Pathways of tumour cell killing by activated macrophages: both tumour cell membrane and nucleus can be the primary target

Plasma membrane and nucleus can be primary targets of tumour cell killing by activated macrophages (AM?). Necrotic-type cytotoxicity with loss of membrane integrity and cytoplasmic swelling was expressed by AM? from normal and from perforin-deficient mice, indicating that perforin was not involved. Incubation with AM? consistently triggered the release of thymidine from prelabelled targets, whereas chromatin condensation and small DNA fragments were only occasionally detected. It is shown by means of Pulsed-Field Gel Electrophoresis that DNA degradation in target cells is a slowly progressing process that may stop at any time, indicating that nuclear-type killing doesnot necessarily lead to the formation of low molecular weight fragments. Neither Fas nor the p55 tumour necrosis factor receptor appear to be involved in signalling nuclear-type killing. Accordingly, AM? do mediate membrane- and nuclear-type killing but the mechanisms differ from those identified in T cell cytotoxicity.     

Tumor target-derived soluble factor synergizes with IFN-gamma and IL-2 to activate macrophages for tumor necrosis factor and nitric oxide production to mediate cytotoxicity of the same target.

Inflammatory mouse peritoneal macrophages were activated by IFN-gamma in synergy with IL-2 or Lipid A to mediate TNF production for autocrine generation of cytotoxic nitric oxide (NO) to kill P815 or L1210 tumor targets. It was determined that for IL-2, but not Lipid A, to effectively trigger activation of IFN-gamma-primed macrophages, the tumor targets must be also present for interaction with effector macrophages to mediate the production of TNF and NO. IFN-gamma- and IL-2-activated macrophages from syngeneic DBA/2 and allogeneic C3H mice had identical MHC-unrestricted requirements for interaction with DBA/2 mouse-derived P815 and L1210 targets to mediate production of TNF and NO for tumor cytotoxicity. To further define the mechanistic requirements for macrophage-tumor target interaction, IFN-gamma- and IL-2-activated macrophages were separated from P815 targets in culture by a semipermeable membrane. Under these conditions, both TNF and NO were produced by the macrophage, which indicated that the requirement for tumor target-macrophage interaction may be due to a soluble factor produced by the target rather than to direct physical contact. This was confirmed by experiments in which 24-h cell-free culture fluids, derived from either P815 or L1210 tumor targets, substituted for the intact tumor cells in the stimulation of TNF mRNA synthesis and secretion with NO generation of TNF mRNA synthesis and secretion with NO generation by IFN-gamma- and IL-2-activated C3H or DBA/2 macrophages. The activity in 24-h culture fluids derived from P815 and L1210 tumor targets was tentatively designated as tumor-derived recognition factor(s) (TDRF) since it was produced constitutively by the tumor targets and synergized with IFN-gamma and IL-2 to induce macrophage production of TNF and NO for death of the same targets. A variety of nontransformed human and mouse fibroblasts, mouse spleen lymphocytes, and two adherent mouse fibrosarcomas did not produce detectable TDRF activity, whereas two mouse T lymphomas, EL4 and EL4.IL-2, produced TDRF activity similar to L1210 mouse leukemia and P815 mastocytoma. The C3H/MCA, a TDRF-nonproducing mouse fibrosarcoma, was susceptible to cytotoxicity mediated by macrophages activated by IFN-gamma and Lipid A, but not by IL-2 triggering. Exogenous TDRF derived from L1210 targets reconstituted the cytotoxic activity for C3H/MCA MCA targets mediated by IFN-gamma- and IL-2-activated macrophages accompanied by the production of TNF and cytotoxic NO.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of fibroblast growth factors in cultured tumor cells and their transplants

Summary The distributions of acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in extracts of various cultured mammalian cells were determined from their elution profiles on heparin-affinity chromatography, and assay of activity as ability to stimulate DNA synthesis in BALB/c3T3 cells. Only aFGF was found in extracts of mouse melanoma B 16 cell and rat Morris hepatoma cell (MH₁C₁) lines. Other tumor cell lines established from solid tumors and some normal cells contained bFGF as a main component, but blood tumor cell lines contained no aFGF or bFGF. The FGFs in extracts of solid tumor tissues derived by transplantations of these cultured tumor cells and various normal tissues of mice were also examined. Tumors formed by all cell lines, regardless of whether they produced aFGF, bFGF, or neither, contained bFGF that was probably derived from host cells including capillary endothelial cells, in addition to the tumor-derived aFGF or bFGF, if produced. The content of bFGF, possibly derived from the host, in these tumor tissues was comparable to those of various mouse organs other than thymus, lung, spleen, and testis, which have higher bFGF contents. Tumor tissues derived from cultured cells producing bFGF had relatively higher bFGF contents. Like bFGF, aFGF was distributed almost ubiquitously in normal mouse tissues.     

Quantification of the strength of cell-cell adhesion: the capture of tumor cells by activated murine macrophages proceeds through two distinct stages

The binding of tumor cells by macrophages activated with Bacillus Calmette-Guerin is a necessary step toward destruction of those cells. Although several characteristics of the interaction have been defined, little is known of how the actual binding process develops. We used a technique to quantify the forces required to disrupt cell-cell interactions. Over a range of applied relative centrifugal forces, the majority of targets that bound to the activated macrophages fell on two distinct plateaus. Approximately 90% of added targets were bound to the monolayers of macrophages over the range of 1 to 100 X G; 25 to 30% remained bound from 1200 X G to 1500 X G. Two strengths of binding, termed weak and strong binding, respectively, were thus defined on the basis of these curves. Strong binding developed only between activated macrophages and tumor cells. By contrast, weak interactions occurred between either activated or nonactivated macrophages and neoplastic or non-neoplastic target cells. The strong binding required time (60 to 90 min), metabolic activity by the macrophages, and trypsin-sensitive surface structures on the macrophages for development, whereas the weak interaction occurred rapidly and required none of these. Additional evidence indicated the weak binding developed into strong when activated macrophages bound neoplastic cells. This stabilization increased the strength of force to separate tumor cells from the macrophages at least approximately 15 fold (i.e., from approximately 16 mu dynes/cell to approximately 240 mu dynes/cell). Of note, the development of strong binding of antibody-coated targets had distinct requirements for establishment. Taken together, the data suggest the stabilization of binding (i.e., the development of weak into strong binding) leading to effective cell-cell interaction is a complex and dynamic process that may vary depending upon the recognition system involved.     

Cell-mediated cytotoxicity in carcinoma of the human urinary bladder

Summary Lymphocytes from patients with tumors of the bladder or other unrelated tissues or with non-malignant genitourinary (GU) conditions and from normal subjects were tested in a microcytotoxicity assay against long- and short-term cultures (T24 and BT) derived from bladder carcinoma and several other target cell types, to determine the validity of the hypothesis that cell-mediated cytotoxicity (CMC) in bladder cancer patients is a specific disease-related phenomenon. At the effector cell level, lymphocytes from bladder cancer patients displayed uniformly greater cytotoxicity for T24 and BT cells than those from normal donors. This proclivity was shared by the lymphocytes of patients with non-GU cancers but not of those with non-malignant GU disorders. At the target cell level, CMC was observed less frequently against non-bladder tumor targets than against T24 and BT cells and the CMC of bladder cancer patients did not differ significantly from that of the other groups. The pattern of CMC observed against the bladder tumor-derived targets was thus one of target cell sensitivity rather than tumor-specificity and disease-related only to the extent that the CMC of patients with cancer was greater overall than of healthy subjects. Abrogation of CMC by passage of lymphocytes through immunoglobulin-coated columns indicated that the effector cells were principally of non-T type, bearing a superficial resemblance to those in normal individuals which induce non-disease related CMC.     

Shark cytotoxic macrophages interact with target membrane amino groups

The types of target structures recognized by cytotoxic macrophages have been described for various microorganisms, but have not been defined for tumor cells. Tumoricidal macrophages are selective in their destructive mechanisms, sparing normal cells while directing their lytic machinery toward neoplastic targets. The cytotoxic activity of macrophages from a primitive vertebrate, the nurse shark, closely resembles the activity of mammalian tumoricidal macrophages. Host defense mechanisms of these animals appear to rely on antigen nonspecific cellular effector systems, and it has been postulated that macrophage-mediated cytotoxicity plays a dominant role in protection during periods of decreased environmental temperatures when lymphocyte responses of poikilothermic vertebrates are compromised. Similar to mammalian tumoricidal macrophages shark macrophages display selective recognition of target cells. Previous studies showed that TNP modification of targets was protective, preventing recognition by the shark spontaneously cytotoxic macrophage. Additionally, it was shown that cytotoxic activity was inhibited in a dose dependent fashion by the addition of excess unlabeled targets. In the present study, similar inhibition experiments with hapten-modified targets have been used to determine the nature of the target structures recognized by the shark cytotoxic macrophage. Cold targets modified with haptens which react covalently with free amino groups on cell membranes, trinitrobenzene sulfonic acid (TNBS) and flourescein isothiocyanate (FITC), are not recognized by the cytotoxic macrophage. The relative amount of membrane bound TNP was correlated with inhibition of cytotoxicity. Conversely, target cells modified with sulfhydryl reacting reagents, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine and dithionicotinic acid, are recognized similarly to untreated targets. Moreover, TNP-containing lipids, permitted to diffuse into target membranes without covalent binding, do not alter target recognition, indicating that TNP itself has no effect on macrophage:target interaction. From these data, it is concluded that the shark cytotoxic macrophage interacts with membrane bound amino, but not sulfhydryl groups. The ability to distinguish between membrane structures may have appeared early in evolution as a means of preserving self cells while retaining protective nonspecific cytotoxic mechanisms.     

Enhancement of selective tumor cell binding by activated murine macrophages in response to phorbol myristate acetate

The fundamental biology of how stable cell-cell bonds develop between activated macrophages and tumor cells, although essential to lysis of the neoplastic targets, remains poorly understood. To investigate whether this phenomenon could be pharmacologically manipulated, we analyzed the effect of phorbol diesters on tumor cell binding by macrophages. Activated murine peritoneal macrophages, treated in vitro with as little as 1 ng/ml of phorbol myristate acetate (PMA), bound significantly more tumor cells than did untreated macrophages. The effect was induced rapidly by PMA (i.e., maximum enhancement was seen within 15 min) and resulted in an average approximately twofold increase in the number of targets bound. The interaction between PMA-treated activated macrophages and tumor cells was completed much more rapidly than by untreated macrophages. The enhanced binding was seen only in macrophages treated with biologically active phorbol esters. Only the selective interaction between activated macrophages and tumor cells was affected (i.e., PMA treatment had no effect on nonselective interactions between activated macrophages and non-neoplastic targets or between nonactivated macrophages and any type of target). Pretreatment of activated macrophages with PMA apparently altered the requirements for microfilaments and microtubules in establishing binding, because cytochalasin B and colchicine, which inhibited control binding, as well as phagocytosis, had no effect on PMA-enhanced binding. PMA treatment did not alter energy requirements for binding, however, because low temperature (4 degrees C) or inhibitors of glycolysis and oxidative phosphorylation blocked both control and PMA-enhanced binding. The enhancement of binding apparently was not due to large quantities of secreted oxygen metabolites but did correlate closely with increased spreading and surface area of the macrophages. PMA treatment resulted in enhanced expression of trypsin-sensitive tumor-cell binding sites on the macrophage surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of macrophage membrane proteins labeled with 125I by the lactoperoxidase method revealed at least four trypsin-sensitive cell surface proteins that were re-expressed after PMA treatment. The data suggest that rearrangement and/or induced expression of surface binding sites may be an important step in the binding of tumor cells and indicate that PMA is a useful pharmacologic probe in dissecting the establishment of such binding into discrete steps.     

Cytostatic and growth-stimulating effects of alveolar macrophages of rats on tumor cells

Cytostatic and growth-stimulating effects of alveolar macrophages (AM) of rats on tumor cells were studied. The experimental results are summarized as follows. 1. The cytotoxicity of AM activated with BCG to tumor cells was increasing with the increase of effector cells/target cells (E/T) ratio. AM without the treatment with BCG expressed slight cytotoxicity to tumor cells at a high E/T, and growth-stimulating effect on tumor cells, at a low E/T. 2. AM after 24-hour culture had a lower manifestation of cytotoxicity to human lung adenocarcinoma cell line than that of AM without 24-hour culture, and had a growth-stimulating effect on B-16 cell line. 3. Cytostatic and growth-stimulating effects of AM without or with 24-hour culture were decreasing with the increase of irradiation doses.     

Sensitivity to macrophage-mediated cytostasis is cell cycle dependent

We have examined the sensitivity of proliferating lymphoid cells in different phases of the cell cycle to macrophage-mediated cytostatic activity. These studies evaluated the ability of target cells enriched in individual cell cycle phases to pass into the next phase during brief (2–6 hr) periods of coculture with activated or nonactivated peritoneal macrophages. Both normal (concanavalin A-stimulated spleen cells) and neoplastic (Gross virus-induced thymic lymphoma) cells were analyzed. Spleen cells or lymphoma cells were first separated by centrifugal elutriation into populations highly enriched for G₁, S, or G₂/M phases of the cell cycle and cultured in the presence of nonactivated or activated macrophages for periods of 2, 4, or 6 hr. The cellular DNA content of recovered nonadherent target cells was then analyzed by flow cytometry after staining with propidium iodide. Macrophage contamination of target cell populations was insignificant under these conditions. Nonactivated macrophages did not affect target cell cycle traverse when compared with target cells cultured alone. Activated macrophage mediated cytostatic activity resulted in complete block of the transition of cells in G₁ phase into S phase and of the further accumulation of DNA by cells in early S phase. Cells already in mid to late S phase were able to continue DNA replication at rates nearly equivalent to control cells. There was no inhibition of the passage of cells through G₂ or mitosis. These effects were seen by as early as 2 hr of macrophage-target cell coculture and both normal and neoplastic cells exhibited identical patterns of cell cycle phase sensitivity.     

Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.     

A microassay for the rapid and selective binding of cells from solid tumors to mouse macrophages

Summary A microassay was developed to study the rapid binding characteristics of murine macrophages activated by gamma interferon and muramyl dipeptide to adherent neoplastic or nonneoplastic target cells. The binding of tumor cells to both activated and nonactivated macrophages was time- and temperature-dependent, and independent of tumor cell type. Activated macrophages bound more tumor cells than nonactivated macrophages. The initial binding of macrophages to target cells did not necessarily lead to lysis. First, primed macrophages bound tumor cells but did not lyse them, and second, nonactivated macrophages bound nontumorigenic cells without subsequent lysis. The rapid binding assay described here could prove useful in investigating the recognition mechanism(s) between macrophages and tumor cells derived from solid primary and metastatic cancers.     

Role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma. II. Down-regulation of macrophage-mediated cytotoxicity by tumor-derived granulocyte-macrophage colony-stimulating factor

Peritoneal elicited macrophages (PEM) from mammary tumor-bearing mice have a decreased capacity to become cytotoxic against syngeneic, allogeneic, and xenogeneic target cells upon in vitro stimulation with LPS, as compared with PEM of normal mice. A regulatory mechanism other than PG release is suggested because the addition of both indomethacin and LPS to macrophage cultures from tumor-bearing mice caused no changes in their cytotoxic capability. Because tumor products have been implicated in the down-regulation of immune responses, we investigated whether pretreatment with supernatants from the tumor cell line DA-3, derived from the in vivo mammary adenocarcinoma D1-DMBA-3, affects the cytolytic capacity of macrophages. This treatment inhibits, in a dose-dependent fashion, the ability of stimulated normal PEM to kill target cells. Partial purification of DA-3 cell line supernatant showed that most of the inhibitory activity was exerted by factors with a molecular mass greater than 10 kDa and less than 30 kDa. However, slight inhibition could also be observed with fractions containing molecules less than 10 kDa. The data suggest that more than one factor released by the mammary tumor cells may be involved in the down-regulation of macrophage-mediated cytotoxicity. Because the DA-3 cells constitutively produce granulocyte-macrophage CSF (GM-CSF), which has a molecular mass of 27 kDa, we pretreated PEM from normal mice in vitro with rGM-CSF for 24 h. This resulted in a dose-dependent decrease in their capacity to kill tumor target cells upon LPS stimulation. Furthermore, PEM from normal mice injected with rGM-CSF for 25 days displayed a profound decrease in their cytolytic ability against DA-3 targets upon in vitro stimulation with increasing amounts of LPS. The pretreatment of PEM from normal mice with a combination of DA-3 cell supernatants and specific anti-GM-CSF partially neutralized the inhibitory effect of the DA-3 supernatant on macrophage tumoricidal capability. These results indicate that tumor-derived GM-CSF is an important factor involved in the decreased macrophage cytotoxicity during mammary adenocarcinoma progression.     

Comparison of monocyte and alveolar macrophage antibody-dependent cellular cytotoxicity and Fc-receptor activity

The cytotoxic potential of rabbit peripheral blood monocytes and alveolar macrophages in antibody-dependent cellular cytotoxicity (ADCC) toward both erythrocyte (RBC_ox) and tumor cell (CEM T-lymphoblast) targets was examined. ADCC was measured in a 4-hr ⁵¹Cr-release assay. Alveolar macrophages were more efficient at killing the tumor cell targets (optimally sensitized with rabbit antisera) than monocytes at similar effector cell/target cell ($\text{E}\text{T}$) ratios. Tumor cell targets sensitized with seven different antisera (anti-CEM) were lysed by alveolar macrophages but not by the monocytes. In marked contrast, the monocytes were more effective at lysing the sensitized erythrocyte target cells. The degree of cytolysis of RBCox and CEM was dependent on the $\text{E}\text{T}$ ratio and the degree of sensitization of these target cells. It was demonstrated that the effector cell selectivity in ADCC was directly related to their ability or inability to bind the sensitized target cells as determined by Fc-receptor rosette formation. The transition from monocyte to macrophage may, therefore, have resulted in an alteration in the criteria necessary for Fc-receptor binding to antibody-sensitized target cells and subsequent ADCC.     

DHFR silence alleviated the development of liver fibrosis by affecting the crosstalk between hepatic stellate cells and macrophages

Liver fibrogenesis is a dynamic cellular and tissue process which has the potential to progress into cirrhosis of even liver cancer and liver failure. The activation of hepatic stellate cells (HSCs) is the central event underlying liver fibrosis. Besides, hepatic macrophages have been proposed as potential targets in combatting fibrosis. As for the relationship between HSCs and hepatic macrophages in liver fibrosis, it is generally considered that macrophages promoted liver fibrosis via activating HSCs. However, whether activated HSCs could in turn affect macrophage polarization has rarely been studied. In this study, mRNAs with significant differences were explored using exosomal RNA-sequencing of activated Lx-2 cells and normal RNA-sequencing of DHFR loss-of-function Lx-2 cell models. Cell functional experiments in both Lx-2 cells and macrophages animal model experiments were performed. The results basically confirmed exosomes secreted from activated HSCs could promote M1 polarization of macrophages further. Exosome harbouring DHFR played an important role in this process. DHFR silence in HSCs could decrease Lx-2 activation and M1 polarization of M0 macrophages and then alleviate the development of liver fibrosis both in vitro and vivo. Our work brought a new insight that exosomal DHFR derived from HSCs had a crucial role in crosstalk between HSCs activation and macrophage polarization, which may be a potential therapeutic target in liver fibrosis.     

Distribution of Deoxyribonucleic Acid Complementary to the Ribonucleic Acid of Avian Myeloblastosis Virus in Tissues of Normal and Tumor-Bearing Chickens

(3)H-labeled 70S ribonucleic acid (RNA) from purified avian myeloblastosis virus (AMV) was used as a probe in deoxyribonucleic acid (DNA)-RNA hybridization experiments to detect the presence of DNA complementary to the AMV genome in various tissues from noninfected normal chickens and from chickens infected with AMV. There was a remarkable constancy in the average cellular concentration of virus-specific DNA found in every tissue from the same uninfected chicken, and even in different chickens from the same strain. In contrast, different tissues from chickens bearing AMV-induced kidney tumors (embryonal nephromas) revealed an unequal distribution in the average virus-specific DNA content per cell. The increase was limited to tumor cells and to tissues that contain target cells for AMV, i.e., red blood cells, kidney cells, and possibly leukocytes. The red blood cells from AMV-infected chickens suffering from acute myeloblastic leukemia, although producing no virus, contained as many viral genome equivalents per cell as did leukemic myeloblasts known to produce large quantities of AMV. An increased viral DNA content was observed in the target cells of chickens that did not show any sign of tumor formation 6 months after infection with AMV. This study demonstrates that vertically transmitted viral DNA is uniformly and stably distributed among all tissues of the offspring, but that horizontal infection after hatching results in an increase in viral DNA content only in some dividing, target tissues that may or may not give rise to neoplasias.     

Macrophage activation by tuftsin and muramyl-dipeptide

Summary Peritoneal macrophages from tuftsin or MDP-treated mice were tested for their cytostatic activity for tumor cell proliferation. Both substances are able to activate macrophages either after intravenous injection or after incubation in vitro with normal macrophages. But a stimulation as well as an inhibition of tumor cell growth can result from macrophage activation depending on the timing and dose injected. Restoration of the impaired cytostatic capacity of macrophages of mice observed with aging, is obtained by repeated administration of tuftsin. Normal and BCG-stimulated macrophages were examined for their regulatory activity on the proliferation of P815 tumor cells. Low density of macrophages per well determines a stimulation of target cell growth whether the macrophages are normal or activated. When the number of macrophages is increased, under conditions in which normal macrophages are not inhibitory. BCG-stimulated macrophages exert already a strong cytostatic activity. At high macrophage content it appears that normal macrophages can also display an inhibitory activity. Macrophage-tumor cell interactions are highly dependent on the concentration and the state of activation of macrophages.Reprint requests should be addressed to M. Bruley-Rosset     

Induction of macrophage-mediated cytolysis of neoplastic cells by mycoplasmas

Unexpected cytolysis was encountered when nonactivated murine peritoneal macrophages were cultured with [³H]TdR-prelabeled syngeneic or allogeneic tumor cells at a 10:1 ratio. The level of specific cytolysis reached 70% within 48 hr of cocultivation. Similar killing was observed whether the macrophages were derived from untreated, thioglycollate-treated, or germ-free mice. Cytolytic activity was also demonstrated when bone marrow-derived or peritoneal macrophages from 9- and 5-day in vitro cultures, respectively, were employed rather than freshly harvested peritoneal macrophages. Thus, the macrophage-mediated killing was neither the result of in vivo preactivation nor a consequence of the presence of lymphocytes in the assay. Moreover, macrophages derived from different strains caused similar effects. Our study revealed that the neoplastic target cell cultures susceptible to cytolysis by nonactivated macrophages were contaminated with mycoplasma. A mycoplasma was isolated from the supernatant of a culture of the A9HT fibrosarcoma line, identified as Mycoplasma orale, and cultivated. Addition of viable mycoplasma from that isolate to mixed cultures of thioglycollate-elicited macrophages and [³H]TdR-prelabeled mycoplasma-free target cells resulted in specific cytolysis of transformed A9 cells, but not of normal mouse fibroblasts. The level of macrophage-dependent cytolysis correlated with the number of viable mycoplasma cells added and was higher than that attained by activation with LPS at optimal concentration. Similar specific cytolysis was observed with heat-killed mycoplasmas. Our results demonstrate that mycoplasmas may cause selective macrophage-mediated cytolysis of neoplastic but not of normal target cells, perhaps via activation of the macrophages. It is suggested that undetected infection of experimental systems by mycoplasmas may account for some reports on lysis of neoplastic cells by nonactivated macrophages.     

The cytolytic function of mononuclear phagocytes. II. Factors that determine the binding and lysis of tumor cells by activated macrophages

The effects of various modifiers upon the interaction of LPS- and BCG-activated macrophages with cells of mastocytoma P815 have been investigated. The efficiency of binding and lysis of the tumor cells is to a great extent determined by activation of the effector-cells, expression of the trypsin-sensitive receptors on the surface of macrophages, and by the type of target-cells. Introduction into the analytical system (effector-target) of unlabeled tumor cells or membrane preparations obtained from them inhibits substantially both binding and lytic activity of cytotoxic macrophages. If nontransformed cells or their membranes are applied, no significant changes in the investigated processes can be detected. Trypsinization of tumor cells as well as of activated but not resident macrophages modifies considerably the interaction of effectors with targets. The quantity of tumor cells bound with macrophages does not depend on the fact, which of the partners is subject to trypsinization, but it is much less than that of target-cells bound in the control. The incubation of activated macrophages with actinomycin D results in a substantial suppression of their lytic activity, whereas treatment of tumor cells with this inhibitor of protein synthesis leads to a considerable decrease in stability of the targets against lytic activity of the factor activated by effectors. The obtained data reveal the ways of selective binding and effective lysis of transformed targets by activated macrophages.     

Systemic Targeting of Liposome-Encapsulated Immunomodulators to Macrophages for Treatment of Cancer Metastasis

and The therapy of cancer is, in reality, the design of therapeutic strategies for therapy of metastatic disease. Metastases consist of unique subpopulations of tumor cells that are derived from the primary tumor, colonize distant target organs, and are able to subvert host immune responses, establish necessary angiogenesis, and obtain a sufficient nutrient supply while evolving to become autonomous from homeostatic mechanisms that function within normal, differentiated tissues. Attempts at eradication of metastases by conventional therapies have generally been unsuccessful due to genetic instability and heterogeneity of metastatic tumors; these properties lead to the emergence of tumor cells that are resistant to most conventional treatments. It may be possible to circumvent this heterogeneity by the activation of tissue macrophages to the tumoricidal state. Activated macrophages are able to kill tumor normals while sparing normal tissues, and efficient activation can be achieved by encapsulation of synthetic muramyl tripeptide analogues into multilamellar vesicles composed of phospholipids. Systemic administration of these liposome-encapsulated compounds leads to tumoricidal activation of alveolar and peritoneal macrophages and eradication of established tumor metastasis in numerous animal tumor models, and this form of therapy is enhanced by combination with parenteral administration of cytokines. Phase III clinical trials of recurrent osteosarcoma are currently in progress. Modulation of the tumor microenvironment by activated macrophages may prove to be an additional modality in treatment strategies that combine the use of biological response modifiers with conventional therapies.     

V gamma 9V delta 2 T cell response to colon carcinoma cells

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vgamma9Vdelta2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-alpha and IFN-gamma secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vgamma9Vdelta2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vgamma9Vdelta2 T cells of various origins, and Vgamma9Vdelta2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vgamma9Vdelta2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vgamma9Vdelta2 T cell agonists in immunotherapies targeting colon tumors.