Danièle Reisser

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 rat. 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.

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.

SummaryDHD/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.

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.

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.

Role of macrophage in the defense against intestinal cancers

The capability of activated macrophages to kill tumor cells in vitro is now well documented. The tumoricidal activation of macrophages against intestinal tumor cells by different agents is described and the main hypothesis on the mechanisms of tumor cell killing in vitro are discussed. These in vitro results suggest that the macrophage can constitute an efficient effector cell in the defense against intestinal tumors. The distribution and ratio of macrophages in normal intestine and intestinal tumors is described. At the moment, potent activators of macrophages studied in vivo on experimental and human intestinal tumors give poor results or even enhance the growth of tumors. Macrophages may also interfere with the specific immune response in two directions by enhancing the immune response or decreasing it by elaboration of mediators such as prostaglandins.

Differences in the Tumoricidal Activation of Rat and Mouse Macrophages by Endotoxins

Conventional and specific pathogen-free rat resident peritoneal macrophages were lytic to tumor cells in the presence of endotoxins even when not elicited or not stimulated in vivo or in vitro. In contrast, conventional mouse resident peritoneal macrophages were not cytolytic in the presence of endotoxins. The induction by endotoxins of rat macrophage-mediated cytolysis was only obtained after the binding of tumor cells by macrophages. Rat resident peritoneal macrophages bound faster and stronger to tumor cells than mouse resident peritoneal macrophages. These differences in binding could explain the species differences in the tumoricidal response to endotoxins.

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

SummaryTreatment 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.

Lipid A OM-174 increases the natural killer activity of peripheral blood cells from breast cancer patients

Natural killer (NK) cells have been implicated in immune surveillance against tumors, but their activity is often impaired in cancer patients. Lipopolysaccharides (LPSs) are known to enhance NK activity, but their toxicity prevents their use in humans. OM-174, a purified 3,3′-O-deacylated Escherichia coli lipid A, is able to cure established tumors in the rat with little toxicity. We tested the ability of OM-174 to stimulate in vitro the lytic potential of peripheral blood cells from patients with non-metastatic breast tumor (n = 48). After 24 h pre-incubation of nylon—non-adherent cells with OM-174, increased antitumor activity was observed toward the NK-sensitive target cells K562 but not the lymphokine-activated killer (LAK)-sensitive target cells Daudi. Both NK cells and a non-NK population are involved in this cytotoxicity. So, although the basal NK activity of blood cells was lower in tumor-bearing patients than in healthy donors, OM-174 may increase their toxicity toward cancer cells.

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

SummaryThe 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.

Strain dependence of rat macrophage natural tumoricidal capacity and its stimulation by endotoxins

Without known stimulation in vivo and in vitro, resident peritoneal macrophages from 5 conventional or specific pathogen-free (SPF) rat strains [Hairless (H), BDIX, Wistar (W), Sprague-Dawley (SD) and Long-Evans (LE)] exhibited an in vitro strain-dependent cytolysis against DHD-K12/TS cancer cells. This natural cytolysis was also observed when polymyxin B was added to the culture medium. The percentage of natural cytolysis varied from one rat to another but was significantly different according to the strain. In the presence of 10 micrograms endotoxin/ml, macrophages from BDIX, W, SD and LE rats were always cytolytic, whilst those of H rats were irregularly cytolytic. Endotoxins induced or increased macrophage-mediated cytolysis from H, BDIX, W and SD rats, but they were without effect for LE rats. The endotoxin effect depended on the level of natural cytolysis. In contrast to mouse resident peritoneal macrophages, which were not naturally cytolytic and not activated in vitro by endotoxins, these results show that rat resident peritoneal macrophages can be naturally cytolytic. This cytolysis can be enhanced by endotoxins as the sole in vitro stimulus. Rat macrophage natural cytolytic activity is strain-dependent.