreshold levels of hybridoma-derived macrophage activating factor (MAF) markedly potentiated this activity. In an experimental metastasis model, subcutaneous or intraperitoneal treatment with FK-565 (1 to 10 mg/kg) markedly inhibited lung metastasis formation when administered 2-4 days prior to i.v. tumor inoculation. Moreover, this protective activity could be abrogated by the selective macrophage inhibitor, 2-chloroadenosine, suggesting that activated macrophage were responsible for the antimetastatic activity of FK-565.     

Murine macrophage cytotoxicity induced by mastocytoma cells, cell-free mastocytoma exudates, and extracts

Peritoneal macrophages obtained from normal CBA mice expressed significant cytotoxicity against the DBA/2-derived P815 mastocytoma but not against DBA/2-derived SL2 tumor or the C57BL-derived tumors TLX9 and EL4. The macrophages also expressed some cytotoxicity against the DBA/2-derived L5178Y tumor. Incubation of normal CBA macrophages with cell-free exudate of intraperitoneally growing P815 cells resulted in cytotoxicity against the SL2 and against the EL4 lymphosarcoma. Incubation of SL2 tumor cells with P815 ascitic fluid before adding the SL2 tumor cells to normal CBA macrophage monolayers also resulted in inhibition of SL2 tumor cell growth on these monolayers. Trypsinization of the macrophages after incubation with ascitic fluid (or tumor extract) but before challenge with tumor cells abolished cytotoxic activity of the macrophages. Incubation of normal macrophages with a comparable amount of trypsin before tumor cells were added had no influence on their activity. Cytotoxicity could be induced after 7 days storage of the exudate at 5 degrees C, but this ability was lost within 72 hr when kept at room temperature. Storage at -20 degrees C had no influence. Gel fractionation of the cell-free exudate showed that the product responsible for the effect is a small molecular weight product (mol wt less than 1000). Furthermore the "product" was dialyzable. The "factor" could not be shown in the supernatant of P815 cell cultures unless the cultures comprised greater than or equal to 40% dead cells.     

槲寄生凝集素腹腔内给药对腹腔巨噬细胞功能的影响

目的：探讨小鼠腹腔内注射槲寄生凝集素对腹腔巨噬细胞功能的影响。方法：16只小鼠分为腹腔内给药组和空白组,分别取腹腔内巨噬细胞并计数,采用ELISA法测定TNF-α表达情况,Griess法测定NO表达,MTT法检测腹腔内巨噬细胞对肠癌HCT116细胞的杀伤作用。结果：腹腔内给槲寄生凝集素后可明显增加腹腔内巨噬细胞数,并增加TNF—α和NO的表达水平;激活的巨噬细胞对HCT116细胞的杀伤作用增强,其杀伤作用与TNF—α和NO表达时间一致。结论：腹腔内注射槲寄生凝集素可激活腹腔内巨噬细胞,激活的巨噬细胞对HCT116细胞有杀伤作用。     

Effect of in vivo administration of T-2 toxin on peritoneal murine macrophages

We investigated the effect of in vivo administration of T-2 toxin, a 12,13-epoxytrichothecene produced by several Fusarium species, on murine macrophage metabolism. Cytoplasmic and lysosomal enzyme levels, generation and release of superoxide anion, phagocytosis and intracellular killing of Salmonella typhi and murine P815 tumour cell lysis were measured under different experimental conditions. When T-2 toxin was administered to mice at sublethal doses (0.50-1.00 mg/kg/24 hr), the levels of lysosomal and cytoplasmic enzyme activity and the generation of superoxide anion were significantly enhanced as compared to controls. This correlated with increased phagocytosis and intracellular killing of S. typhi. Cytotoxic activity against murine P815 mastocytoma cells exhibited by macrophages isolated from mice treated with T-2 toxin was inhibited in a dose-dependent manner. In vivo administration of T-2 toxin may result in the activation of specific metabolic pathways of peritoneal macrophages, while inhibiting other paths.     

Effect of low-level lead exposure on antibody-dependent and natural killer cell-mediated cytotoxicity

Splenic antibody-dependent, cell-mediated cytotoxicity against chicken red blood cell targets [ADCC (CRBC)] and natural killer cell-mediated cytotoxicity (NKMC) were determined in C57BL/6 mice given 1300 ppm lead acetate (827 ppm lead) in their drinking water for 8 weeks. ADCC in lead exposed mice was significantly lower than controls at effector to target cell ratios of 100:1, 50:1, and 25:1. In contrast, no significant difference in unstimulated NKMC was seen in lead exposed mice and control mice at effector to target cell ratios of 200:1, 100:1, and 50:1. Moreover, in vivo poly I:C-enhanced NKMC in lead-treated mice was similar to controls at an effector to target cell ratio of 200:1. Blood lead levels averaged 40.5 +/- 1.2 micrograms% in lead-exposed mice and 1.9 +/- 0.3 micrograms% in controls. These findings suggest that chronic low-level lead exposure in mice results in significant suppression of ADCC (CRBC) but does not alter natural killer activity.     

Macrophage activation by polysaccharide biological response modifier isolated from Aloe vera L. var. chinensis (Haw.) Berg

A mannose-rich polysaccharide biological response modifier (BRM), derived from Aloe vera L. var. chinensis (Haw.) Berg., was demonstrated to be a potent murine B- and T-cell stimulator in our previous study. We here report the stimulatory activity of PAC-I on murine peritoneal macrophage. The polysaccharide when injected into mice enhanced the migration of macrophages to the peritoneal cavity. Peritoneal macrophage when treated by PAC-I in vitro had increased expression of MHC-II and FcgammaR, and enhanced endocytosis, phagocytosis, nitric oxide production, TNF-alpha secretion and tumor cell cytotoxicity. The administration of PAC-I into allogeneic ICR mice stimulated systemic TNF-alpha production in a dose-dependent manner and prolonged the survival of tumor-bearing mice. PAC-I is thus a potent stimulator of murine macrophage and the in vitro observed tumoricidal properties of activated macrophage might account for the in vivo antitumor properties of PAC-I. Our research findings may have therapeutic implications in tumor immunotherapy.     

Comparison of the in vitro cytolytic effect of hepatic, splenic and peritoneal macrophages from glucan-treated mice on sarcoma M5076

Our previous results have demonstrated that glucan will significantly modify the course of syngeneic murine tumors. Additionally, results denote that glucan increases macrophage-mediated lysis of syngeneic tumor cells. The present study was undertaken to more closely examine the effect of parenteral glucan administration on the tumoricidal activity of hepatic, splenic and peritoneal macrophage populations. C56B1/6J mice were injected intravenously with glucan (0.45 mg/mouse) on days 1,3,6,9,12 and 15. Hepatic, splenic and peritoneal macrophages were isolated on days 8, 12 and 16. The macrophages were co-incubated for 72 h with syngeneic reticulum cell sarcoma M5076 at a target: effector ratio of 1:50. A significant increase in the cytolytic activity of all three macrophage populations was observed. Kupffer cell tumoricidal activity was increased (p less than 0.01) on day 8, but was not significantly different from control on days 12 and 16. Peritoneal exudate macrophages showed increased (p less than .001) tumoricidal activity on days 12 and 16, respectively. Splenic macrophages showed an enhanced (p less than 0.01) increase in lytic activity only on day 16. The present results indicate that: 1) glucan will enhance macrophage-mediated tumoricidal activity of three distinct macrophage populations and 2) a temporal relationship exists between glucan administration and expression of lytic activity by the macrophage populations. These observations further define the mechanism by which glucan exerts its potent anti-tumor activity.     

Dissociative effects of a novel immunomodulating agent (CP-20, 961) on host defenses of mice

The antimicrobial and antitumor effects of CP-20,961, a synthetic lipoid amine with immunomodulating properties, were investigated. Mice given CP-20,961 ip seven or three days before challenge with ip Listeria monocytogenes had a lower mortality than control mice. By contrast, CP-20,961 did not protect against lethal challenges of either Salmonella typhimurium or Toxoplasma gondii. In parallel with the in vivo studies, peritoneal macrophages from CP-20,961-injected mice inhibited multiplication of L. monocytogenes but not T. gondii. Further studies demonstrated that CP-20,961 protected mice against an ip challenge of P815 tumor cells as measured by survival time. This correlated with the ability of stimulated peritoneal macrophages to inhibit (3H-TdR uptake inhibition) and kill (Cr51 release) P815 cells in vitro. These data indicate that CP-20,961 affords protection against an ascitic mastocytoma tumor line and at least one, but not all, intracellular pathogens. The dissociation of the immunomodulating effect, which was reflected in peritoneal macrophage function, may be characteristic of this new class of immunomodulators.     

A small molecular weight peptide from P815 mastocytoma cells induces macrophage cytotoxicity

In a previous article we have shown the presence of a "factor" in the cell free exudates of intraperitoneally growing P815 mastocytoma cells as well as in cell-free tumor cell extracts. This "factor" is able to induce nonspecific macrophage cytotoxicity against tumor target cells. The "factor" was sensitive to heating at 100 degree C for 1 min, destroyed by pronase treatment, and dialyzable. On the other hand, the "factor" was not removed by centrifugation (25,000 x g), and was resistant to chloroform extraction, ultraviolet irradiation, heating at 56 degree C (for 20 min), or RNAase treatment. Sephadex gel fractionation of the mastocytoma exudate showed that the "factor" has an approximate molecular weight of 650-700 daltons. Normal peritoneal macrophages incubated with an extract of murine mast cells, purified histamine or serotonin did not show detectable cytotoxicity against tumor cells. The results of our experiments suggest that the effect of the P815 cell-free exudate and tumor cell extract is due to a dialyzable small molecular weight peptide.     

Tumor cytotoxicity of peritoneal macrophages induced by OK-432

In the present study we investigated the enhancement of cytotoxicity of peritoneal macrophages induced by OK-432. Rats received an i.p. injection of OK-432 at doses of 0.1, 0.5 or 1.0 KE/rat. Two days later, rats were sacrificed and peritoneal macrophages were isolated. Then the number of macrophages was counted, and the macrophages were analyzed for their lactic dehydrogenase (LDH) activity, acid phosphatase (ACP) activity, phagocytic activity, secretion of nitric oxide (NO) and cytotoxicity. The number of peritoneal macrophages, the activity of LDH and ACP, phagocytic activity, NO secretion, and cytotoxicity were increased with the increasing doses of OK-432. The results suggested that OK-432 enhanced tumor cytotoxicity of peritoneal macrophages by three steps. The first step is to attract a great number of macrophages into the peritoneal cavity. The second step is to enhance the phagocytic and eliminating function of these macrophages. The last step is to increase the non-contact cytotoxicity of macrophages.     

Liposomal Induction of a Heat-stable Macrophage Priming Factor to Induce Nitric Oxide in Response to LPS

Purpose. The effects of liposomes on nitric oxide (NO) production from mouse peritoneal macrophages following intraperitoneal injection of liposomes were investigated. Methods. Mouse peritoneal macrophages were collected following intraperitoneal injection of liposomes and cultured with and without lipopolysaccharide (LPS). Peritoneal washing fluid was also collected from the mice injected with liposomes. NO production was evaluated by measuring the concentration of nitrite in the macrophage culture supernatant by Griess reagent. Results. NO production stimulated by LPS was observed in peritoneal macrophages obtained from the liposome-treated mice, but liposomes did not activate macrophages directly to induce NO in response to LPS. NO production was higher in the liposomes composed of phospha-tidylcholine than that of negatively charged liposomes composed of phosphatidylserine. Peritoneal washing fluid obtained from mice injected with liposomes has a capacity to induce NO production in the macrophages from naive mice. This capacity was not diminished by heat-treatment at 100°C for 5 min. Conclusions. Peritoneal macrophages were activated to produce NO in response to LPS following intraperitoneal injection of liposomes. They did not activate macrophages directly, and the induction of heat-stable macrophage priming factor, but not cytokines, is suggested.     

Mechanism of antiviral action of quercetin against cardiovirus infection in mice

Oral treatment with quercetin protected ABD2F1/Jena mice significantly against intraperitoneal encephalomyocarditis, Col, SK, MM, Mengo M,L and MengoM virus infections, but not against intracerebral challenge with MengoM virus. Enhanced resistance to MengoM virus were induced in the genetically different DBA 2/Jena, C57BL/Jena, C57BL/Lati and ABC2F1/Jena mice, C57BL6/Jena nu/nu mice were also protected, indicating that the thymus was non-essential to the protective effects of quercetin. In AB/Jena and Lati:CFLP mice the drug failed to be effective. Quercetin was not virucidal and did not interfere with Mengo virus replication in L cells. Interferon was not detected (less than 1 : 8) in sera of ABD2F1/Jena mice 1-48 h after oral administration of the drug. The virus spread from the site of injection to the lymph nodes and other target organs were impaired. Silica treatment, to suppress macrophage function, did not evidently increase the susceptibility of ABC2F1/Jena mice to Mengo virus. However, this treatment abolished the antiviral activity of quercetin, indicating the requirement for macrophages for quercetin to be effective. Virus replication could not be demonstrated in cultures of adherent peritoneal macrophages from either untreated or quercetin-treated ABD2F1/Jena mice.     

Involvement of resident macrophages and mast cells in the writhing nociceptive response induced by zymosan and acetic acid in mice

Intraperitoneal administration of zymosan and acetic acid induced a dose-dependent nociceptive writhing response in mice. Lavage of the peritoneal cavities with saline reduced the number of total resident peritoneal cells and caused a proportional decrease in the nociceptive responses induced by these stimuli. Furthermore, the specific reduction of the peritoneal mast cell population by intraperitoneal administration of compound 48/80 also reduced the nociceptive responses induced by zymosan and acetic acid. In contrast, enhancement of the peritoneal macrophage population by pretreatment of the cavities with thioglycollate caused an increase in the number of writhes induced by both stimuli. These data suggest that the nociceptive responses induced by zymosan and acetic acid are dependent upon the peritoneal resident macrophages and mast cells. These cells modulate the nociceptive response induced by zymosan and acetic acid via release of tumour necrosis factor alpha (TNF-alpha), interleukin 1beta and interleukin 8. This suggestion is supported by the following observations: (a) pretreatment of the peritoneal cavities with antisera against these cytokines reduced the nociceptive responses induced by these stimuli; (b) peritoneal cells harvested from cavities injected with zymosan or acetic acid released both interleukin 1beta and TNF-alpha; (c) although individual injection of TNF-alpha, interleukin 1beta or interleukin 8 did not induce the nociceptive effect, intraperitoneal injection of a mixture of these three recombinant cytokines caused a significant nociceptive writhing response. In conclusion, our results suggest that the nociceptive activity of zymosan and acetic acid in the writhing model is due to the release of TNF-alpha, interleukin 1beta and interleukin 8 by resident peritoneal macrophages and mast cells.     

Immunopharmacological actions of neurotropin (4). Effect of neurotropin on mouse peritoneal macrophages

It was reported that neurotropin (NSP), an extract isolated from the inflamed skins of rabbits inoculated with vaccinia virus, activates murine T cell functions participating in cell-mediated immunity. The present study was undertaken to examine the effect of NSP on plastic dish-adherent macrophages (M phi) from ddY mice in vitro. Total activities of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase in resident peritoneal M phi was slightly enhanced when the M phi were cultured with NSP (10-1000 micrograms/ml) for 48 and 96 hr, but no enhancement was noted in 24 hr culture. Intracellular activity of lactate dehydrogenase (LDH) was also strongly enhanced in a dose-dependent manner by culturing with NSP for 48 and 96 hr. The enhanced LDH activity in the M phi cultured with NSP for 96 hr was completely inhibited by cycloheximide, an inhibitor of protein synthesis. In addition, consumption of glucose in the culture media by the M phi was also enhanced by culturing with NSP for 96 hr. Intracellular activity of LDH and glucose consumption of plastic dish-nonadherent cells from normal mouse peritoneal cells, however, was not enhanced by NSP in 96 hr culture. In regard to allogeneic M phi-mediated cytostatic activity to P815-X2 mastocytoma, NSP had no effect on cytostatic activities of the resident and thioglycollate-induced M phi, although NSP by itself dose-dependently inhibited the growth of P815-X2 mastocytoma without affecting cell viability. These results suggest that NSP biochemically activates mouse peritoneal M phi in vitro, but the M phi activated by NSP can not inhibit the growth of P815-X2 mastocytoma.     

Effect of alpha-mercaptopropionylglycine (alpha-MPG) and sodium dipropylacetate (DPA) on antibody formation (III) (author's transl)]

Mechanisms of the immunostimulative activity. In the present paper, an investigation was carried out to clarify the mechanisms regarding immunostimulative activity of alpha-MPG and DPA. Phagocytosis of peritoneal macrophage was enhanced by high concentration of alpha-MPG, but was unaffected with DPA in vitro. Mice were immunized with an i.v. injection of sheep erythrocytes and 3 days later spleen cells were isolated and incubated with alpha-MPG and DPA FOR 24 HR. Although neither drug affected the number of recovered and living cells, hemolytic plaque forming cells were increased with alpha-MPG and there was a tendency to increase with DPA. Phytohemagglutinin-P and/or lipopolysacchride E. coli-induced blast formation was not reinforced by either drug in spleen cells of mice, and no mitogenic activity was found in the spleen cells. In the early stage of immuno-response to polyvinylpyrrolidone as a T cell independent antigen, the titre of antibody showed no increase after either drug. alpha MPG and DPA apparently act as potentiators in antigen modifications of macrophages.     

商陆多糖Ⅰ联合白细胞介素2对小鼠脾细胞杀瘤活性的影响

商陆多糖Ⅰ(PAP-I),0.3～3μg·ml^-1和小鼠脾细胞培养3～5d可显著增强其杀伤P815肿瘤细胞活性及IL-2(250～500IU·ml^-1)诱导的LAK细胞活性,最适浓度为1μg·ml^-1。PAP-I及IL-2和脾细胞培养的上清液对P815肿瘤细胞无细胞毒作用,但能增强脾细胞及LAK细胞杀瘤活性。PAP-I,5,10及50mg·kg-1,ip可增强脾细胞杀伤P₈₁₅和L₉₂₉细胞的活性及IL-2诱导的LAK细胞活性。