Differential expression of metastasis-associated cell surface glycoproteins and mRNA in a murine large cell lymphoma

A metastatic variant cell subline of the Abelson virus-transformed murine large lymphoma/lymphosarcoma RAW117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117-H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117-P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus-associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117-P and -H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one-half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.     

Computerized analysis of tumor cell interactions with extracellular matrix proteins, peptides, and endothelial cells under laminar flow

Arrest and formation of stable adhesive interactions between circulating cells and the endothelium or exposed subendothelial matrix are important processes in many biological situations. We have developed a highly sensitive hydrodynamic assay that utilizes a parallel-plate flow chamber, video microscopy, and digital image processing to separate and measure the primary arrest and adhesion stabilization of flowing cells. Our data indicate that primary cell contact triggers secondary adhesion stabilization, and the secondary events are likely to be critical to metastasis formation. To study the relationship between tumor cell adhesion stabilization and organ-specific blood-borne metastasis, we investigated the adhesion stabilization of metastatic murine RAW117 large-cell lymphoma cells to the extracellular matrix proteins fibronectin and vitronectin, several Arg-Gly-Asp (RGD) containing peptides, and microvascular endothelial cells from the liver or lung. The highly liver metastatic RAW117-H10 subline showed the fastest stabilization to fibronectin, vitronectin, and RGD peptides. Poorly metastatic RAW117-P cells had stabilization times 3-10 times longer than for RAW117-H10 cells, while the lung- and liver-metastatic RAW117-L17 subline failed to stabilize at all. The adhesion stabilization of the RAW117-H10 cells to the extracellular matrix proteins and RGD peptides was inhibited by anti-beta(3) integrin monoclonal antibodies and RGD peptides. In contrast, the RAW117-L17 subline had the shortest stabilization time to unstimulated microvascular endothelial cells of the lung and hepatic sinusoids, followed by RAW117-H10 cells and RAW117-P cells. Monoclonal antibodies against the beta(3) integrin subunit and RGD peptides did not inhibit adhesion stabilization of RAW117-H10 cells to endothelial cells, suggesting that different metastatic variants of large-cell lymphoma cells use differing mechanisms to adhere to organ-specific endothelial cells. (c) 1996 John Wiley & Sons, Inc.     

Differential growth properties of metastatic large-cell lymphoma cells in target organ-conditioned medium

Metastatic variant sublines of the murine RAW117-P large cell lymphoma have been sequentially selected in vivo for enhanced liver (RAW117-H10) or lung (RAW117-L17) colonization. Such cell sublines were tested for their survival and growth in vitro in medium conditioned by soluble factors released from mouse kidney, brain, liver, or lung tissues. Liver-colonizing H10 and L17 sublines were growth-stimulated by target liver tissue-derived factors at concentrations that inhibited the growth of the parental cells. Lung-colonizing L17 as well as liver-colonizing H10 cells were stimulated by lung tissue factors at concentrations that growth-inhibited the parental cells. In contrast, there was no significant growth stimulation by factors from kidney or brain tissues. In general, the metastatic patterns of RAW117 cells correlated with their abilities to be stimulated by medium from target organ tissues, but other factors, such as organ-specific adhesion mechanisms [10-12], must also be involved in the specificity of blood-borne metastatic organ colonization.     

Butanol-extractable and detergent-solubilized cell surface components from murine large cell lymphoma cells associated with adhesion to organ microvessel endothelial cells.

Metastatic variant cell lines of the murine RAW117 large cell lymphoma were used to study the cell surface components involved in syngeneic tumor cell/microvessel endothelial cell interactions. Poorly liver-metastatic parental RAW117-P cell line adhered to murine hepatic sinusoidal endothelial cell monolayers at significantly lower rates than the liver-selected, highly liver-metastatic RAW117-H10 line and both cell lines were poorly adherent to lung microvessel and bovine aorta endothelial cells. Viable, 2% 1-butanol-treated RAW117-H10 tumor cells formed fewer liver tumor nodules in experimental metastasis assays than untreated H10 cells and were significantly less adherent to murine hepatic sinusoidal endothelial cell monolayers. When 2% 1-butanol extracts of metabolically labeled or CHAPS detergent lysates of cell surface-labeled tumor cells were analyzed for their ability to bind to fixed microvessel endothelial cell monolayers, quantitative differences were found in the extractable tumor cell surface components that bound to the different organ-derived microvessel endothelial cells. Cell surface components (1-butanol extractable), of Mr approximately 85,000-90,000 and approximately 37,000-40,000 bound to hepatic sinusoidal endothelial cell monolayers to a greater extent than to murine lung microvessel endothelial or bovine aortic endothelial cell monolayers, whereas tumor cell surface components of Mr approximately 45,000, approximately 33,000, and approximately 25,000 bound similarly to endothelial cells regardless of origin. The results suggest but do not prove that tumor cell/endothelial cell adhesion involves multiple tumor cell surface components, some of which commonly bind to various endothelial cells and others for which binding may be dictated by the tissue origin and type of endothelial cell. Particular RAW117 butanol-extractable cell membrane components were associated with tumor cell-endothelial cell adhesion, and these components could be responsible, in part, for the preferential adhesion of RAW117 cells to liver sinusoidal endothelial cells and metastasis to liver.     

Sialoglycoproteins of murine RAW117 large cell lymphoma/lymphosarcoma sublines of various metastatic colonization properties

A metastatic model for large-cell lymphoma/lymphosarcoma has been developed by sequential selection in vivo of the murine RAW117 cell line for enhanced liver metastasis or in vitro for loss of lectin-binding properties. The metastatic variants obtained from such selections show alterations in cell surface lectin-binding components, such as the wheat germ agglutinin (WGA)-reactive sialoglycoproteins. Detergent lysates from RAW117 cells were analyzed by polyacrylamide gel electrophoresis (PAGE) followed by reaction with 125I-labeled WGA. The [125I]WGA became bound to a diffuse band of Mr 120 000-200 000 in the gels that overlapped with the major sialoglycoprotein band revealed by the periodate-sodium borotritide labeling. However, the [125I]WGA reactivity diminished when gels were pretreated with mild acid to remove sialic acid in situ. The binding of [125I]WGA to the glycoprotein(s) was greater in the high liver-colonizing RAW117-H10 subline than in the parental RAW117-P line. Another lectin with different saccharide specificity, Ricinus communis agglutinin I (RCAI), became bound to a similar class of sialoglycoproteins, as well as to glycoproteins of lower Mr, but only when the gels were pretreated with mild acid to remove sialic acid. These differences in the relative RCAI-binding intensities after chemical removal of sialic acid were similar to those seen with WGA and indicate that differences in WGA reactivity of this class of sialoglycoproteins were not due to increased sialylation of the carbohydrate chains. Sialic acid was removed from RAW117 cells by neuraminidase treatment, and lysates were analysed for [125I]RCAI reactivity after electrophoresis. The migration of the glycoproteins was not affected by neuraminidase, indicating that the diffuseness of the major sialoglycoprotein band was not due to differences in sialylation. [125I]WGA reactivity to the sialoglycoprotein components, before and after Smith degradation in situ, strongly suggests that the oligosaccharide back-bones are highly branched and asparagine-linked. Only the high Mr portion of the diffuse sialoglycoprotein band was stained with peanut agglutinin (PNA) after in situ removal of sialic acid. To determine whether the expression of the sialoglycoprotein was causally related to liver metastasis, the amounts of sialoglycoproteins in RAW117 cells obtained by in vitro selection for increased or decreased metastasis were examined. Binding of [125I]WGA to intact cells and affinity chromatography of vectorially radiolabeled cell surface proteins on WGA-agarose were performed, and the results indicated that the in vitro selected high liver-colonizing RAW117 variants possesses high WGA r     

Expression of galectins on microvessel endothelial cells and their involvement in tumour cell adhesion

Lactoside-binding lectins (galectins) with molecular weights of about 14.5 kDa (galectin-1) and 29–35 kDa (galectin-3) bind preferentially to polylactosaminoglycan-containing glycoconjugates and have been found on the surface of tumour cells and implicated in cell-cell and cell-extracellular matrix adhesion and metastasis. We have demonstrated by immunoblotting that both galectin-1 and galectin-3 are present in extracts of endothelial cells cultured from bovine aorta, rat lung, mouse lung and mouse brain microvessels, whereas mouse hepatic sinusoidal endothelial cells expressed primarily galectin-1. These galectins were also localized by indirect immunofluorescent labelling on the surface of the different endothelial cells in culture and by immunohistochemical staining in human tissuesin vivo. Anti-galectin-1 antibodies inhibited the adhesion of liver-preferring murine RAW117-H10 large-cell lymphoma cells to hepatic sinusoidal endothelial cells or lung microvessel endothelial cellsin vitro. The data indicate that galectin-1 is expressed on the extracellular surface of endothelial cells and can mediate in part the adhesion of RAW117-H10 cells to liver microvessel endothelial cells.     

Proteome analysis of human androgen-independent prostate cancer cell lines: variable metastatic potentials correlated with vimentin expression

To better understand the molecular mechanisms of prostate cancer (PCA) dissemination and to develop new anti-metastasis therapies, key regulatory molecules involved in PCA metastasis were identified in two human androgen-independent PCA cell lines, highly metastatic 1E8-H and lowly metastatic 2B4-L cells. Through 2-DE and MS analyses, 12 proteins with different expression levels in the two cell lines were identified. The following proteins were found to be significantly up-regulated in 1E8-H cells compared with 2B4-L cells: gp96 precursor, calreticulin precursor, vimentin (VIM), Hsp90alpha, peroxiredoxin 2, HNRPH1, ezrin, T-complex protein 1, alpha subunit, and hypothetical protein mln2339. In contrast, heart L-lactate dehydrogenase H chain, annexin I, and protein disulfide isomerase were notably down-regulated in 1E8-H cells compared with 2B4-L cells. To our knowledge, this study is the first to demonstrate that up-regulation of VIM expression positively correlates with the invasion and metastasis of androgen-independent PCA.     

Suppression of GD1alpha ganglioside-mediated tumor metastasis by liposomalized WHW-peptide

GD1alpha ganglioside-replica peptides were recently isolated from a phage-displayed random pentadecapeptide library by assaying for inhibition of adhesion of RAW117-H10 lymphosarcoma cells to hepatic sinusoidal microvessel endothelial (HSE) cells. We show here that the Trp-His-Trp (WHW) peptide was identified as a minimal sequence of the GD1alpha-replica peptide WHWRHRIPLQLAAGR. The addition of WHW peptide-attached liposomes displayed efficient inhibition of liver metastasis of RAW117-H10 cells as well as of GD1alpha-mediated adhesion of RAW117-H10 cells to HSE cells in vitro. These results suggest that engineered liposomes for peptide delivery are applicable to treatment for metastasis.     

Modulation of macrophage function by gamma-irradiation. Acquisition of the primed cell intermediate stage of the macrophage tumoricidal activation pathway

Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. Although this synergistic response of normal macrophages to sequential incubation with activation signals has been well established, characterization of the intermediate stages in this pathway has been difficult, due in large measure to the instability of the intermediate cell phenotypes. We have developed a model system for examination of macrophage-mediated tumor cell lysis, with the use of the murine macrophage tumor cell line RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both interferon-gamma (IFN-gamma, the priming signal) and bacterial lipopolysaccharide (LPS, the triggering signal) in the development of tumor cytolytic activity. In this system, the priming effects of IFN-gamma decay rapidly after withdrawal of this mediator and the cells become unresponsive to LPS triggering. We have recently observed that gamma-irradiation of the RAW 264.7 cells also results in development of a primed activation state for tumor cell killing. The effects of gamma-radiation on the RAW 264.7 cell line are strikingly similar to those resulting from incubation with IFN-gamma, with the exception that the irradiation-induced primed cell intermediate is stable and responsive to LPS triggering for at least 24 hr. Treatment with gamma-radiation also results in increased cell surface expression of major histocompatibility complex-encoded class I antigens; however, class II antigen expression is not induced. Irradiation-induced development of the primed phenotype is not solely the result of cytostatic effects as treatment of the cells with a radiomimetic drug, mitomycin C, results in decreases in [3H]thymidine incorporation that are similar to those observed after irradiation, without concomitant development of cytolytic potential. In addition, priming by gamma-radiation does not appear to be mediated by the release of soluble autoregulatory factors. This alternate pathway for induction of the primed macrophage activation state should serve as a useful tool for identification of molecules important to the functional potential of primed cells, and for elucidation of the biochemical mechanisms of the priming event in tumoricidal activation.     

Decrease of the major surface glycoprotein gp160 in activated macrophages

The quantity of surface-radioiodinated gp160, the previously described trypsin- and plasmin-sensitive surface glycoprotein of guinea pig macrophages was reduced 70% in macrophages activated in vivo in comparison to elicited macrophages. The reduction of gp160 was also detected by Coomassie blue staining, demonstrating that the absolute number of gp160 molecules is lower in activated macrophages. Gp160 was present on activated macrophages as single-chain intact molecules; no proteolytically cleaved gp160 was detected. Biosynthesis of gp160 was compared for activated and elicited macrophages by culturing with [³⁵S]methionine. Whereas biosynthetically labeled gp160 was readily detected in elicited macrophages, negligible [³⁵S]methionine-labeled gp160 was detected in parallel cultures of activated macrophages, suggesting that a major cause of decreased surface gp160 on activated macrophages lies at the level of synthesis.     

Co-localization of beta1,6-branched oligosaccharides and coarse melanin in macrophage-melanoma fusion hybrids and human melanoma cells in vitro

Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of beta1,6-N-acetylglucosaminyltransferase V and beta1,6-branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced 'coarse melanin'- autophagic vesicles with multiple melanosomes. As beta1,6-branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin- and immunohistochemistry, we analyzed cell lines producing coarse melanin for beta1,6-branched oligosaccharides: gp100/pmel-17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94-H48, a highly metastatic, macrophage-melanoma experimental fusion hybrid; (ii) 6(neo) mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel-23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel-23 cells, and co-localized with stains for beta1,6-branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co-expression of beta1,6-branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.     

Lectin-like molecules on the murine macrophage cell surface

Lectin-like molecules on the surface of murine peritoneal exudate macrophages induced by thioglycolate or an anti-tumor streptococcal preparation, OK-432, were investigated and isolated. Furthermore, their sugar-binding specificities and their role in macrophage-mediated tumor cytotoxicity were examined. A neoglycoprotein, D-galactose (Gal)-bovine serum albumin, bound to these murine peritoneal macrophages. This binding of Gal-bovine serum albumin was inhibited by D-galactose, and by complex-type oligosaccharides (unit B) and high mannose-type oligosaccharides (unit A) prepared from porcine thyroglobulin. When thioglycolate-elicited macrophages were activated by lipopolysaccharide and/or the culture supernatant of concanavalin A-activated mouse spleen cells, they became tumoricidal and the number of the lectin-like molecules on the macrophage surface was found to increase. Since the binding and cytotoxic activities of these tumoricidal macrophages toward tumor cells were partially inhibited by D-galactose, the D-galactose-binding lectin-like molecules on the surface of tumoricidal macrophages might play an important role in macrophage-mediated cytotoxicity. These lectin-like molecules were then isolated from solubilized murine peritoneal exudate cells labeled with pyridoxal 5'-phosphate and sodium [3H]borohydride by affinity chromatography on columns of asialo unit B oligosaccharide-Sepharose 4B and/or beta-D-galactose-Bio-Gel P-100. The proteins bound to the asialo unit B oligosaccharide-Sepharose 4B column and eluted specifically were found to have approximate molecular weights of 79 000 and 18 000, and the protein bound to and eluted from the beta-D-galactose-Bio-Gel P-100 column had an approximate molecular weight of 77 000. These isolated proteins bound to the surface of glutaraldehyde-fixed tumor cells, and their binding was inhibited by D-galactose and also by D-mannose. Since most of the 77 kDa protein bound to the asialo unit B oligosaccharide-Sepharose 4B, this protein was assumed to be identical with the 79 kDa protein. These results suggest that the lectin-like molecules on murine macrophages have wide specificity and that one lectin-like molecule can bind both D-galactose and D-mannose.     

Induction of tumor cell resistance to macrophage-mediated lysis by preexposure to non-activated macrophages

Thioglycollate-elicited peritoneal exudate (non-activated) macrophages do not lyse tumor cells and in contrast to activated macrophages bind less target cells. However, a non-lethal encounter of tumor cells with non-activated macrophages resulted in a pronounced effect on the subsequent tumor cell binding to and lysis by activated macrophages. Our results have shown that binding of tumor cells by non-activated macrophages was Ca2+ and temperature dependent; had a requirement for a Pronase-sensitive structure on macrophage surface membranes; was saturable; and was 2-3X less than that observed for activated macrophages. Experiments were conducted in which syngeneic tumor cells were incubated with a monolayer of non-activated macrophages and then assayed for selective binding and sensitivity to lysis. The important observations were that as a result of a 3-hr incubation with non-activated macrophages at an EC: TC ratio of 5:1 there was an increase in the number of tumor cells that bound to both activated and non-activated macrophages; a loss of selective binding in which the ratio of tumor cells bound to activated/non-activated macrophages (normally greater than 2) was lowered to 1.0; and a concomitant decrease in the susceptibility of tumor cells to macrophage-mediated cytolysis. The induction of tumor cell resistance to macrophage kill required an exposure to an excess number of non-activated macrophages, was reversible upon culturing with or without macrophages for 24 hr and required cell-cell contact. Our results reinforce the importance of selective binding between tumor cells and activated macrophages as an initial phase in tumor cell killing and also illustrates an active role for non-activated macrophages in vivo in allowing tumor cells to escape the immune surveillance by activated macrophages.     

Sensitivity to macrophage-mediated cytostasis is cell cycle dependent

We have examined the sensitivity of proliferating lymphoid cells in different phases of the cell cycle to macrophage-mediated cytostatic activity. These studies evaluated the ability of target cells enriched in individual cell cycle phases to pass into the next phase during brief (2–6 hr) periods of coculture with activated or nonactivated peritoneal macrophages. Both normal (concanavalin A-stimulated spleen cells) and neoplastic (Gross virus-induced thymic lymphoma) cells were analyzed. Spleen cells or lymphoma cells were first separated by centrifugal elutriation into populations highly enriched for G₁, S, or G₂/M phases of the cell cycle and cultured in the presence of nonactivated or activated macrophages for periods of 2, 4, or 6 hr. The cellular DNA content of recovered nonadherent target cells was then analyzed by flow cytometry after staining with propidium iodide. Macrophage contamination of target cell populations was insignificant under these conditions. Nonactivated macrophages did not affect target cell cycle traverse when compared with target cells cultured alone. Activated macrophage mediated cytostatic activity resulted in complete block of the transition of cells in G₁ phase into S phase and of the further accumulation of DNA by cells in early S phase. Cells already in mid to late S phase were able to continue DNA replication at rates nearly equivalent to control cells. There was no inhibition of the passage of cells through G₂ or mitosis. These effects were seen by as early as 2 hr of macrophage-target cell coculture and both normal and neoplastic cells exhibited identical patterns of cell cycle phase sensitivity.     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Somatic cell hybrids between macrophages from Bcgr and Bcgs mice: characterization of MHC class II expression

In order to gain a better understanding of the regulation of MHC class II expression related to the Bcg gene, we have produced macrophage-macrophage somatic cell hybrids by fusing the RAW 309 macrophage cell line derived from BALB/c.Bcgs mice with peritoneal macrophages from Bcgr C3H/HeN mice. The differential screening of the hybrids was based on the differential sensitivity of Ia expression to suppression with cycloheximide. We found that most of the hybrids expressed Ia without further stimulation with rIFN-gamma. Cycloheximide suppressed the expression of Ia by some of the hybrids. Treatment of these cells with rIFN-gamma resulted in a cycloheximide resistant Ia expression of both parental haplotypes. The macrophage hybrids produced IL-1 beta, IL-1 alpha, and TNF-alpha when stimulated with LPS. There was no correlation between the levels of monokines produced and the persistence of Ia expression. The results of this investigation indicate that the product of the Bcg gene contributed by macrophages from C3H/HeN mice will affect the expression of the I-Ad glycoprotein that is normally transiently expressed by the RAW 309 cell line.     

The effect of phorbol esters on tumor cell sensitivity to macrophage-mediated cytostasis

Macrophage-mediated cytostasis was measured in a mouse syngeneic system where EL4 thymoma cells were found to be inhibited by C57B1/6 mouse macrophages. When tumor cells were pretreated with TPA, they became resistant to macrophage-mediated stasis. Nonelutriated as well as elutriated cells enriched in G1/early S and late S were sensitive to macrophage-mediated stasis. However, when elutriated cells were treated with TPA, cells enriched in G1/early S were rendered resistant to the cytostatic activity of macrophages whereas cells enriched in late S were not. The TPA effect on tumor cell susceptibility to stasis was found to be reversible and a nontumor-promoting phorbol ester, alpha-PDD, was ineffective.     

The interaction of Naegleria fowleri amoebae with murine macrophage cell lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappa B activation: role of H(2)O(2) and nitric oxide in inducible nitric oxide synthase expression in macrophages.

Reactive molecules O(-)(2), H(2)O(2), and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N(G)-monomethyl-L-arginine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappa B was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O(-)(2) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H(2)O(2) induced NF-kappa B activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H(2)O(2) enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H(2)O(2) was inhibited by catalase and PDTC. These findings suggest that H(2)O(2) production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappa B activation and that NO is a negative feedback inhibitor of iNOS protein expression.     

Suppression of macrophage cytostatic activation by serum retinoids: a possible role for transglutaminase

Mouse resident peritoneal macrophages, activated in vitro with murine recombinant interferon-gamma and lipopolysaccharide in the presence of sera from different sources, showed marked differences in their abilities to inhibit murine adenocarcinoma cell growth, and in induced activity of the enzyme, tissue transglutaminase. The extraction of lipids from the serum abolished its ability to induce tissue TGase activity and to inhibit cytostatic activity, but these capabilities were fully restored by readdition of all trans-retinol or all trans-retinoic acid at physiological concentrations. Addition of dansylcadaverine, a competitive inhibitor of TGase, resulted in complete recovery of macrophages from retinoid-induced suppression of cytostatic activity. These results suggest that endogenous retinoids play an important role in the regulation of macrophage-mediated cytostatic activity in a process that is independent of prostaglandin secretion but seems to involve the protein cross-linking enzyme, tissue transglutaminase.