Irradiated tumor cells adenovirally engineered to secrete granulocyte/macrophage-colony-stimulating factor establish antitumor immunity and eliminate pre-existing tumors in syngeneic mice

The specific aim of this study was to examine the prophylactic as well as the therapeutic efficacies of irradiated mouse CT26 colon cancer cells, infected with recombinant adenoviruses harboring cDNAs specific for granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN-γ) and monocyte chemotactic protein1 (MCP-1). Results showed that tumor cells secrete the respective cytokines for several days after infection and subsequent irradiation. Vaccination with irradiated GM-CSF-secreting CT26 cells protected 90% of syngeneic mice challenged with live parental cells. On the other hand, vaccination with irradiated IFNγ or MCP-1-secreting CT26 cells totally failed to protect mice from tumor development after challenge with parental cells. None of the tumor-free mice initially vaccinated with irradiated GM-CSF-producing CT26 cells developed tumor upon repeated challenge with parental cells during the entire observation period. The establishment of specific and long-lasting antitumor immunity following vaccination with GM-CSF-producing tumor cells requires the simultaneous presence of GM-CSF and tumor antigen at the vaccine site. Depletion of CD8⁺ cells, but not CD4⁺ cells, blocked the vaccine efficacy of GM-CSF-producing tumor cells. Subcutaneous injection of irradiated GM-CSF-producing CT26 cells also effectively prevented the growth of a small load of parental tumor that was implanted 3 days earlier or the development of metastatic foci in the lung from intravenously injected parental cells either 7 days before or 3 days after vaccination. Our data thus show that, in these experimental tumor models, subcutaneous injection of irradiated tumor cells adenovirally, transduced with the GM-CSF gene leads not only to prevention of growth of subsequently implanted tumor but also to elimination of pre-existing and metastatic tumors.     

Regulation of Nitric Oxide Production by Murine Peritoneal Macrophages Treated in Vitro with Chemokine Monocyte Chemoattractant Protein 1

Monocyte chemoattractant protein 1 (MCP-1) is an important mediator of monocyte/macrophage recruitment and activation at the sites of chronic inflammation and neoplasia. In the current study, the role of nitrogen monoxide (NO) in the activation of murine peritoneal macrophages to the tumoricidal state in response to in vitro MCP-1 treatment and the regulatory mechanisms involved therein were investigated. Murine peritoneal macrophages upon activation with MCP-1 showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. N-monomethyl- -arginine (L-NMMA), a specific inhibitor of the -arginine pathway, inhibited the MCP-1-induced NO secretion and generation of macrophage-mediated tumoricidal activity against P815 (NO-sensitive, TNF-resistant) cells but not the L929 (TNF-sensitive, NO-resistant) cells. These results indicated -arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of MCP-1-treated macrophages. Supporting this fact, expression of iNOS mRNA was also detected in the murine peritoneal macrophages upon treatment with MCP-1. Investigating the signal transduction pathway responsible for the NO production by the MCP-1-activated murine peritoneal macrophages, it was observed that the pharmacological inhibitors wortmannin, H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride), and PD98059 blocked the MCP-1-induced NO production, suggesting the probable involvement of phosphoinositol-3-kinase, protein kinase C, and p42/44 MAPkinases in the above process. Various modulators of calcium and calmodulin (CaM) such as EGTA, nifedipine, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester), A23187, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide) were also found to modulate the in vitro macrophage NO release in response to MCP-1. This observation indicated the regulatory role of calcium/CaM in the process of MCP-1-induced macrophage NO production. Similarly, the role of serine/threonine and protein tyrosine phosphatases in the above pathway was suggested using the specific inhibitors of these phosphatases, okadaic acid and sodium orthovanadate.     

Role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice

Intratracheal instillation of the monocyte chemoattractant JE/monocyte chemoattractant protein (MCP)-1 in mice was recently shown to cause increased alveolar monocyte accumulation in the absence of lung inflammation, whereas combined JE/MCP-1/lipopolysaccharide (LPS) challenge provoked acute lung inflammation with early alveolar neutrophil and delayed alveolar monocyte influx. We evaluated the role of resident alveolar macrophages (rAM) in these leukocyte recruitment events and related phenomena of lung inflammation. Depletion of rAM by pretreatment of mice with liposomal clodronate did not affect the JE/MCP-1-driven alveolar monocyte accumulation, despite the observation that rAM constitutively expressed the JE/MCP-1 receptor CCR2, as analyzed by flow cytometry and immunohistochemistry. In contrast, depletion of rAM largely suppressed alveolar cytokine release as well as neutrophil and monocyte recruitment profiles upon combined JE/MCP-1/LPS treatment. Despite this strongly attenuated alveolar inflammatory response, increased lung permeability was still observed in rAM-depleted mice undergoing JE/MCP-1/LPS challenge. Lung leakage was abrogated by codepletion of circulating neutrophils or administration of anti-CD18. Collectively, rAM are not involved in JE/MCP-1-driven alveolar monocyte recruitment in noninflamed lungs but largely contribute to the alveolar cytokine response and enhanced early neutrophil and delayed monocyte influx under inflammatory conditions (JE/MCP-1/LPS deposition). Loss of lung barrier function observed under these conditions is rAM independent but involves circulating neutrophils via beta(2)-integrin engagement.     

Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.     

Induction of chemokine secretion and enhancement of contact-dependent macrophage cytotoxicity by engineered expression of granulocyte-macrophage colony-stimulating factor in human colon cancer cells

We investigated the role of tumor cell-derived GM-CSF in recruitment and tumoricidal activation of tissue macrophages. Transfection of the murine GM-CSF gene into KM12SM human colon cancer cells decreased the tumorigenicity of transfected cells and nontransfected bystander colon cancer cells in nude mice. Sequential tissue sections from sites injected with high GM-CSF-producing tumor cells (but not from nontransfected or low GM-CSF-producing cells) demonstrated a dense infiltration of polymorphonuclear cells (PMN), followed by infiltration of macrophages, which correlated with expression of the macrophage-inflammatory protein-1alpha and the monocyte chemoattractant protein-1 (MCP-1) in mouse PMN and macrophages. GM-CSF-producing KM12SM cells were highly sensitive to lysis by mouse macrophages and also increased macrophage-mediated lysis of bystander nontransfected KM12SM cells. The incubation of macrophages with GM-CSF induced expression of the CD11b surface adhesion molecule, which was associated with increased attachment to tumor cells. All KM12SM cells were sensitive to macrophage-mediated lysis in the presence of rGM-CSF and recombinant MCP-1. Collectively, the results demonstrate that tumor cell-derived GM-CSF stimulates PMN and macrophages to secrete macrophage-inflammatory protein-1alpha and MCP-1, which triggers recruitment of mononuclear cells, induces expression of adhesion molecules on macrophages, and enhances contact-dependent cytolysis of tumor cells.     

Suppression of tumor formation in vivo by expression of the JE gene in malignant cells.

The early growth response gene JE encodes a monocyte chemoattractant, MCP-1. The JE/MCP-1 protein attracts and stimulates human monocytes and induces monocyte-mediated inhibition of tumor cell growth in vitro. Expression of human or murine JE/MCP-1 in Chinese hamster ovary (CHO) cells completely suppressed their ability to form tumors in nude mice. Coinjection of JE/MCP-1-expressing cells with nonexpressing CHO cells or with HeLa cells also prevented tumor formation. Since JE/MCP-1 expression had no discernible effect on the tranformed phenotype of these cells in vitro, the suppressive effect depends on host animal factors. These factors are likely to be components of the inflammatory response, because JE/MCP-1-expressing cells elicited a predominantly monocytic infiltrate at the site of injection. Our results suggest that JE/MCP-1 protein may be useful in cancer therapy.     

Regulation of nitric oxide production by murine peritoneal macrophages treated in vitro with chemokine monocyte chemoattractant protein 1.

Monocyte chemoattractant protein 1 (MCP-1) is an important mediator of monocyte/macrophage recruitment and activation at the sites of chronic inflammation and neoplasia. In the current study, the role of nitrogen monoxide (NO) in the activation of murine peritoneal macrophages to the tumoricidal state in response to in vitro MCP-1 treatment and the regulatory mechanisms involved therein were investigated. Murine peritoneal macrophages upon activation with MCP-1 showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. N-monomethyl-l-arginine (L-NMMA), a specific inhibitor of the l-arginine pathway, inhibited the MCP-1-induced NO secretion and generation of macrophage-mediated tumoricidal activity against P815 (NO-sensitive, TNF-resistant) cells but not the L929 (TNF-sensitive, NO-resistant) cells. These results indicated l-arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of MCP-1-treated macrophages. Supporting this fact, expression of iNOS mRNA was also detected in the murine peritoneal macrophages upon treatment with MCP-1. Investigating the signal transduction pathway responsible for the NO production by the MCP-1-activated murine peritoneal macrophages, it was observed that the pharmacological inhibitors wortmannin, H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride), and PD98059 blocked the MCP-1-induced NO production, suggesting the probable involvement of phosphoinositol-3-kinase, protein kinase C, and p42/44 MAPkinases in the above process. Various modulators of calcium and calmodulin (CaM) such as EGTA, nifedipine, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester), A23187, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide) were also found to modulate the in vitro macrophage NO release in response to MCP-1. This observation indicated the regulatory role of calcium/CaM in the process of MCP-1-induced macrophage NO production. Similarly, the role of serine/threonine and protein tyrosine phosphatases in the above pathway was suggested using the specific inhibitors of these phosphatases, okadaic acid and sodium orthovanadate.     

Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells

Many acute and chronic lung diseases are characterized by the presence of increased numbers of activated macrophages. These macrophages are derived predominantly from newly recruited peripheral blood monocytes and may play a role in the amplification and perpetuation of an initial lung insult. The process of inflammatory cell recruitment is poorly understood, although the expression of inflammatory cell-specific chemoattractants and subsequent generation of chemotactic gradients is likely involved. Although immune cells such as macrophages and lymphocytes are known to generate several inflammatory cell chemoattractants, parenchymal cells can also synthesize and secrete a number of bioactive factors. We now demonstrate the generation of significant monocyte chemotactic activity from tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta-treated pulmonary type II-like epithelial cells (A549). The predominant inducible monocyte chemotaxin had an estimated molecular mass of approximately 14-15 kDa and was neutralized by specific antibody to human monocyte chemotactic protein-1 (MCP-1). Induction of activity was accompanied by increases in steady-state mRNA level for MCP-1. These data are consistent with the induction of MCP-1 expression from A549 cells by TNF and IL-1. MCP-1 production from A549 cells could be induced by lipopolysaccharide (LPS)-stimulated alveolar macrophage (AM)-conditioned media, but not by LPS alone. The inducing activity in AM-conditioned media was neutralized with specific antibodies to IL-1 beta, but not TNF-alpha. Our findings suggest that the alveolar epithelium can participate in inflammatory cell recruitment via the production of MCP-1 and that cytokine networking between contiguous alveolar macrophages and the pulmonary epithelium may be essential for parenchymal cell MCP-1 expression.     

Secretion by human fibroblasts of monocyte chemoattractant protein-1, the product of gene JE

We recently purified human monocyte chemoattractant protein-1 (MCP-1) from culture fluids of either human glioma cell lines or mitogen-stimulated human peripheral blood mononuclear leukocytes. It has now been shown that MCP-1 is the product of the gene JE, which was first recognized by its expression in fibroblasts stimulated with platelet-derived growth factor (PDGF). We therefore studied secretion of MCP-1 by three human fibroblast cell lines. Monocyte chemotactic activity was found in culture fluids of all three lines after growth to confluence in DMEM-10% FCS, and the amounts secreted per cell were comparable for the three lines. The MRC-5 line was chosen for further study. Monocyte chemotactic activity secretion by confluent MRC-5 cultures continued after a switch to serum-free medium and was not inhibited by anti-PDGF antibody, indicating that secretion may not have been caused by autocrine release of PDGF. When concentrated serum-free MRC-5 culture fluid was injected into an HPLC gel filtration column, only one chemotactic activity peak was observed, which was in the same location as glioma-derived MCP-1. The activity was completely absorbed out by an anti-MCP-1 affinity column, which indicates that all the chemotactic activity in MRC-5 culture fluid was accounted for by MCP-1. PDGF caused a marked increase in chemotactic activity over that found in serum-free culture fluid of MRC-5 or 501T cells. Immunoprecipitation by anti-human MCP-1 showed two bands, corresponding to the two forms of MCP-1 previously described (MCP-1 alpha and beta); and the amounts increased in response to PDGF stimulation. Thus, the reported increase in human fibroblast JE mRNA in response to PDGF-containing serum stimulation is reflected in increased secretion of the MCP-1 gene product.     

The elaboration of leukotactic mediators during the interaction between parental-type lymphocytes and F1 hybrid cells.

Mixed leukocyte cultures consisting of white blood cells from (Lewis times BN) F1 hybrids and Lewis parents produced monocyte chemotactic factor. Elaboration of this material preceded incorporation of 3H-thymidine. Local graft vs host (GVH) reactions were induced by subcapsular injection of parental thoracic duct cells into F1 hybrids. Homogenates from these kidneys, but not from kidneys injected with syngeneic thoracic duct cells, contained monocyte chemotactic factor. Little or no neutrophil chemotactic factor was present. Ultracentrifugal analysis of the monocyte chemotactic factor indicated a distribution similar to that found previously in culture fluids of lymphoid cells stimulated by soluble antigens. Differential counts of inflammatory cells extracted in suspension form from kidneys undergoing a GVH reaction indicated the majority of cells to be lymphocytic in type, but with a significant proportion of monocytes. Virtually no neutrophils were present. These findings indicate that a monocyte chemotactic factor is produced by cultures of parental leukocytes stimulated by semiallogeneic cells and that a similar factor appears in GVH reactions in the rat kidney. This chemotactic factor may be relevant to the character of the cellular exudates.     

Prostaglandin E2 does not inhibit tumoricidal activity of mouse macrophages against adherent tumor cells

We determined whether endogenously produced PGE2 can down-regulate the tumoricidal properties of macrophages by a negative feedback mechanism. Peritoneal exudate macrophages or resident peritoneal macrophages of mice were incubated in medium (control) or in medium containing IFN-gamma and LPS. Activated macrophages were highly tumoricidal against syngeneic melanoma cells and secreted high levels of PGE2. Treatment with indomethacin or diclofenac sodium (voltaren) completely inhibited the production and secretion of PGE2 but not the tumoricidal activity of activated macrophages measured either immediately after activation or 1 to 3 days thereafter. Finally, the addition of exogenous PGE2 did not alter the ability of peritoneal exudate macrophages to respond to IFN-gamma or of LPS to produce high levels of tumor cell lysis. Collectively, these results show that PGE2 produced by activated macrophages is not a down-regulator of their tumoricidal activity against adherent tumor cells.     

Transforming growth factor-beta-induced gene expression of monocyte chemoattractant JE in mouse osteoblastic cells, MC3T3-E1

A recent study demonstrated that PDGF-inducible JE is an inflammatory cytokine that directs chemotactic activity of monocytes. Accumulation of monocyte/macrophage lineage cells at site of bone tissue sites is very important for formation of multinucleate osteoclasts, which mediate bone resorption. Since transforming growth factor-beta (TGF-beta) is a potent regulator in bone remodeling, we examined whether TGF-beta induced JE gene expression in mouse osteoblastic cells, MC3T3-E1. TGF-beta induced a maximum JE mRNA expression at 3 hr after initiation of the cytokine treatment. This maximal expression was observed in when TGF-beta was used at a concentration of 1 ng/ml. The chemotactic activity for human monocytes was detected in conditioned medium of TGF-beta-treated cells, and the chemotactic activity was neutralized by anti-JE serum treatment.     

Identification of the increased expression of monocyte chemoattractant protein-1, cathepsin S, UPIX-1, and other genes in dystrophin-deficient mouse muscles by suppression subtractive hybridization

The lack of dystrophin results in muscular dystrophy characterized by degeneration, inflammation, and partial regeneration of skeletal muscles. The fate of these muscles may be determined by the extent of adaptation to the defect and the efficiency of regeneration that is affected by inflammatory cells. We have used suppression subtractive hybridization and quantitative Northern blot analysis to identify differentially expressed genes. Increased expression of murine monocyte chemoattractant protein-1 (JE/MCP-1), cathepsin S, UPIX-1, nmb, cathepsin B, and lysozyme M mRNAs were identified in 2-month-old mdx mouse leg muscles. UPIX-1 is a novel gene. Although it was not expressed in control muscles, it was expressed in control brain, heart, and spleen. JE/MCP-1 and cathepsin S proteins in mdx muscles, as well as JE/MCP-1 protein in the serum of mdx mice were also detected. JE/MCP-1 may be responsible for attraction of inflammatory cells, and cathepsin S, a potent elastolytic protease, may contribute to the remodeling of the extracellular matrix that is required for the migration of these cells to the injured muscles.     

Cytokine-induced production of monocyte chemoattractant protein-1 by cultured human mesangial cells.

The infiltration of the glomerulus by monocyte-derived macrophages is an important step in the pathogenesis of glomerular injury. The factors regulating glomerular leukocyte traffic remain unknown. We postulated that the glomerular mesangial cell (MC) may participate in the development of glomerular inflammation through the production of the monocyte-specific chemotactic factor, monocyte chemoattractant protein-1 (MCP-1). Using a cell culture system, we found that human MC produced a basal level of monocyte chemotactic activity, which was significantly increased by the inflammatory cytokines IL-1 beta and TNF-alpha. This increase in bioactivity correlated with the increased expression of MCP-1 mRNA by cytokine-conditioned MC. The total chemotactic activity of MC-conditioned supernatants was reduced by more than 80% after immunoadsorption with a specific anti-MCP-1 antibody. Thus, MC could play a role in inflammatory glomerular conditions through the production of MCP-1.