Characterization of a cytotoxic factor induced in mouse peritoneal fluid by OK-432

A cytotoxic factor (peritoneal cytotoxic factor, PCF) was strongly induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with OK-432. In order to clarify characteristics of PCF, physicochemical and immunological studies were conducted. When incubated with LPS, the macrophages from mice primed with OK-432 induced PCF whereas the lymphocytes did not. These results indicate that PCF is different from lymphotoxin. PCF appears to be quite similar to tumor necrosis factor (TNF) in the serum for the following reasons: The two factors are similar in the mode of cytotoxic action in vitro; both factors have a tumor necrotizing effect when injected into tumor bearing mice; both are produced from macrophages; they are similar in physicochemical characteristics; and the cytotoxic activity of PCF is totally abolished by anti-TNF serum.     

Therapeutic Effect of Endogenous Tumor Necrosis Factor on Ascites Meth A Sarcoma

The therapeutic effect of endogenous tumor necrosis factor (TNF) on Meth A ascites fibrosarcoma in mice was investigated. Serum and peritoneal fluid from tumor bearing mice treated with OK-432 and LPS were cytotoxic to tumor cells in vitro. The peak of cytotoxicity in both the serum and peritoneal fluid was found in the fraction corresponding to a molecular weight of approximately 54,000-56,000 on HPLC and the PI was found to be 4.9-5.1 by is oelectric focusing. These results are consistent with previously reported findings on TNF, and indicate that endogenous TNF has a satisfactory life-prolonging effect.

The tumor necrosis factor (TNF) is considered to be one of the clinically most promising anti-cancer cytokines because of its potent and very specific antitumor effect on target cells (Carswell, Old, Kassel, Green, Fiore & Williamson, 1975; Matthews & Watkins, 1978; Niitsu, Watanabe & Urushizaki, 1984).

TNF as an anti-cancer cytokine for the treatment of cancer may be applied in one of the two following ways: 1) by administration of purified TNF or 2) by endogenously inducing TNF in cancer bearing individuals. The antitumor effects of TNF administered exogenously have been examined using crude preparations or serum containings TMF (tumor necrosis serun, TNS) (Carswell et al., 1975; Watanabe, Niitsu, Sone, Neda, Ishigaki & Urushizaki, 1984).

In a previous paper we reported that mice primed with OK-432 and challenged with endotoxin produced a soluble cytotoxic factor in peritoneal fluids (Yamamoto, Nagamuta, Usami, Sugawara, Watanabe, Niitsu & Urushizaki, 1985; Nagamuta, Yamamoto, Usami, Sugawara, Watanabe, Niitsu & Urushizaki, 1985).

This peritoneal cytotoxic factor (PCF) had cytostatic and/or cytotoxic effect not only on mouse tumor cell lines but also on human tumor cell lines without species specificity. Normal cell lines were not affected. Here we report the endogenous production of TNF in tumor bearing mice and its antitumor effects.     

Therapeutic Effect of Ok-432 Induced Endogenous Tnf on Tumor Bearing Mice and Cancer Patients

The therapeutic effect of OK-432 induced endogenous TNF on tumor bearing mice and cancer patients was investigated.

OK-432 (10 KE/mouse) was administered intraperitoneally to Balb/c mice 7 days prior to the transplantation of Meth A cells (1×10⁶/mouse) into the abdominal cavity. And at day 1 of tumor inoculation, 1 KE/mouse of OK-432 was administered intraperitoneally.

The significant prolongation of life span was observed in these mice.

On the basis of these observation, therapeutic effect of endogenous TNF on cancer patients was clinically evaluated. OK-432 was administered intraperitoneally or intrapleurally to cancer patients with peritonitis carcinomatosa or pleuritis carcinomatosa 4 times (10KE each) every other day and 50KE of OK-432 was readministered with the interval of 7 days.

An appreciable activity of TNF was detected in peritoneal fluids or pleural effusion, and the significant decreasing of these fluids was observed. It is therefore concluded that these therapeutic approach may well be taken into account in treatment of cancer.     

Enhanced adherence activity of OK-432-induced peritoneal neutrophils to tumor cells correlates to their increased expression of CD11b/CD18.

We previously found that activated peritoneal neutrophils adhered to tumor cells and destroyed them in the cancer ascites of patients who had received intraperitoneal (ip) OK-432 injection therapy. Since tight adhesion to the tumor cell is essential for effective neutrophil-mediated tumor cell destruction, we investigated the mechanism of peritoneal neutrophil adhesion to tumor cells, using a microplate adhesion assay. An in vitro study demonstrated that the adherence activity of the peritoneal neutrophils of patients who received OK-432 injection therapy to tumor cells increased greatly compared to that of blood neutrophils. The expression of the adhesion molecules (CD11a,b,c/CD18) of peritoneal neutrophils, which was determined by an immunofluorescence study, was about four times as much in CD11b and twice as much in CD11c and CD18 compared to that in blood neutrophils. In vitro OK-432 stimulation of normal blood neutrophils increased neither the adhesion to PLC nor the CD11b expression. The enhanced adherence activity of peritoneal neutrophils to tumor cells was significantly inhibited by pretreatment of the neutrophils with anti-CD11b and anti-CD18 monoclonal antibodies (mAb), but not by pretreatment with anti CD11a or anti-CD11c mAb. These results indicated that the increased adhesiveness of OK-432-induced peritoneal neutrophils to tumor cells was due to the enhanced expression of CD11b/CD18. We concluded that CD11b/CD18 molecules on OK-432-induced peritoneal neutrophils play a crucial role in the neutrophil adherence activity against tumor cells, and these results are the first demonstration in the field of human neutrophil function.     

In vitro activation of murine bone marrow-derived macrophages with cisplatin and mitomycin-C

Peritoneal macrophages from BALB/c mice after treatment for 24 h in vitro with cisplatin, lipopolysaccharide (LPS) or mitomycin-C were rendered significantly cytotoxic against L-929 tumor target cells. In a similar experiment none of these agents could induce tumoricidal activity of fresh non-adherent bone marrow cells (NABMC). NABMC when incubated in medium alone or in medium containing L-929 culture medium (L-929 CM), a form of macrophage colony stimulating factor (M-CSF), for three days matured to macrophages which were positive for non-specific esterase staining. These bone marrow-derived macrophages cultured with medium alone did not respond to cisplatin. LPS or mitomycin-C for induction of tumoricidal activity whereas bone marrow derived macrophages with that were incubated with L-929 CM showed also significantly enhanced cytotoxicity after treatment with cisplatin, LPS and mitomycin-C. Culturing of NABMC with L-929 CM significantly enhanced cell survival as compared to the cells incubated in medium alone. These results suggest that bone marrow cells not only mature in L-929 CM but also are primed by L-929 CM for induction of tumoricidal activity.     

Neutrophil-mediated tumor cell destruction in cancer ascites. II. A OK-432 attracts killer neutrophils through activation of complement C5

When a streptococcal preparation, OK-432, was administered intraperitoneally to patients with malignant ascites, the number of neutrophils with cytotoxic activity against tumor cells was increased in the peritoneal cavity immediately after the OK-432 injection. In order to investigate the underlying mechanisms of such neutrophil accumulation, a possible neutrophil chemotactic activity in ascitic fluid was assayed by a modified Boyden method. The chemotactic activity for neutrophils was found significantly higher 6 hr after the OK-432 injection. OK-432 along had no direct chemotactic activity for neutrophils. The chemotactic activity was generated in vitro when ascitic fluid from patients without OK-432 treatment was incubated with OK-432 for 30 min at 37 degrees C. However, preheating of the fluid at 56 degrees C for 30 min or the addition of EDTA to the fluid resulted in the failure of generation of the chemotactic activity after the incubation with OK-432. The addition of EGTA did not show a significant effect. The chemotactic activity in ascitic fluid was found near cytochrome c marker (MW 12,400 D), when fractionated by Sephadex G-200 gel chromatography. The chemotactic activity was heat stable, nondialyzable, and neutralized completely with anti-human complement C5 antibodies. These results suggest that C5a generated via the alternative pathway activated by OK-432 may be responsible for the infiltration of killer neutrophils in the peritoneal cavity in patients with malignant ascites when they are treated by the intraperitoneal injection of OK-432.     

PSK and OK-432-Induced Immunomodulation of Inducible Nitric Oxide (NO) Synthase Gene Expression in Mouse Peritoneal Polymorphonuclear Leukocytes and NO-Mediated Cytotoxicity

Abstract

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

PSK and OK-432-induced immunomodulation of inducible nitric oxide (NO) synthase gene expression in mouse peritoneal polymorphonuclear leukocytes and NO-mediated cytotoxicity

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

Effects of Inactivated Streptococci (OK-432) on macrophage functions in Mice

The effects of inactivated streptococci (OK-432) on murine macrophage functions were investigated, in viva treatment of peritoneal macrophages with OK-432 augmented the direct cytotoxic activity against TU5 tumor cells in a 48 h tritiated thymidine release assay. OK-432 also stimulated the rapid (6 h, ⁵¹Cr release) macrophage-mediated killing of Actinomycin D-sensitized WEHI 164 sarcoma cells. Moreover, the expression of la antigens on peritoneal macrophages was found to be greatly enhanced after in vivo treatment with OK-432. The immunomodulatory effects of OK-432 on macrophages functions may contribute to the antitumor activity of inactivated streptococci.     

Effects of Inactivated Streptococci (OK-432) on macrophage functions in Mice

Abstract

The effects of inactivated streptococci (OK-432) on murine macrophage functions were investigated, in viva treatment of peritoneal macrophages with OK-432 augmented the direct cytotoxic activity against TU5 tumor cells in a 48 h tritiated thymidine release assay. OK-432 also stimulated the rapid (6 h, ⁵¹Cr release) macrophage-mediated killing of Actinomycin D-sensitized WEHI 164 sarcoma cells. Moreover, the expression of la antigens on peritoneal macrophages was found to be greatly enhanced after in vivo treatment with OK-432. The immunomodulatory effects of OK-432 on macrophages functions may contribute to the antitumor activity of inactivated streptococci.     

Tumor necrosis factor and macrophage activation are important in clearance of Nocardia brasiliensis from the livers and spleens of mice.

The roles of tumor necrosis factor (TNF) and macrophage activation in clearance of Nocardia brasiliensis from BALB/c mouse livers and spleens were evaluated. TNF activity was detectable in sera from animals at all stages of infection. Treatment of infected mice with an antiserum against TNF significantly enhanced the experimental infection as judged by enumeration of CFU in the spleens and livers of infected mice. In another set of experiments, a population of activated macrophages from the peritoneal cavities of N. brasiliensis-infected mice was studied by using a cytostatic assay. The observed cytotoxic activity of these activated macrophages against L929 cells was mediated by TNF, since this activity was inhibited by anti-TNF antiserum treatment. The level of TNF activity generated in vitro in the presence of lipopolysaccharide (LPS) by peritoneal macrophages from infected mice was higher than that of adherent peritoneal cells obtained from normal mice after challenge with LPS. When the nocardiacidal activity of peritoneal cells from N. brasiliensis-infected mice was estimated in vitro, a significant decrease in the number of CFU recovered was observed. Moreover, nocardiacidal activity of peritoneal cells obtained from N. brasiliensis-infected mice previously treated with anti-TNF antiserum was significantly reduced compared with the activity of cells obtained from infected mice previously treated with normal rabbit serum and that of cells from uninfected mice. These data suggest a role for TNF in resistance to N. brasiliensis infection.     

Augmentation of interleukin 1 and interleukin 2 production by OK-432

Intraperitoneal (i.p.) administration of OK-432 augmented both interleukin 1 (IL-1) and interleukin 2 (IL-2) production to the rechallenge of OK-432 in vitro. Peritoneal exudate cells (PEC) of mice 8 days after i.p. injection with OK-432 (1 KE/mouse) showed maximum IL-1 production to the restimulation with OK-432 in vitro. OK-432-induced IL-1 was consisted of three molecular weight species (two major peaks: 85 K and 15 K daltons and one minor peak: 67 K daltons) on Sephadex G-100 chromatography. Splenocytes of mice 4 days after i.p. injection with OK-432 (1 KE/mouse) demonstrated maximum IL-2 production to the in vitro rechallenge of OK-432, however, in vivo OK-432 administration failed to enhance ConA-induced IL-2 production in vitro. From gel filtration analysis, OK-432 induced IL-2 had an unique molecular weight (approximately 70 K daltons). From these results, OK-432-induced augmentation of cellular immunity against tumor cells might be due to the activation of so-called lymphokine cascade reaction mediated by IL-1 and IL-2.     

Effect of immunostimulants and antitumor agents on tumor necrosis factor (TNF) production

OK-432, a lyophilized preparation of Streptococcus pyogenes, showed a priming activity for TNF production in mice, associated with an increase of spleen weight. PSK, a protein-bound polysaccharide preparation from Coriolus versicolor, did not show such activity. Both OK-432 and PSK potentiated the TNF production in mice primed with Corynebacterium parvum (CP) and challenged with Escherichia coli endotoxin (LPS). Cytotoxic antitumor agents of 5-fluorouracil (5-FU), cyclophosphamide (CY) and bleomycin (BLM) suppressed TNF production in mice primed with CP and challenged with LPS. TNF production suppressed by 5-FU, CY and BLM was partially restored by the combined treatment with OK-432 or PSK. These results suggest that the administration of cytotoxic antitumor agents suppresses the intrinsic TNF production in cancer patients, and the combined use of immunostimulants such as OK-432 and PSK is advantageous in restoring TNF production suppressed by cytotoxic antitumor agents.     

Augmentation of natural killer (NK) cell activity by a streptococcal preparation,OK-432, in patients with malignant tumors

Recently, a streptococcal preparation, OK-432 has been used successfully as an immunopotentiator for immunotherapy in patients with malignant tumors in Japan. In this paper, we report that the administration of OK-432 augments the cytotoxic activity of peripheral blood lymphoid cells against a natural killer (NK) cell-sensitive erythroleukemic cell line, K562, in tumor patients. In patients before or after surgery, sufficient amounts of OK-432 strongly augmented the cytotoxic activity within 3 days after the initial administration of OK-432. Thereafter the levels of cytotoxicity declined rapidly. The administration of a lower dose of OK-432 gave a lower increase in cytotoxicity. Enhanced cytotoxicity occurred with the reintroduction of OK-432 but remained at lower levels of activity. Characterization and fractionation of OK-432-induced effector cells revealed that the augmented cytotoxicity seemed to be carried mainly by NK cells. A low titer of interferon was detected in 3 of 10 patients within 72 hr after the first inoculation of the agent. Furthermore, we discuss the potency of OK-432 for the induction of interferon in detail.     

Treatment of cancer ascites by intraperitoneal administration of a streptococcal preparation OK-432 with fresh human complement--role of complement-derived chemotactic factor to neutrophils

The role of complement in the polymorphonuclear leukocyte (PMN)-mediated tumor cell destruction in cancer ascites was investigated in relation to a streptococcal preparation OK-432, a so-called biological response modifier. Incubation of OK-432 with fresh human serum at 37 degrees C for 60 min resulted in the generation of C3a and C5a chemotactic factors. Intraperitoneal (i.p.) injection of the mixture to a patient with cancer ascites revealed an accumulation of PMNs in the ascitic fluid for a longer period with a rapid reduction of the ascitic fluid, than an intraperitoneal injection of OK-432 alone examined in the same patient. PMNs were found to invade clusters of the tumor cells and then form rosettes followed by the destruction of tumor cells. These findings induced by OK-432 continued over 10 days in the presence of fresh serum, while diminished within 3-4 days when OK-432 alone was injected. When fresh human plasma or fresh frozen plasma was used instead of serum and i.p. injected with OK-432 avoiding preincubation, the same cytological and clinical changes were observed in other patients. These data strongly indicate that OK-432 activates human complement either in vitro or in the peritoneal cavity, and induces PMNs to accumulate in the ascitic fluid. Although the mechanism of killing of tumor cells by PMNs is obscure, addition of human serum or plasma to i.p. use of OK-432 seems to be valuable for the management of patients with malignant ascites.     

Adoptive immunotherapy of poorly immunogenic tumors with in vitro sensitized cells generated by intratumoral administration of biological response modifiers

We investigated the efficacy of intratumoral administration of biological response modifiers (BRM) in induction of in vitro sensitized (IVS) cells for adoptive immunotherapy of the poorly immunogenic MCA 102 sarcoma and B16-BL6 (BL6) melanoma. We used the bacterial immunoadjuvant Nocardia rubra cell wall skeleton (N-CWS), and a streptococcal preparation, OK-432, for MCA 102 and BL6, respectively. After C57BL/6 (B6) mice were inoculated subcutaneously (s.c.) with viable MCA 102 or BL6 tumor cells in the foot-pad, mice were injected intratumorally (i.t.) with N-CWS ranging from 10 to 400 gg or OK-432 ranging from 1 to 100 μg. Draining popliteal lymph nodes (LN) were harvested 7 days after i.t. administration of BRM, and LN cells were cultured with irradiated tumor cells in the presence of IL-2 for 11 days. These IVS cells (7.5 × 10⁶ or 2 × 10⁶) were transferred intravenously (i.v.) to B6 mice with 4 day pulmonary metastases established by i.v. injection of viable MCA 102 cells (1 × 10⁶) or viable BL6 cells (3 × 10⁵). The mice were also received intraperitoneally 4 × 10⁴ IU/day of IL-2 for 4 days after adoptive transfer. The transfer of IVS cells from mice immunized by i.t. injection of 100 μg of N-CWS 1 week after inoculation of tumor cells significantly reduced MCA 102 pulmonary metastases, compared with control IVS cells without administration of N-CWS. Moreover, the transfer of IVS cells from mice immunized by i.t. injection of 10 gg of OK-432 3 days after inoculation of tumor cells significantly reduced BL6 pulmonary metastases compared with control IVS cells without administration of OK-432. The administration of N-CWS resulted in no enhancement of in vitro cytotoxicity. Although the administration of 10 gg of OK-432 augmented in vitro cytotoxicity of 1VS cells against BL6, cytotoxic activity was lower than that of IVS cells immunized with N-CWS. The major phenotype was CD8⁺ cells in IVS cells immunized with N-CWS or OK-432. These results suggest that i.t. administration of N-CWS and OK-432 facilitates the production of sensitized T-cells, and this administration route of BRM may be useful in the adoptive immunotherapy of human cancer.     

Inhibition of autoimmune diabetes in NOD mice with serum from streptococcal preparation (OK-432)-injected mice.

We have recently reported that systemic and chronic administration of recombinant tumour necrosis factor alpha (TNF-alpha), as well as streptococcal preparation (OK-432), inhibits development of insulin-dependent diabetes mellitus (IDDM) in NOD mice and BB rats, models of IDDM. In this study we examined whether serum containing endogenous TNF induced by OK-432 injection could inhibit IDDM in NOD mice. Treatment twice a week from 4 weeks of age with OK-432-injected mouse serum, which contained endogenous TNF (75U), but not IL-1, IL-2 and interferon-gamma (IFN-gamma) activity, reduced the intensity of insulitis and significantly inhibited the cumulative incidence of diabetes by 28 weeks of age in NOD mice, as compared with the incidence in non-treated mice (P less than 0.01) and in mice treated with control serum (P less than 0.02). This inhibitory effect of the serum was diminished, although not significantly, by neutralization of serum TNF activity with anti-mouse TNF antibody. In the mice treated with the serum from OK-432-injected mice, Thy-1.2+ or CD8+ spleen cells decreased (P less than 0.01) and surface-Ig+ (S-Ig+) cells increased (P less than 0.05), whereas the proliferative response of spleen cells to concanavalin A (P less than 0.01) and lipopolysaccharide (P less than 0.05) increased. The results indicate that the inhibition by OK-432 treatment of IDDM in NOD mice was partially mediated by serum factors including endogenous TNF.     

Strain-dependent cytotoxic effects of endotoxin for mouse peritoneal macrophages.

The cytotoxic effects of bacterial lipopolysaccharides (LPS) on mouse leukocytes have been examined in vivo and in vitro. Intraperitoneal injection of LPS into C57BL/6 mice greatly reduced the recovery of mononuclear cells; LPS was cytotoxic for macrophages, but had a mitogenic effect on lymphocytes. Similar effects of LPS on peritoneal leukocytes were observed in vitro. When monolayers of adherent peritoneal cells were studied in vitro, cytotoxicity was also observed, suggesting that the effect of LPS on macrophages is direct and does not require participation by lymphocytes. Entirely different results were obtained when peritoneal macrophages from LPS-resistant C3H/HeJ mice were studied. LPS failed to activate lymphocytes and was not cytotoxic for macrophages in vitro or in vivo. The effect of LPS on polymorphonuclear leukocytes appeared to be the same in all mouse stains studied. Lipid A was shown to be the most biologically active portion of the LPS molecule. Whereas polysaccharide-deficient endotoxins extracted from rough mutants of Salmonella typhimurium were cytotoxic for macrophages in vitro, polysaccharides that lacked esterified fatty acids did not exhibit this activity. Since LPS may mediate its effects through affinity for mammalian cell membranes, the cellular unresponsiveness of C3H/H3J mice to LPS may reflect an inability of cells from LPS-resistant strains to interact with LPS at the membrane level.     

Oral administration of a streptococcal agent OK-432 activates peritoneal macrophages in mice

The effect of orally administered OK-432, a streptococcal preparation, on the function of peritoneal macrophages in mice was examined. The administration of OK-432 orally (1 KE or 2 KE, four times every three days) did not affect the numbers of both total peritoneal cells and macrophages recovered five days after the final administration. However, the macrophages exhibited an increase in their spreading ability. Other functions of the peritoneal macrophages including lysosomal enzyme activity, phagocytic activity and interleukin 1 (IL-1) production were also enhanced significantly by the oral administration of OK-432 (1 KE or 2 KE). The production of H2O2, however, was not affected by the same treatment with OK-432. The activation of peritoneal macrophages by orally administered OK-432 reported here may contribute to expansion of the clinical application of this drug.     

Incorporation of LPS in liposomes diminishes its ability to induce tumoricidal activity and tumor necrosis factor secretion in murine macrophages

We investigated the effect of lipopolysaccharide (LPS) incorporated into phospholipid vesicles (liposomes) on the induction of macrophage-mediated tumor cytotoxicity and tumor necrosis factor (TNF) secretion. The incorporation of Salmonella minnesota rough (Re)-LPS into multilamellar or small unilamellar vesicles (liposomes) resulted in an 100- to 1,000-fold reduction in its potency to activate both the macrophage cell line RAW 264.7 and murine thioglycolate elicited peritoneal macrophages to become cytotoxic for L929 and P815 tumor cells. Liposomal LPS was also a 100- to 1,000-fold less potent inducer of TNF secretion from RAW 264.7 cells. Cytokines secreted by the activated macrophages contributed to the cytotoxic effect on the L929 cells but not the P815 cell line. Human recombinant TNF was not cytotoxic for either cell line but was cytostatic for the L929 cell line. Morphological examination of the cells after uptake of fluorescent, free, and liposomal LPS revealed that both forms were internalized by the endocytic pathway. This, together with the considerably reduced potency of liposomal LPS to induce tumor cytotoxicity and TNF secretion, suggests that the interaction of the hydrophobic part of the lipid A moiety of LPS with the macrophage plasma membrane is needed to optimally activate these cells. Incorporation of LPS into liposomes effectively abrogates this interaction.