Nitric oxide inhibits proliferation but increases life-span of T lymphocytes in tumour-bearing rats.

Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression.

IL-13 restores an in vitro specific cytolytic response of spleen cells from tumor-bearing rats.

In a model of colon cancer, spleen cells from tumor-bearing rats are neither cytotoxic nor proliferative in vitro in the presence of tumor cells. Interleukin-13 (IL-13) induced an in vitro cytolytic activity of spleen cells from tumor-bearing rats in response to the tumor they bore, but had no effect on spleen cells from normal rats. This cytotoxic response was dependent on both adherent and non- adherent cells, involving both an antigen-presenting activity that was enhanced by IL-13 and a cytolytic activity of lymphocytes. So, IL-13 reversed the tumor-induced immunosuppression.

Interleukin-8 antitumour effect is associated with a local infiltration but not with a systemic activation of T lymphocytes.

In a model of colon cancer in rats (peritoneal carcinomatosis), IL-8 was found to have a highly reproducible antitumoural effect. During IL-8-induced tumour regression the infiltration of nodules by CD4+ T lymphocytes was enhanced. However, splenic lymphocytes did not proliferate in response to tumour cells in vitro. IL-8 antitumour effect was associated with a local but not with a systemic activation of T lymphocytes.

CD4+ T cells recovered from a mixed immune lymphocyte-tumor cell culture induce thymidine incorporation by naive rat lymphocytes in response to tumor cells.

Spleen cells from tumor-immune rats incorporate thymidine when co-cultured for 4 days with syngeneic cancer cells. Non-adherent cells, recovered from a 7-day mixed culture with cancer cells, had lost their capacity for incorporating thymidine when exposed again to the same tumor cells; however, in these conditions, the non-adherent cells induce thymidine incorporation by fresh naive spleen cells. This response is restricted to tumor lines which originate from the same tumor as the cells used for immunization and is due to memory and/or activated CD45RC- CD4+ T cells. Our results indicate that tumor-specific T cells maintain their capacity to respond to tumor antigens, even after an extended culture time with tumor cells. In another study, in the same conditions, spleen cells from normal or tumor-bearing rats did not evoke significant responses.

Interleukin-8 has antitumor effects in the rat which are not associated with polymorphonuclear leukocyte cytotoxicity.

The antitumor effect of lipopolysaccharides (LPS) has been observed in several experimental models and is likely to be mediated by macrophages. Stimulation of macrophages with LPS results in the release of several cytokines, including tumor necrosis factor, interleukin-1 and neutrophil-activating peptide-1/interleukin-8 (IL-8), which activates polymorphonuclear leukocytes (PMN) in vitro. Since PMN have an antitumor activity, we tested the in vivo effect of IL-8 on the growth of peritoneal carcinomatoses induced by PROb colon cancer cells in syngeneic rats. IL-8 induced a significant regression of tumors measuring 1-5 mm, and a complete regression was observed in 8 out of 40 rats in four independent experiments. IL-8 was not directly cytotoxic in vitro for tumor cells and was effective in vivo in a narrow range of doses. IL-8 had a significant chemotactic effect for peritoneal PMN in both normal and tumor-bearing rats. PMN taken from the peritoneum of tumor-bearing rats during IL-8 treatment had the same cytotoxic activity against PROb tumor cells as PMN from untreated control rats. Microscopic examinations of tumors during the treatment showed poor infiltrating by PMN. We conclude that the antitumor activity of IL-8 in this model is not mediated by PMN cytotoxicity.

In vivo and in vitro reactivity of rat spleen cells against regressor and progressor colon-cancer cell variants.

Previous reports demonstrated that progressor and regressor tumor-cell variants isolated from the same colon carcinoma chemically induced in a BD-IX rat differed in their capacity to induce an immune response. The present study was aimed at analyzing the characteristics of the responses to the regressor REGb and progressor PROb clones. Spleen cells from rats bearing early REGb tumors neutralized PROb cell tumorigenicity in a Winn-type local transfer assay, but responded occasionally to REGb and PROb cells in vitro. However, spleen cells from rats immunized by several injections of REGb and PROb cells strongly proliferated when cultured with PROb or REGb cells. This response was selective for the cell lines generated from the individual tumor at the origin of PROb and REGb lines, was dependent on CD4+ spleen cells, and was partially inhibited by an antibody against major histocompatibility complex class-II molecules. Although PROb cells shared tumor-rejection antigen(s) with REGb cells, splenocytes from PROb tumor-bearing rats did not neutralize PROb-cell tumorigenicity in vivo, nor did they proliferate when cultured with PROb or REGb cells in vitro. The unresponsiveness of spleen cells from PROb tumor-bearing rats was not due to the presence of immune suppressive cells, and a defect of antigen-presenting cells was shown to be unlikely. This unresponsiveness was limited to a lymphocyte subpopulation, since spleen cells from tumor-bearing rats responded normally to stimulation by PHA or allogeneic lymphocytes. These results strongly suggest that unresponsiveness of spleen cells from tumor-bearing rats is due to a tumor-specific anergy of the lymphocyte clones able to respond to tumor-associated antigens.

Mechanism of the cytostasis mediated by blood unstimulated neutrophils on syngeneic rat colon cancer cells.

Non-activated polymorphonuclear neutrophils (PMNs) isolated from the blood of non-stimulated rats were spontaneously and selectively cytostatic in vitro against a syngeneic colon cancer cell line. In this experimental model, the mechanism of PMN-mediated cytostasis did not depend on oxidative metabolites, but involved soluble factor(s), possibly granule proteases.

Influence of the injection site on the tumorigenicity of a cloned colon tumor cell line in the rat.

The cloned DHD/K12/TSb line obtained from a chemically-induced rat colon carcinoma, presents tumors which always regress when injected subcutaneously to the syngeneic animal. This study reports that an intraperitoneal injection of the DHD/K12/TSb cells induces a progressive carcinomatosis in a high proportion of the syngeneic rats. This result underlines the effect that the local environment has on tumorigenicity.

Non-activated rat neutrophils kill syngeneic colon tumor cells by the release of a low molecular weight factor.

The mechanisms of cancer cell destruction by unelicited peripheral blood neutrophils has never been reported in a syngeneic model. We demonstrated that in vitro, unelicited polymorphonuclear neutrophils isolated from rat blood were toxic against syngeneic colon cancer cells. The tumor cell lysis was not due to oxygen metabolites released by PMNs, but was due to a cytolytic factor. This factor was spontaneously secreted by PMNs, was heat-stable and had a low molecular weight (less than 10 kD). Its partial inhibition by chymotrypsin and/or chymotrypsin-like proteases suggested a peptidic structure of this factor.

Involvement of effector cells in the treatment with endotoxins of peritoneal carcinomatosis induced by colon tumour cells.

Peritoneal carcinomatoses, an ordinary way for human colon carcinoma to spread, are incurable. When rat peritoneal carcinomatoses of colon origin were treated with endotoxins i.p. administered 3 days after the tumour cell injection, 70% of the BDIX rats were alive 30 weeks after the PROb tumour cell injection whereas all the untreated rats died of their tumour within 14 weeks. The study of the effector cells involved in the antitumour effect of endotoxins suggests that T lymphocytes are required for this effect. The endotoxin effect is local and is not reflected by the cytolytic activity of peritoneal cells.

Comparative effect of rat and fetal calf serum on measurement of the natural tumoricidal activity of rat lymphocytes, macrophages and polymorphonuclear cells.

The effect of rat serum versus fetal calf serum on the in vitro natural cytolytic activity of rat lymphocytes, macrophages and polymorphonuclear cells against syngeneic tumour cells was compared. The cytolysis level mediated by the three varieties of effector cells was lower when rat serum was used instead of fetal calf serum to supplement the culture medium. This could explain in part the discrepancies found between in vitro and in vivo studies.

Correlation between the synergistic effect of liposomes and endotoxins on the activation of macrophage tumoricidal activity and the effect of liposomes on the rough endoplasmic reticulum of macrophages.

Treatment of resident peritoneal macrophages of rats with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV) potentiated their activation for tumor cell lysis by endotoxins. The fluorescence polarization of diphenylhexatriene (DPH) embedded in rough endoplasmic reticulum membranes isolated from DPPC SUV-treated macrophages was enhanced. The average fluorescence lifetime of DPH and the rotational correlation time deduced from anisotropy decay were unchanged, whereas the residual anisotropy and hence the order parameter were increased. The measurement of the fluorescence anisotropy of DPH as a function of the temperature showed a phase transition. No phase transition was observed in the rough endoplasmic reticulum membranes of macrophages either treated or not treated with cholesterol/DPPC SUV (1/1; mol/mol). The synergistic effect of DPPC SUV on the tumoricidal activity of macrophages induced by endotoxins appears to be correlated with the changes in the properties of the rough endoplasmic reticulum membranes. Both effects were transient; they had the same kinetics of induction and reversion, and they were both inhibited by cholesterol.

In vivo and in vitro effects of fish-containing diets on colon tumour cells in rats.

Polyunsaturated n-3 fatty acids, abundant in sea fish, can inhibit the growth of chemoinduced or transplanted mammary tumours in the rat. Since mammary and colonic cancers have both been linked to a high fat consumption, we studied the effect of 2 diets moderately (7% fish meal) or strongly (9% fish oil) enriched in fish fatty acids on the growth of colon cancer cells subcutaneously inoculated into syngeneic rats. The diets had no effect on the in vivo tumor growth and on the in vitro tumouricidal activity of peritoneal macrophages or splenic lymphocytes.

Regression induced by lentinan, of peritoneal carcinomatoses in a model of colon cancer in rat.

Lentinan has been tested in a model of colon cancer in rats. Peritoneal carcinomatoses were induced in BDIX rats by i.p. injections of syngeneic cells isolated from a colon carcinoma, and established in a permanent cell line. The treatment consisted of five i.p. injections, 2 days apart, of 2 mg lentinan/kg at a concentration of 200 micrograms/ml. This was started on day 14 after tumor cell injection, when the rats bore numerous nodules of 1-5 mm. Lentinan significantly inhibited the growth of carcinomatoses. Eleven out of the 20 rats treated with the best lentinan therapy were tumor free at autopsy on day 42. Lentinan significantly increased the life span of carcinomatous rats. The half life following tumor cell injection was 42 days in the control and 70 days in the treated group. Four out of 10 treated rats were still alive on day 210. They were tumor free at autopsy, whereas all the controls died between the 40th and the 70th day. The effectiveness of lentinan was dependent on the number and frequency of the injections. A dose effect was obtained and a strong influence of the concentration was shown.

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells.

DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h 51Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants.

In vitro natural killer activity against progressive and regressive variants of a rat colon adenocarcinoma. Effect of treatments with anti-asialo GM1 plus complement.

In a previous work, a cell line (DHD/K12) was established from a colon adenocarcinoma induced in a BDIX rat by 1,2-dimethylhydrazine. From this line, two cloned sublines, PROb and REGb, were then isolated. When subcutaneously inoculated into syngeneic rats, PROb cells yield progressive tumors, whereas REGb cells yield tumors which regress. In this study, in a 16-h 51Cr release assay, natural cytotoxicity mediated by BDIX splenic nonadherent lymphoid cells (NK cells) was shown to be much higher against REGb cells than against PROb cells. Whatever the target cells, NK cytotoxicity was always higher when the effector cells were obtained from males rather than from females. Treatment of BDIX splenic lymphocytes by anti-asGM1 serum plus complement revealed that both anti-asGM1 sensitive and non-sensitive NK cells exist. The activity of anti-asGM1 non-sensitive NK cells appeared to be minor and to be detected only when the level of cytotoxicity before treatment was sufficiently high. The difference between PROb and REGb tumor growth appears to be linked, at least in part, to a higher sensitivity of REGb cells to NK cells and especially to anti-asGM1-sensitive NK cells.

Treatment with endotoxins of peritoneal carcinomatosis induced by colon tumor cells in the rat.

Peritoneal carcinomatosis, a common spreading of human colon carcinoma, can be obtained by intraperitoneal injection of colon tumor cells in rats. When BDIX rats are injected with 10(6) syngeneic tumor cells, isolated and cloned from a chemically induced colon carcinoma, they die within 2-3 months with solid peritoneal tumors and hemorrhagic ascites. Repeated intraperitoneal injections of 20 micrograms endotoxins (Escherichia coli W0128:B12) from day 3 after tumor cell challenge inhibited tumor growth. This effect was long-lasting since 7 out of 10 treated rats were still alive and tumor-free 6 months after tumor cell challenge. When the endotoxins were administered from day 15 after the tumor cell challenge, in rats with established tumors visible with the naked eye, the survival times were significantly increased, and 6 out of 30 treated rats were still alive and tumor-free 6 months after tumor cell challenge. The optimum effect was obtained with 5 repeated injections. The different frequencies of injection tested, i.e. 1, 3 or 5 days apart were equally effective. Endotoxins were ineffective when administered intravenously. No side effect was observed.

Lack of effect of a high polyunsaturated fat diet on the growth of transplantable colon tumors and on the cytolytic activity of macrophages in rats.

The effect of a high dietary level of polyunsaturated fat was tested on the growth of three different colon cancer cell lines injected subcutaneously into syngeneic rats. This effect was also tested on the in vitro cytolytic activity of resident peritoneal macrophages, natural or endotoxin and/or indomethacin-modulated. A 12% corn oil dietary supplement had no effect in any of the cases tested.

[In vitro tumoricidal activity of peritoneal macrophages and splenic lymphocytes of rats under diet with or without fish]. / Activité tumoricide in vitro de macrophages péritonéaux et de lymphocytes spléniques de rats soumis à un régime avec ou sans poisson.

Rats were fed with or without a diet supplemented with fish meal over a 4-week period. The moderate fish fatty acids intake had no influence on the tumoricidal activity of peritoneal and splenic lymphocytes.