Activated Mouse Peritoneal Macrophages Research Articles

Growth-stimulating phase of macrophage response to activation: the phenomenon and its implications for tumour growth and immunotherapy.

The dynamics of growth-stimulating and cytotoxic activity of mouse peritoneal macrophages (PMo) in response to in vivo and in vitro bacillus Calmette-Guérin (BCG) or bestatin treatment was studied. It was shown that BCG and bestatin induce cytotoxicity in PMo, and that after the cytotoxic response strong growth-stimulating activity develops. PMo, rendered cytotoxic in vivo and afterwards cultivated in vitro, displayed the same switch from a cytotoxic to a growth-stimulating phase. These results suggest that the growth-stimulating phase is the obligatory PMo response to biological response modifiers (BRM) at least to BCG and bestatin. The growth rate of tumours, transplanted into mice during the cytotoxic phase of the response to BCG, was suppressed, whereas tumours transplanted during the growth-stimulating phase were stimulated. It appears that the development of a growth-stimulating phase after the cytotoxic phase of response to activation by BRM could be one of the reasons for the limited effectiveness of immunotherapy based on the application of macrophage activators.

A cytochemical study of the interaction between Tritrichomonas foetus and mouse macrophages.

Light and electron microscopy were used to analyse the process of interaction of normal and antibody-coated Tritrichomonas foetus with resident and activated mouse peritoneal macrophages. Activated macrophages ingest more parasites than do resident macrophages. Previous incubation of the parasites in the presence of sub-agglutinating concentrations of a polyclonal anti-T. foetus antibody significantly increased their ingestion by the macrophages. Adherence of the parasites to the surface of activated macrophages triggers the respiratory oxidative burst as revealed by reduction of nitroblue tetrazolium. This process was more evident in antibody-coated parasites. Transmission electron microscopy showed the presence of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase in the portions of the macrophage plasma membrane that were in contact with the parasites as well as in the phagocytic vacuoles. Fusion of macrophage lysosomes with parasite-containing phagocytic vacuoles was observed in macrophages labeled with Lucifer yellow and gold-labeled peroxidase as well as by localisation of acid phosphatase.

Inhibition of lipoxygenase activity and HL60 leukemic cell proliferation by ursolic acid isolated from heather flowers ( Calluna vulgaris)

A compound was isolated and purified from heather flowers ( Calluna vulgaris) based on its ability to inhibit lipoxygenase activity. This molecule was characterized as ursolic acid by GC-MS. Ursolic acid was found to be an inhibitor of both potato tuber 5-lipoxygenase and soybean 15-lipoxygenase with IC 50 values of 0.3 mM. Ursolic acid also inhibits lipoxygenase activity in mouse peritoneal macrophages at 1 μM and HL60 leukemic cells growth ( IC 50 = 0.85 μM) as well as their DNA synthesis ( IC 50, = 1 μM). The possible role of lipoxygenase inhibition in the proliferation of leukemic cells is discussed.

Murine alpha 1,3-galactosyltransferase. A single gene locus specifies four isoforms of the enzyme by alternative splicing.

We have reported the characterization of a cDNA (clone 31A) encoding bovine alpha 1,3-galactosyltransferase (alpha 1,3-GT) (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). With the goal of isolating a full-length cDNA encoding murine alpha 1,3-GT we screened a cDNA library with clone 31A and isolated a 3.4-kilobase (kb) alpha 1,3-GT clone (4A). The murine coding sequence is 78% similar to that of the bovine alpha 1,3-GT cDNA, but the "stem" region (defined as the region that links the single transmembrane domain to the catalytic domain) of the murine alpha 1,3-GT encoded by clone 4A, is 31 amino acids shorter than the corresponding region of the bovine alpha 1,3-GT. To screen for heterogeneity in the murine alpha 1,3-GT transcripts, we carried out a polymerase chain reaction (PCR) analysis on mouse C127 cDNA. Four distinct transcripts were detected, which predict four isoforms of the alpha 1,3-GT polypeptide that differ only in the length of their stem region. To determine how the four different transcripts are generated from a single gene, we have established the genomic organization for murine alpha 1,3-GT. The full-length mRNA spans at least 35 kb of genomic DNA and is distributed over nine exons that range in size from 36 base pairs (bp) to approximately 2600 bp. The protein coding region is distributed over six exons, and the 5'-untranslated sequence is distributed over three exons. Comparison of the genomic DNA sequence with that of the four different mRNAs indicates that these transcripts are produced by alternative splicing of the murine pre-mRNA according to a cassette model. A tissue survey using RNA-PCR revealed the presence of four different alpha 1,3-GT transcripts in all mouse tissues and cell lines examined to date, with the notable exception of male germ cells. Additionally, although alpha 1,3-GT levels increased upon thioglycollate-induced activation of mouse peritoneal macrophages, the ratio of the alpha 1,3-GT isoforms was essentially unchanged. Similar results were obtained upon retinoic acid-induced differentiation of murine F9 teratocarcinoma cells. Lastly, a similar PCR analysis of bovine cDNA produced only a single DNA fragment, corresponding to bovine cDNA clone 31A.

Action of danazol on 5′-nucleotidase activity of mouse peritoneal macrophages

Action of danazol on 5′-nucleotidase activity of mouse peritoneal macrophages

Ｌ‐９２９細胞のコロニー形成試験を用いた水中微量汚染物質の毒性評価

Colony formation assay using L-929 cells was applied to 40 chemicals which were reported to occur in drinking water. In the 40 chemicals including 4 of specified chemical substances, 7 of pesticides, 19 of industrial chemicals and 10 of halogenated hydrocarbons, some of pesticides and halogenated hydrocarbons inhibited strongly the colony formation of L-929 cells, and the number and position of halogen in the chemicals influenced their cytotoxicities. The results were compared with those by the other cytotoxicity tests involving viability of HL-60 cells, phagocytic activity of mouse peritoneal macrophages, and LDH release or glycogenolytic activity of rat hepatocytes, and cytotoxicity matrix were obtained from the comparison of these four cytotoxicity tests. Availability of the cytotoxicity matrix is discussed to estimate the micropollutants in water.

Induction of cationic amino acid transport activity in mouse peritoneal macrophages by lipopolysaccharide

The transport of cationic amino acids has been investigated in mouse peritoneal macrophages cultured in vitro. The transport activity for lysine was rather low in cells cultured for 1 h and increased slightly in cells cultured for 12 h. This increase varied with the serum lot used in the culture medium and was suppressed by polymyxin B, suggesting that the transport activity is induced by endotoxins in the serum. When the macrophages were cultured in the medium containing 1 ng/ml lipopolysaccharide, the transport activity for lysine increased by more than 10-fold. The transport activity for lysine induced by lipopolysaccharide has been characterized. Lysine was transported mainly by a Na +-independent, saturable system. The uptake of lysine was potently inhibited by extracellular cationic amino acids, but not by neutral amino acids tested. In addition, transport of lysine showed trans-stimulation. From these results, we have concluded that the transport activity for cationic amino acids is potently induced by lipopolysaccharide and that the characteristics of the induced activity is consistent with those of system y +.

Stimulation of antimycobacterial activity in mouse peritoneal macrophages by priming with trehalose dimycolate (TDM)

We examined the potential of two bacterial immunomodulators, trehalose dimycolate (TDM) and lipopolysaccharide (LPS), to stimulate the capacity of mouse peritoneal macrophages to control the growth of the intracellular bacterium, Mycobacterium tuberculosis BCG. Macrophages were obtained from mice innately susceptible (Bcgs) or resistant (Bcgr) to BCG infection. In all mouse strains tested (Bcgr and Bcgs), with the exception of BALB/c (Bcgs), TDM was sufficient to elicit macrophages with strong antimycobacterial activity in vitro. In BALB/c mice, the induction of anti-BCG activity required two signals, TDM and LPS, given in sequence. Our data suggest that additional gene(s), besides the Bcg locus, control macrophage resistance to BCG.

Stimulation of antimycobacterial activity in mouse peritoneal macrophages by priming with trehalose dimycolate (TDM)

Stimulation of antimycobacterial activity in mouse peritoneal macrophages by priming with trehalose dimycolate (TDM)

Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages.

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.

Chemical synthesis and biological activities of 6,6′-di- O-mycoloyl-β,β- and -α,β-trehalose

6,6′-Di- O-mycoloyl-β,β-trehalose (β,β-TDM) and 6,6′-di- O-mycoloyl-α,β-trehalose (α,β-TDM) were synthesized and their toxicity and ability to activate peritoneal macrophages in situ were examined in mice, in comparison with 6,6′-di- O-mycoloyl-α,α-trehalose (TDM). Both β,β-TDM and α,β-TDM caused a decrease in body weight two days after injection, however the weights reverted to a normal level. No deaths were caused by either analog. On the other hand, TDM showed potent toxicity, causing decrease in body weight and death of all animals injected. β,β-TDM and α,β-TDM were effective in the in situ activation of mouse peritoneal macrophages.

Bioactivity of skeletal muscle proteolysis-inducing factors in the plasma proteins from cancer patients with weight loss

We determined the circulating level of bioactivity for skeletal muscle proteolysis-inducing factors (PIF) in the blood samples from cancer patients whose body weight loss was greater than 10%. The level of bioactivity was estimated by measurement of tyrosine release from isolated 1at diaphragm muscles incubated with an ultrafiltered fraction of plasma or serum proteins containing molecules from 0 to 25 kDa in molecular weight. Significant levels of bioactivity were detected in 25 of the 50 cancer samples. No activity was found in 18 of the samples from healthy human blood donors. The ability of 13 of the cancer samples to induce muscle proteolysis was significantly inhibited by incubation of muscles in presence of indomethacin (10 microM). The neutralisation of 12 of the cancer samples with the antibodies to recombinant human interleukin-1 (IL-1), alpha and beta forms, partially abrogated the activity in five samples. These results suggest that the accelerated breakdown of proteins induced by the cancer plasma factors is at least in part mediated by IL-1 in cooperation with other active factors not yet defined. Additionally, we have shown that the increased breakdown of proteins induced by PIF in the crude supernatant derived from activated mouse peritoneal macrophages is prevented by the treatment of muscles with either indomethacin or quin-2 (1 microM). These observations provide indirect evidence for a possible causal relationship between the production of PIF and the body-weight loss of cancer patients.

Activation of Peritoneal Macrophages by Orally Administered Ether Analogues of Lysophospholipids

Oral administration of dodecylglycerol, inflammatory product of cancerous tissues, and the alkyl lysophospholipid derivative, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3-choline), greatly activated mouse peritoneal macrophages. The activation was dose related and was assessed as increased Fc-mediated ingestion of red blood cells, superoxide production, chemiluminescence activity, and incorporation of radioactive thymidine and leucine. Furthermore, the data show that dodecylglycerol or ET-18-OCH3-choline was capable of inducing equally high levels of macrophage activation and cytotoxic action on tumor cells, just as occurs with intraperitoneal administration. Dodecylglycerol appeared to activate the macrophages at a relatively lower dose (5 micrograms/mouse) than ET-18-OCH3-choline (15 micrograms/mouse). The optimal oral doses required to activate macrophages for ingestion and cytotoxic activities were relatively higher than previously observed when these agents were administered intraperitoneally. Thus, the dose difference provided crucial information for correlating oral dosages with in vivo concentration of these agents as bioassayed by macrophage activation. These observations have extended and further support our earlier findings that these agents are effective immunopotentiators and thus could therapeutically be used to activate macrophages for cytotoxic effects on tumor cells via the oral route.

Effect of uptake of albumin muspheres on cellular activity of mouse peritoneal macrophages

Empty and drug-containing albumin muspheres (2–5 μm) produced by an emulsion cross-linking process were presented to mouse peritoneal macrophages. Empty muspheres caused a 36.7 and 43.3% release of β-galactosidase and β-glucuronidase, respectively. The inclusion of prednisolone (19μg/mg), prednisolone succinate (26μg/mg) and methotrexate (50μg/mg) caused a reduction in lysosomal release. Similarly the presence of incorporated anti-inflammatory drugs caused a depression in glycolytic activity, illustrated by monitoring levels of 14CO 2. Both effects were found to be dose dependent. This report demonstrates that the possible toxicity of the albumin matrix can be suppressed by inclusion of an anti-inflammatory drug into the musphere. Potentially, this system could be administered by the intra-articular route to deliver steroidal drugs to the synovial joint in order to treat rheumatoid arthritis.

Microbicidal Activities of <i>Salmonella typhimuriu</i><i>m</i>- and Interferon-Gamma-Activated Mouse Peritoneal Macrophages

Activation of mouse peritoneal macrophages during infection of mice by various facultative intracellular bacteria and after intravenous injection of recombinant interferon-gamma (rIFN-gamma) was studied. Macrophage activation was demonstrated on the basis of three different criteria, i.e. inhibition of Toxoplasma gondii proliferation, enhanced release of H2O2 and increased expression of Ia antigen. Macrophages activated during an infection with Salmonella typhimurium showed no enhanced salmonellacidal or listericidal activity relative to control macrophages, whereas Listeria-activated macrophages killed Listeria but not Salmonella faster than control macrophages. The rate of proliferation of Salmonella in spleen and liver of activated mice was comparable to the proliferation in the organs of control mice. rIFN-gamma-activated macrophages displayed neither an enhanced salmonellacidal nor an enhanced listericidal activity. When high numbers of Listeria were injected intravenously the proliferation in spleen and liver of rIFN-gamma-treated and control mice was similar. The proliferation of Listeria in the liver of rIFN-gamma-treated mice was less than in control mice when 1 LD50 or lower numbers of bacteria were injected. It is concluded that peritoneal macrophages become activated during infections of mice with various intracellular pathogens. However, these activated macrophages do not show enhanced bactericidal activity against all bacteria. Furthermore, rIFN-gamma is not sufficient to enhance the listericidal activity of macrophages.

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

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

Picolinic acid, a catabolite of tryptophan, as the second signal in the activation of IFN-gamma-primed macrophages.

Abstract We have studied the effects of picolinic acid, a product of tryptophan degradation, on the activation of mouse peritoneal macrophages (M phi). Picolinic acid acts synergistically with IFN-gamma in activating M phi from C57BL/6 mice. Moreover, M phi from C3H/HeJ mice and C3H/HeN that do not become cytotoxic in response to IFN-gamma alone could be fully activated by exposure to picolinate plus IFN-gamma. These results indicate that picolinic acid is a potent costimulator of M phi activation that functions as a second signal. Inasmuch as we have previously demonstrated that the activation of cytotoxic M phi correlates with specific changes in ribosomal RNA (rRNA), we investigated whether picolinic acid could modify M phi RNA metabolism. Picolinic acid inhibited the synthesis of total M phi RNA, the accumulation of newly synthesized 28S rRNA, and augmented the steady state levels of rRNA precursors (pre-rRNA). These changes in RNA metabolism were similar to those previously described in murine M phi activated in vitro or in vivo to express tumoricidal activity. These results demonstrate that picolinic acid is a potent, biologic M phi second signal, suggest that the changes in rRNA are causally connected with the expression of tumoricidal activity, and suggest the existance of an autocrine effect mediated by picolinic acid.

Stimulation of tumor necrosis factor secretion by purified influenza virus neuraminidase

We showed that purified neuraminidase (NA) of influenza virus, but not hemagglutinin (HA), possessed the potential to increase in vitro and in vivo the interleukin 1 (IL 1) activity of mouse peritoneal macrophages. In this study, we report the effect of NA and HA on the secretion of tumor necrosis factor (TNF) activity by murine peritoneal macrophages, TNF being a cytokine sharing many related and overlapping biological functions with IL 1. The two glycoproteins of the strain A/USSR/90/77 (H1N1) were purified electrophoretically and were tested in vitro at doses ranging from 0.5 to 5.0 μg using the adherent peritoneal macrophages of C3H/HeN mice elicited with thioglycolate. The TNF activity of culture supernatants, collected 24 hr after stimulation with viral protein, was evaluated by the standard cytolytic assay using L929 and WEHI.164 cells. No increase of the TNF activity was observed at 0.5 μg of NA (4.8 Units (U)/ ml in the L929 assay and 20.4 U/ml in the WEHI assay) but further increase of NA to 1.0 μg had a significant effect on the TNF activity (39.7 and 88.8 U/ml, respectively). Higher concentrations of NA (2.0 and 5.0 μg) did not improve the TNF activity. The addition of a rabbit anti-TNF-α serum to the assay system reduced the lysis of L929 cells by 85%, suggesting that the observed activity was due to TNF. In parallel, the enhancement of IL 1 activity due to NA was reverified using D10.G4.1 cells instead of the C3H/HeJ thymocytes assay used previously. NA augmented the IL 1 activity up to 1.0 μg (25.8 U/ml). The addition of monoclonal anti-IL 1 antibodies (100 neutralizing units) to the supernatants reduced the incorporation of [ 3H]-thymidine by 90 to 95%, suggesting that the observed activity was due to IL 1. Comparative results of NA and HA showed that only NA stimulated the TNF and IL 1 activities of murine macrophages.

Activation of mouse peritoneal macrophages during infection with Salmonella typhimurium does not result in enhanced intracellular killing.

Our study was performed to investigate whether macrophages become activated during an infection with Salmonella typhimurium and, if so, whether these activated macrophages kill S. typhimurium faster than resident macrophages. Mice received i.v. injections with a sublethal number of S. typhimurium; on about day 12 of the infection the numbers of bacteria in the liver and the spleen were maximal. During the infection, activation of peritoneal macrophages could be demonstrated on the basis of three criteria, i.e., the ability to inhibit the proliferation of Toxoplasma gondii, an enhanced production of H2O2 and an increased expression of Ia Ag. The rate of in vitro intracellular killing of S. typhimurium by these activated macrophages was not increased compared to that for resident macrophages. To determine the growth of S. typhimurium in activated mice a nalidixic acid-resistant mutant strain, called S. typhimurium 510R, was used. The net growth rates of the mutant S. typhimurium 510R in the spleen of S. typhimurium 510-activated and normal mice were similar. However, in the liver of S. typhimurium 510-activated mice the number of S. typhimurium 510R did not change during 3 to 48 h after injection. The role of specific antibodies during the initial phase of the infection was negligible, because only low levels of antibodies were detected during the first 15 days of infection and the growth rates of S. typhimurium 510 in the spleen and liver of mice with high titers of antibodies were not significantly different from the rates in normal mice. The results of this study demonstrate that although macrophages become activated during an infection with S. typhimurium, these cells do not display an enhanced bactericidal activity in vitro and in vivo no significant effect on the growth rate of S. typhimurium in the spleen and a bacteriostatic effect in the liver is found. Hence macrophage activation is probably not very important in the host defense against S. typhimurium.

Purification of macrophage deactivating factor.

Macrophage deactivation factor (MDF) in P815 tumor cell-conditioned medium was assayed by its suppression of the ability of activated mouse peritoneal macrophages to release hydrogen peroxide. MDF displayed properties of a soluble protein(s) associated with both low (8-25,000) and high (greater than 450,000) Mr fractions. MDF was purified 6,140-fold by a seven-step procedure: extraction with acid-ethanol; precipitation with ether; and fractionation on gel filtration, anion-exchange, diphenyl reversed-phase and C4 reversed-phase HPLC columns, the last column twice. The final preparation contained two species: (a) a approximately 13,000 Mr band on reducing or nonreducing SDS-PAGE and on autoradiograms after radioiodination with chloramine T, and (b) a 66,000 Mr species ranging from approximately 5% to approximately 50% of the protein detectable by silver strain. The 66,000 Mr species was identified as albumin from its NH2-terminal amino acid sequence. However, no amino acid sequence could be obtained for the approximately 13,000 Mr species, either in fluid phase or after electroelution of the corresponding SDS-PAGE band. Thus, approximately 13,000 Mr MDF associates tightly with albumin through a variety of separation techniques, and may have a blocked NH2 terminus. Purified MDF afforded 50% inhibition of activated macrophage H2O2 releasing capacity at a concentration of 1-10 nM. Separation of MDF from most higher Mr moieties was associated with disproportionately small increases in specific activity, suggesting MDF might be partially inactivated by purification. As purified, MDF was approximately 1,000-fold less potent at deactivating macrophages than TGF-beta. Antibodies that neutralized the macrophage-deactivating effect of TGF-beta did not inhibit deactivation by MDF.