Activation in vitro of mouse macrophages by syngeneic, allogeneic, or xenogeneic lymphocyte supernatants.

Macrophages from normal C57BL/6 mice, those with a subcutaneous B16 melanoma, and mice immunized against the tumor were examined for in vitro cytotoxicity to B16 tumor cells. Macrophages were treated by incubation with supernatants from B16 cells grown either in unmixed cultures or in cultures containing syngeneic, normal, or sensitized allogeneic (A mouse), or xenogeneic (rat) lymphocytes. The various treated and untreated macrophages were then cultured for 5 days with viable B16 cells prelabeled with 125I-5-iodo-2'-deoxyuridine; the cultures were terminated, and the extent of destruction of the B16 target cells was determined from the amounts of radioactivity remaining in adherent tumor cells. Of the untreated macrophages, only those from immunized mice were cytotoxic to the tumor cells; macrophages from normal and tumor-bearing mice became cytotoxic by incubation with supernatants from cultures containing lymphocytes from immunized syngeneic mice, sensitized allogeneic mice, or sensitized rats; and macrophages incubated with supernatants from cultures containing normal nonsensitized allogeneic or xenogeneic lymphocytes showed no cytotoxicity. Thes results suggested that macrophages from tumor-bearing animals are potentially cytotoxic to their syngeneic tumors and can be activated by mediators released from sensitized syngeneic, allogeneic, and/or xenogeneic lymphocytes in vitro.     

Establishment of transplantable tumor lines and in vitro cell lines of malignant thymoma developed in a BUF/Mna rat.

A spontaneous malignant thymoma was found in an 18-month-old female BUF/Mna rat and serially transplanted subcutaneously in both syngeneic BUF/Mna rats (designated as MTH-R) and KSN nude mice (MTH-NM) for more than 5 years. Both tumors shared the histological appearance of sarcomatoid carcinoma as seen in the original tumor. However, MTH-NM grew faster than MTH-R in the respective hosts. The MTH-NM grew in both KSN-nude mice and BUF/Mna-rnu/rnu rats but not in BUF/Mna rats, the host of the original tumor. Three continuous tissue culture cell lines (MTHC-1, MTHC-2 and MTHC-3) were established from the MTH-NM tumors at the 2nd, 15th and 17th transplantation generations, respectively. The MTH-NM tumors and latter two tissue culture cell lines carried one or more mouse chromosomes, probably acquired by cell fusion with mouse cells during passages in vivo. The presence of the mouse chromosomes was confirmed by the presence of mouse DNA and of antibodies to the MTHC-2 and MTHC-3 cells in the sera of BUF/Mna rats transplanted with MTH-NM.     

Establishment of Transplantable Tumor Lines and in vitro Cell Lines of Malignant Thymoma Developed in a BUF/Mna Rat

A spontaneous malignant thymoma was found in an 18-month-old female BUF/Mna rat and serially transplanted subcutaneously in both syngeneic BUF/Mna rats (designated as MTH-R) and KSN nude mice (MTH-NM) for more than 5 years. Both tumors shared the histological appearance of sarcomatoid carcinoma as seen in the original tumor. However, MTH-NM grew faster than MTH-in the respective hosts. The MTH-NM grew in both KSN-nude mice and BUF/Mna- rnu/rnu rats but not in BUF/Mna rats the host of the original tumor. Three continuous tissue culture cell lines (MTHC-1, MTHC-2 and MTHC-3) were established from the MTH-NM tumors at the 2nd, 15th and 17th transplantation generations, respectively. The MTH-NM tumors and latter two tissue culture cell lines carried one or more mouse chromosomes, probably acquired by cell fusion with mouse cells during passages in vivo. The presence of the mouse chromosomes was confirmed by the presence of mouse DNA and of antibodies to the MTHC-2 and MTHC-3 cells in the sera of BUF/Mna rats transplanted with MTH-NM.     

Freshly isolated bone marrow cells induce death of various carcinoma cell lines

In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.     

Decreased macrophage-mediated cytotoxicity in mammary-tumor-bearing mice is related to alteration of nitric-oxide production and/or release

Peritoneal-exudate macrophages (PEM) from mammarytumor-bearing mice have impaired cytotoxic activity against syngeneic and allogeneic tumor targets. The ability of PEM from normal and tumor-bearing mice to bind tumor targets was found to be similar in the presence or the absence of surrogate receptors, which enhanced the binding but not the killing of tumor targets by PEM from tumor-bearing mice, suggesting that other mechanisms are involved in their impaired cytolytic activity. Soluble and membrane-bound TNF-α, as well as H₂O₂, were found in higher amounts in PEM from tumor bearers upon stimulation with LPS, as compared with PEM from normal mice. However, tumor-bearers' macrophages displayed decreased capacity to produce and/or release nitric oxide, which could be reversed by the addition of increasing levels of IFN-γ. These results indicate that the lack of macrophage cytotoxicity in mammary-tumor-bearing mice is related to impaired production and/or release of NO by these effector cells, possibly aggravated by the insufficient IFN-γ production previously reported in these animals. Moreover, mammary-tumor progression results in dis-regulation of synthesis of macrophage-mediators, with over-production of molecules to which mammary-tumor cells are insensitive and deficient production of NO, the crucial molecule to which these cells appear to be highly sensitive.     

Effect of interleukin 2 on cytotoxic effectors: I. Short-term culture of the cytotoxic effectors and the in vivo anti-tumor activity of the cultured effectors isolated from tumor site

The effects of interleukin 2 (IL2) on the in vitro and in vivo activity of cytotoxic T cells have been studied. IL2 was produced by W/Fu rat spleen cells cultured with concanavalin A. The IL2 thus prepared gave an optimal T-cell growth-promoting effect at a concentration of 5-20% equivalents of the original preparation. In the primary syngeneic mixed lymphocyte/tumor cell cultures (MLTC) against FBL-3 tumor cells, the addition of IL2 failed to generate a cytotoxic response. However, the cytotoxic response could be generated in MLTC by addition of exogenous macrophages. On the other hand, IL2 could maintain the growth of performed cytotoxic T cells for 3 to 5 weeks. These cytotoxic T cells were generated either by in vitro sensitization (MLC or MLTC) or by in vivo sensitization of B6 mice against a syngeneic tumor FBL-3. In short-term cultures, augmentation of the cytotoxic activity was seen after 10 days' culturing with IL2. The antigenic specificity of the cytotoxic reaction was altered after 28-35 days in culture, and the effectors broadly reactive. When growing a nonadherent population of lymphocytes isolated from FBL-3 ascites tumor, supplementation with IL2 selectively promoted the growth of a T-cell population, resulting in the elimination of the contaminating tumor cells. These purified T cells were highly cytotoxic for FBL-3 cells in vitro and also possessed strong in vivo anti-tumor activity against FBL-3 cells in the adoptive transfer experiments. The present study demonstrates that short-term culture (2-3 weeks) in IL2 promotes the growth of T cells and augments their cytotoxic activity with the appropriate antigenic specificity. IL2 also promoted the selective growth of T cells isolated from tumor site and these T cells showed augmented in vitro and in vivo anti-tumor activity.     

In vitro sensitization to embryonic antigens

Lymphoid cells from virgin, untreated BALB/c mice cultured 5 days on 12- or 13-day syngeneic mouse embryo fibroblasts (MEF) or on cells from a syngeneic 3-methylcholanthrene-induced mouse sarcoma (1315) were cytotoxic for 1315 tumor target cells when tested with a microcytotoxicity assay. They did not kill embryo or skin fibroblast target cells. Lymphoid cells cultured with 11-, 14- or 17-day MEF were not cytotoxic for cells from the 1315 tumor, from embryo or from adult skin. Lymphoid cells from multiparous BALB/c mice cultured on 11-, 12- or 13-day MEF, on line 1315 sarcoma cells or on skin fibroblasts were cytotoxic for tumor target cells. MEF were killed by multipara lymphoid cells that had been cultured on 11-, 12- or 13-day MEF or on the 1315 tumor line. Multipara lymphoid cells cultured on 1315 cells or 12-day MEF were cytotoxic for adult skin fibroblasts, while multipara lymphoid cells cultured on 14- or 15-day MEF were not cytotoxic for any of the target cells. The data thus indicate that lymphoid cells can mediate both primary and secondary immune reaction in vitro to antigens shared by neoplastic and normal embryonic cells, and that the primary reactions appear to be more specific to putatively embryonic tumor antigen(s) than the secondary ones.     

Effects of presensitization in vivo on cell-mediated responses to embryonic antigens in vitro

Lymphoid cells specifically reactive with antigens shared by rat bowel carcinomas and mid-term embryo cells were generated by in vitro culture on monolayers of embryo cells. Spleen cells from WF females were cultured for 5 days on monolayers of syngeneic embryo cells or adult cells and assayed for cytotoxic activity on syngeneic embryo, bowel carcinoma, or adult fibroblast target cells in microcytotoxicity and 51Cr-release assays. After culture on embryo monolayers, spleen cells were cytotoxic for embryo and tumor but not for adult fibroblast target cells. Enhanced cytotoxicity was recorded when the spleen cells were cultured from females after interstrain (WF X BN) pregnancy rather than from virgin females. In contrast, previous intrastrain (WF X WF) pregnancy appeared to depress the generation of spleen cells cytotoxic for target cells bearing embryonic antigens.     

Macrophage arginase promotes tumor cell growth and suppresses nitric oxide-mediated tumor cytotoxicity

Macrophages use L-arginine to synthesize nitric oxide (NO) and polyamines through the inducible NO synthase (iNOS) and arginase, respectively. The released NO contributes to the tumoricidal activity of macrophages, whereas polyamines may promote the growth of tumor cells. Both the tumoricidal and growth-promoting activities from macrophages have been reported; however, the underlying mechanisms for switching between this dual function of macrophages remain unclear. Here, we test the hypothesis that arginase participates in the switching between the cytotoxic and growth-promoting activities of macrophages toward tumor cells. To alter arginase activity in macrophages, cells (murine macrophage cell line J774A.1) were transfected with the rat liver arginase gene or treated with an arginase inhibitor, L-norvaline. The effects of macrophage arginase activity on the growth-promoting and cytotoxic activities of macrophages toward breast tumor cells (ZR-75-1) were investigated in a coculture system. The results demonstrated that overexpression of arginase in macrophages enhanced L-ornithine and putrescine production and consequently promoted tumor cell proliferation. This proliferative effect was down-regulated by the arginase inhibitor L-norvaline. Furthermore, increases in arginase activity also attenuated NO production by the lipopolysaccharide-activated macrophages and thus reduced the cytotoxic effect on cocultured tumor cells. Inhibiting arginase activity by L-norvaline effectively reversed the suppression of NO-mediated tumor cytotoxicity. Together, these results suggest that arginase induction in macrophages can enhance tumor cell growth by providing them with polyamines and suppress tumor cytotoxicity by reducing NO production. It appears that L-arginine metabolism through the arginase and iNOS pathways in macrophages can have very different influences on the growth of nearby tumor cells depending on which pathway is prevailing.     

An intrinsic thymic epithelial abnormality is responsible for the spontaneous development of predominantly lymphocytic thymomas in BUF/Mna rats.

The nature of tumorigenesis of predominantly lymphocytic thymoma was examined using an animal model. Rats of the inbred BUF/Mna strain were found spontaneously to develop predominantly lymphocytic thymomas, histologically indistinguishable from their human counterparts, at an incidence of virtually 100%. Thymic rudiments of BUF/Mna rats grafted 17 months previously under the renal capsule of young athymic ACI/NMs-rnu/rnu rats also gave rise to similar lesions. The lymphocytes in the thymomas expressed T-cell antigens (rat Lyt-1 and Lyt-2.3), as in the normal case, and ACI rat specific antigen. When BUF/Mna rats of thymoma age were irradiated with a lethal dose of 12 Gy and then received a single injection of bone marrow cells (8 x 10(7)) from BALB/c-nu/nu mice, thymomas were re-formed three weeks later (in 2 of 5 rats) with the replacement lymphocytes expressing mouse Thy-1.2 antigen. These results indicate that an intrinsic thymic epithelial abnormality is responsible for the development of predominantly lymphocytic thymomas in BUF/Mna rats.     

An Intrinsic Thymic Epithelial Abnormality Is Responsible for the Spontaneous Development of Predominantly Lymphocytic Thymomas in BUF/Mna Rats

The nature of tumorigenesis of predominantly lymphocytic thymoma was examined using an animal model. Rats of the inbred BUF/Mna strain were found spontaneously to develop predominantly lymphocytic thymomas, histologically indistinguishable from their human counterparts, at an incidence of virtually 100%. Thymic rudiments of BUF/Mna rats grafted 17 months previously under the renal capsule of young athymic ACI/NMs- rnu/rnu rats also gave rise to similar lesions. The lymphocytes in the thymomas expressed T-cell antigens (rat Lyt-1 and Lyt-2.3), as in the normal case, and ACI rat specific antigen. When BUF/Mna rats of thymoma age were irradiated with a lethal dose of 12 Gy and then received a single injection of bone marrow cells (8 × 10⁷) from BALB/c- nu/nu mice, thymomas were re-formed three weeks later (in 2 of 5 rats) with the replacement lymphocytes expressing mouse Thy-1.2 antigen. These results indicate that an intrinsic thymic epithelial abnormality is responsible for the development of predominantly lymphocytic thymomas in BUF/Mna rats.     

Evidence for the production of nitric oxide by activated macrophages treated with the antitumor agents flavone-8-acetic acid and xanthenone-4-acetic acid

Activated peritoneal macrophages, obtained from mice pretreated with Bacillus Calmette-Guérin, after exposure in vitro to flavone-8-acetic acid (FAA; NSC 347512) at a concentration of 890 microM, produce nitrite (3.7 nmol/10(6) cells), as measured 20 h later by the Griess reaction. Stimulation of nitrite production was inhibited at least 90% by NG-monomethylarginine (125 microM), suggesting that nitrite was formed via nitric oxide as a product of arginine metabolism. Stimulation was only partially inhibited by dexamethasone (0.1 microM). The ability of xanthenone-4-acetic acid (XAA) and three of its analogues to stimulate nitrite production was also investigated. 5,6-Dimethyl-XAA stimulated nitrite production (12.6 nmol/10(6) cells) at an optimal concentration of 80 microM, 8-methyl-XAA was without effect, and XAA and 5-methyl-XAA showed intermediate activity. The optimal in vitro drug concentrations for stimulation by FAA, XAA, and active XAA analogues correlated with the optimal in vivo dose required for the induction of either hemorrhagic necrosis or growth delay of s.c. Colon 38 tumors. These results strongly imply that FAA and active XAA derivatives function as low molecular weight stimulators of nitric oxide formation in macrophages, possibly acting on the same differentiation pathway as do endotoxin and tumor necrosis factor alpha. We suggest that nitric oxide, which is known to be toxic to tumor cells, contributes to the cytotoxic action of FAA and its analogues.     

Chromosomal Mapping of Genetic Locus Associated with Thymus-size Enlargement in BUF/Mna Rats

The thymoma-prone rat of the BUF/Mna strain is a useful model for human thymoma. In this strain thymoma development is regulated by a single autosomal susceptible gene, Tsr-1. At pre-thymoma age, BUF/Mna rats have extremely large thyrauses, when compared to those of other strains of rats. Genetic studies in crosses between BUF/Mna rats with large thymuses and WKY/NCrj rats with small thymuses suggested the presence of a major autosomal gene, Ten-1 , which contributes to thymus enlargement in a backcross population. Linkage studies between Ten-1 and microsatellite markers in backcross rats of (WKY/NCrj×BUF/Mna)Fl×BUF/Mna have led to the localization of Ten-1 in chromosome 1. This result may provide an approach to clone Tsr-1 , which could be allelic to Ten-1.     

A single dominant susceptible gene determines spontaneous development of thymoma in BUF/Mna rat

Rats of the BUF/Mna strain developed spontaneous epithelial thymomas morphologically indistinguishable from human homologues at virtually 100% incidence. Segregation of thymoma development among crosses between BUF/Mna and ACI/NMs, which has 0% thymoma incidence, indicated that thymoma susceptibility was determined principally by a single autosomal dominant gene Tbm-1 (thymoma in BUF/Mna rats). In these crosses, another autosomal dominant or semidominant gene(s) contributed by ACI/NMs parents moderately reduced the thymoma incidence.     

Cytotoxic activity of guinea-pig lymph-node cells stimulated in vitro

Guinea-pig lymph-node cells (LNC) cultured for 5 days in medium containing fetal calf serum (FCS) were cytotoxic to various target cells. LNC cultured in medium supplemented with fresh, autologous guinea-pig serum (GPS) instead of FCS were not detectably cytotoxic unless agents that stimulate lymphocyte proliferation were added to the culture medium. The stimulating agents we studied were 2-mercaptoethanol (2-ME), syngeneic tumor cells, allogeneic peritoneal exudate cells (PEC) and the T-cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA). LNC cultured in the presence of these agents were cytotoxic to normal syngeneic fibroblasts and to syngeneic, allogeneic or xenogeneic tumor cells but not to PHA-induced lymphoblasts. Potentiation of cytotoxicity in vitro was accompanied by a marked proliferation of the cultured LNC; the combination of several stimulatory agents had an additive effect on the generation of cytotoxicity in culture.     

Nitric oxide induces oral squamous cell carcinoma cells apoptosis with p53 accumulation

Nitric oxide has been reported to have cytotoxic effects in several tumor cells. The objective of this study was to investigate the effects of exogenous nitric oxide on apopotosis in oral squamous cell carcinoma cells and to reveal its possible mechanism. Tca8113 cells were cultured with various concentrations of nitric oxide that were released from sodium nitroprusside (SNP). Nitrite/nitrate levels in the culture supernatant were determined using a commercial available nitric oxide kit. Cellular proliferation was determined by MTT assay. Apoptosis was detected by flow cytometry. Expression of inducible nitric oxide synthase (iNOS) was determined by immunocytochemistry. p53 expression was assessed by Western blot. SNP can release nitric oxide into the culture medium in a dose-dependent manner. Nitric oxide remarkably inhibits proliferation in a dose and time-dependent manners and lead to apoptosis of the Tca8113 cell. The p53 expression was elevated accompanying by the increased apoptotic cells. No difference of iNOS was found whether or not the cells were treated with SNP. Exogenous nitric oxide had an inhibitory effect on Tca8113 cells proliferation in a dose and time-dependent manners and possibly via p53 dependent apoptosis pathway. Exogenous nitric oxide had no significant effect on cellular iNOS protein.     

In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L.

Saffron corms contain a proteoglycan that is highly cytotoxic on human tumor cells. The present work was undertaken to study the possible immunomodulatory and anti-invasive properties of this compound. Non-cytotoxic concentrations of this glycoconjugate promoted significant macrophage activation, detected by the release of nitric oxide. A rapid activation of protein kinase C and NF-kappaB was obtained after proteoglycan treatment, which could explain the induction of nitric oxide synthase. Proteoglycan concentrations ranging from 10-1000 ng/ml specifically promoted apoptosis of macrophages, probably triggered by their activation. This molecule did not inhibit in vitro migration or invasion of human tumor cells. Altogether these results support a plausible immuno-modulating activity for this saffron Crocus compound.     

Human Alveolar Macrophages Augment Natural Killer Cell Stimulatory Factor (Interleukin-12)-inducible Killer Activity from Autologous Blood Lymphocytes

Interleukin-12 (IL-12), also known as natural killer cell stimulatory factor (NKSF), was found to induce cytotoxic activity from human blood T cells and NK cells. The present study was undertaken to examine the effect of human alveolar macrophages (AM) on induction by IL-12 cytotoxic cells from blood lymphocytes. AM were obtained by bronchoalveolar lavage from healthy donors. Highly purified lymphocytes (>99%) and monocytes (>90%) were also isolated by centrifugal elutriation from peripheral blood of the same donors. Cytotoxicity of lymphocytes was measured by 4-h ⁵¹Cr release assay. IL-12 stimulated blood lymphocytes to produce interferon γ (IFNγ) and tumor necrosis factor α (TNFα), and this effect was augmented by co-cultivation with monocytes or AM. AM-upregulated induction of cytotoxic lymphocytes was stimulated with IL-12, and this effect was significantly abrogated by addition of antibodies against IFNγ and TNFα. Induction by IL-12 of IFNγ production and cytotoxic activity of CD8⁺ cells was also augmented by co-cultivation with monocytes or AM. AM were more effective than monocytes in augmenting the cytotoxic activity of IL-12-stimulated lymphocytes and CD8⁺ cells. These observations suggest that in situ induction of IL-12-stimulated cytotoxic cells in the lung may be regulated by complex cytokine networks, depending on participation of monocytes and alveolar macrophages.     

Destruction of experimental malignant melanoma by mediators of cellular immunity.

Listeria monocytogenes (LM) in admixture with B-16 melanoma suppresses local tumor development in syngeneic C57BL/6 mice. In vitro, LM-immune peritoneal and splenic cells are cytotoxic to B-16. Induction of cell-mediated immunity to LM antigens are required for the killing effect, since effector cells from LM-"immune" athymic nude mice are unable to kill tumor cells in vitro. Further, elimination of macrophages by a specific antiserum plus complement abrogates the cytotoxic effect of peritoneal cells. Peritoneal or splenic adherent or nonadherent cells are not cytotoxic, whereas combination of these two cell populations in the presence of the specific antigen can kill the B-16 target cells. A factor, probably lymphotoxin, released by the intact effector cells in the culture fluid mediates tumor cell destruction in vitro. Production of this factor requires cooperation of macrophages with specifically sensitized thymus-derived cells.     

Possible single dosage effects of the nude gene: suppression of spontaneous development of thymoma and nephropathy in BUF/Mna-rnu/+ rats

Single dosage effects of the rat nude gene (rnu) on spontaneous development of epithelial thymoma, muscle atrophy and nephrotic syndrome were studied by comparing littermates of rnu /+ and +/+ rats on a high thymoma strain, BUF/Mna, background. Heterozygous rnu/+ rats had a significantly smaller thymus than the +/+ littermates at 6 weeks of age. The incidence of thymoma at 12 months of age was extremely low in the female rnu/+ rats (3%) as compared with that of the +/+ rats (94%). Development of the nephrotic syndrome but not of the muscle atrophy was also suppressed in the heterozygotes. The results suggest that a recessive mutant gene, rnu, in a single dosage, interfered with critical steps of the disease processes of the thymoma and nephrotic syndrome in BUF/Mna-background rats.