Laminin receptor expression on murine tumor cells: correlation with sensitivity to natural cell-mediated cytotoxicity

Previous studies have identified a relationship between the presence of cell surface laminin receptors on murine tumor cells and sensitivity to killing by natural killer (NK) cells. On the basis of these observations, we suggested that laminin and laminin receptors may function to facilitate the interaction of NK-sensitive murine target cells with NK cells. Our original studies were conducted with a number of genetically unrelated tumor cell lines. In order to extend these earlier observations, studies have now been conducted in which sensitivity to NK-mediated lysis and responsiveness to laminin were compared in a number of variant populations derived from the NK-sensitive cell lines Yac-1 and RL-1 and from the NK/NC-resistant line P815. All of the lines which interacted with murine NK cells as indicated by sensitivity to killing and/or by ability to "cold-target" compete with the killing of sensitive Yac-1 cells were able to bind 125I-laminin and to respond to laminin in an aggregation assay. Of 4 NK-resistant populations identified in these studies, 3 failed to respond to laminin. These studies indicate that even among genetically related tumor cell lines there is a relationship between laminin receptor expression and interaction with NK cells.     

Natural cytotoxicity of mouse, rat, and human lymphocytes against heterologous target cells.

Natural cell-mediated cytotoxicity by a particular subpopulation of lymphocytes [designated natural killer (NK) cells] in NIH Swiss nude and CBA/N mice, WF rats, and humans was demonstrated against tumor cells in 4-hour 51Cr release assays. In most studies, only reactivity against target cells of the homologous species was examined. In the present studies, mouse NK activity also was found against a rat lymphoma tissue culture cell line and against human tissue culture lines. Rat NK cells reacted not only against syngeneic tumor cells but also against heterologous tumor cell lines. In contrast to the heterologous NK activity in rodents, no significant NK activity of human peripheral blood lymphocytes against heterologous targets was found in the present studies. In mice and rats the effector cells that mediated the cytotoxicity against heterologous target cells were indistinguishable from NK cells, the effector cells being nonadherent and nonphagocytic. In addition, the mouse effector cells for heterologous activity as well as mouse NK cells were sensitive to repeated treatment with anti-Thy 1.2 serum plus complement. The specificities of these reactions were indicated by a cold target inhibition assay. The results indicated a sharing of specificities between homologous and heterologous tumor cells recognized by mouse and rat NK cells. In contrast, only the human cell lines were able to appreciably inhibit the cytotoxicity of human peripheral blood lymphocytes.     

Natural cytotoxicity to human leukemia mediated by mouse non-t cells

Natural killer (NK) cells cytotoxic for certain syngeneic or allogeneic tumor cells have been described in various species, including mouse, rat and man, as a new type of effector cell. In this paper, we show that mouse NK cells also function in a xenogeneic system. Established human hematopoietic cell lines were used as targets for normal mouse spleen effector cells in a short-term ⁵¹Cr release assay. Nine malignant lymphoma, two myeloma and four leukemia lines as well as six lympho-blastoid cell lines of presumed non-neoplastic origin were tested. Specific and reliable killing was obtained with the T leukemias MOLT-4, JM, CCRF-H-SB-2 and the myeloid leukemia K 562. Most other lines were sensitive to some degree but their susceptibility and the specificity of the reactions were of doubtful significance. The heterologous cytotoxic reactivity was shown to be caused by the same kind of murine effector cells that mediate NK activity against certain mouse lymphomas by the following criteria: (1) the differences in killing capacity found between spleen cell preparations from genetically “high” or “low” NK-reactive inbred mouse strains did apply in this heterologous system; (2) cytotoxicity was related in a typical manner to the age of the effector cell donor; (3) spleen cells from T-deficient nude mice were highly cytotoxic; (4) removal of T cells, B cells and phagocytic cells using standard fractionation procedures did not abrogate the lytic ability of the remaining spleen cell population. Specificity of lysis was assessed using unlabelled cells as competing targets together with labelled target cells. When competitor cells were identical to the target cells, good competition was achieved. Mouse or human cell lines insensitive to lysis by mouse NK cells had no competitive capacity for target cells of either species. Using NK sensitive lines, competition between mouse and human cells was found to be unidirectional with the most sensitive tumors being the best inhibitors. Thus, unlabelled mouse T lymphoma YAC-1 cells were efficient competitors for lysis of human T leukemia lines. Conversely, susceptible human cells used as competitors did not significantly inhibit killing of YAC-1 targets. The implications of these findings are discussed.     

Non-T-cell-mediated cytotoxicity in mice with tumors induced by Moloney murine sarcoma virus (M-MuSV). II. Granulocyte-mediatd cytotoxicity against autochthonous target cells isolated from M-MuSV-induced tumors

Experiments were performed in A/Sn and A/WySn mice to determine the specificity, organ distribution, and further characteristics of the small non-T-cells that infiltrate Moloney murine sarcoma virus (M-MuSV)-induced tumors and are capable of killing the autochthonous M-MuSV-infected tumor target cells. Continuous bovine serum albumin density gradient separation proved to be the most effective method of enriching the cytotoxic cells. Increase in cytotoxic activity, measured by both the 51Cr release assay and the microcytotoxicity test, paralleled the increase in the proportion of polymorphonuclear leukocytes in mice bearing M-MuSV-induced tumors also contained myeloperoxidas-positive cytotoxic effector cells. The cytotoxic activity appeared to be nonspecific. Various mouse tumor lines as well as allogeneic fibroblasts were sensitive to these effector cells. The only target cell type not affected was the syngeneic fibroblast. In addition to the tumor mass, cytotoxic cells were found in the bone marrow, blood, and spleens of mice bearing M-MuSV-induced tumors. Only bone marrow cells from normal mice exhibited cytotoxic activity. Thus cells of the myelopoietic series may be important in fighting M-MuSV-induced tumors by way of their direct cytotoxic effects on the infected cells.     

Disease-related lymphocyte cytotoxicity in rats bearing transitional cell carcinoma of bladder. effect of immunomodulators

Blood lymphocytes from rats bearing transitional cell carcinoma (TCC) of the bladder were studied for their cytotoxicity in vitro against xenogeneic YAC-1 target and against syngeneic TCC cells. Control lymphocytes were obtained from age and sex-matched syngeneic rats. The following differences were observed: (I) lymphocytes from TCC-bearing rats were cytotoxic to syngeneic TCC target cells whereas those from control rats were not; (2) lymphocytes from TCC-bearing and control rats were cytotoxic to NK sensitive YAC-I cells; however, NK cells from TCC-bearing rats were more adherent to nylon wool-columns than NK cells from control rats. The adherent and non-adherent cells from TCC-bearing rats were both cytotoxic to syngeneic TCC target cells. Levamisole treatment of effector cells from TCC-bearing rats did not affect the NK activity, yet it increased the cytotoxicity of non-adherent cells on TCC target cells. Treatment of the adherent cells with poly-l:poly-C increased slightly their NK activity on YAC-I cells and their anti-TCC cytotoxicity. However, a marked increase in the cytotoxicity by both adherent and non-adherent cell fractions was observed on TCC target cells pretreated with poly-l:poly-C. A disease-related cytotoxicity of lymphocytes from rats bearing TCC has been observed. Treatment of TCC target cells with poly-l:poly-C increased their susceptibility to lysis by the activated effector cells.     

Lymphokine-activated killer cells in rats: analysis of progenitor and effector cell phenotype and relationship to natural killer cells

The progenitor and effector cell phenotype of lymphokine-activated killer (LAK) cells generated in F344 rats by recombinant human interleukin 2 (IL-2) (rIL-2) were analyzed. Highly purified populations of peripheral blood large granular lymphocytes (LGL) exhaustively depleted of T-cells were fully capable of generating high levels of LAK activity by 3 to 5 days in culture while purified populations of resting T-cells devoid of LGL could not generate LAK activity. This pure population of LGL expressed surface markers characteristic of rat natural killer (NK) cells [i.e., OX8+, asialomonoganglioside (asialo-GM1+), laminin+, OX19-, R1-3B3-, W3/25-, Ia-, surface immunoglobulin negative (SIg-)]. Further evidence that NK cells were the progenitors of cells with LAK activity was obtained by treatment of spleen or peripheral blood lymphocytes with anti-laminin or anti-asialo-GM1 antibodies plus complement or with the lysosomotropic agent L-leucine methyl ester. These treatments effectively depleted LGL/NK cell activity and the subsequent generation of rIL-2-induced LAK activity. Analysis of the LAK effector phenotype by cell sorting demonstrated that the majority of cells with LAK activity were OX8+, asialo-GM1+, laminin+, OX6+, OX19-, R1-3B3-, W3/25-, and SIg-. Furthermore, treatment of LAK cells with L-leucine methyl ester also significantly reduced their cytolytic activity. Thus, the LAK effector cells were also LGL and expressed surface marker characteristic of activated NK cells and not those of mature T- or B-cells. The proliferative response of rat spleen or blood lymphocytes to rIL-2 appeared to be primarily associated with LGL/NK cells since depletion of NK cells by anti-asialo-GM1 or anti-laminin antibody plus complement or by L-leucine methyl ester significantly (P less than 0.001) reduced the incorporation of [3H]thymidine into DNA. In contrast, depletion of T-cells (by anti-T-cell antibody plus complement) did not significantly affect rIL-2-induced proliferation. Similarly, T-cell-depleted, highly purified populations of LGL gave substantial proliferative responses to rIL-2. These studies clearly indicate that in the rat, the major cell population activated by rIL-2 is the LGL/NK cell and these cells appear to represent the major population of cells in blood or spleen which generate broad antitumor (LAK) cytotoxicity.     

Increased sensitivity of murine leukemia virus-infected tumor cells to lymphocyte-mediated cytotoxicity

The cytotoxic sensitivity of murine leukemia virus (MuLV)-infected and noninfected fibrosarcoma cells in syngeneic inbred WKA/Hok rats was compared by in vitro cell-mediated 51Cr release cytotoxicity assay. A highly significant increase in cytotoxic sensitivity of target cells was observe in MuLV-infected tumor cells as compared with noninfected cells when spleen cells from syngeneic tumor-bearing hosts (TBH) were used as a source of effector lymphocytes. The cytotoxicity of spleen cells against MuLV-infected tumor cells was specifically directed to the tumor-associated antigen (TAA), but not to the virus-associated antigen. However, there was no quantitative difference in the amount of TAA on the cell membranes between virus-infected and noninfected tumor cells as measured by a quantitative absorption test of anti-TAA serum. The cytotoxic activity of spleen cells from TBH against MuLV-infected tumor cells was abrogated by the treatment of anti-T-serum plus complement and significantly decreased after trypsin treatment. Spleen cells from normal rats given injections of immune sera from TBH acquired the cytotoxic activity against MuLV-infected tumor cells.     

Natural killer cell activity in reticulum-cell sarcomas of SJL/J mice. II. Analysis of RCS-associated NK activity

Lymphoid cells taken from the grossly enlarged lymph nodes (LN) and spleens of SJL/J mice bearing transplantable reticulum-cell sarcomas (RCS), showed increased levels of natural killer (NK) cell activity, when compared to LN and spleen cells taken from normal SJL mice. The NK susceptible target cells used for these studies included RLmale-1 and YAC-1. Target cells that were not susceptible to NK lysis by CBA/J effector cells (high NK strain) were also resistant to lysis by RCS tumor cells. Although significant lysis of target cells was detectable at 4 h, optimal NK cytotoxicity was observed in a 16-h 51Cr-release assay. In terms of lytic units (LU)/10(7) effector cells, greater NK activity was observed in RCS effectors than was apparent in normal CBA/J lymphoid cells. Depletion of macrophages from RCS cell preparations had no effect on the observed NK activity. Purified Mu-IFN-alpha stimulated a small increase in the NK activity expressed by SJL lymphoid cells; however RCS-derived supernatants (containing Mu-IFN-gamma) did not augment NK activity of SJL effector cells, nor were they cytotoxic for NK target cells, Therefore, tumor-derived soluble factors appear not to be involved in the observed RCS-associated NK activity. The data presented in this report further define the parameters of the NK activity manifested by RCS tumor cell preparations. The accompanying article characterizes the nature of the NK effector cells present within RCS tumors of SJL mice. Taken together, these data indicate that the RCS tumors represent either neoplastic expansion of conventional NK cells with somewhat modified properties, or else an entirely new class of cytotoxic effector cells.     

Restoration of impaired T cell functions in tumor-bearing mice by the administration of interleukin 1

The effect of in vivo administration of recombinant human interleukin 1 (IL-1) on T cell functions in tumor-bearing mice was studied using an in vitro assay system. The in vitro induction of trinitrophenyl (TNP)-specific cytotoxic T cell and proliferative T cells responses from spleen cells was impaired in X5563 plasmacytoma-bearing C3H/He mice. However, the administration of IL-1 alpha or IL-1 beta to tumor-bearing mice restored T cell functions in a dose-dependent manner. Antigen-presenting activities of spleen cells in tumor-bearing mice for T cell activation were not restored by the administration of IL-1. The activities of cytotoxic T cells and cytostatic T cells specific for X5563 cells were also enhanced by the administration of IL-1. Furthermore, in IL-1-treated mice, NK cell activity of spleen cells detected in terms of the killing of Yac-1 cells was also restored. In accordance with these results, the growth of X5563 cells was significantly inhibited and the lymphocytes from IL-1-treated mice specifically inhibited the growth of tumor cells. These results suggest that the in vivo administration of IL-1 restored the impaired T cell and NK cell functions in tumor-bearing mice and activated protective immunity against tumor cells. Thus, recombinant IL-1 can be applied for tumor immunotherapy.     

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.     

Specific (EMT6) and non-specific (WEHI-164) cytolytic activity by host cells infiltrating tumour spheroids

The development of a serum-free, low-protein culture medium has allowed the detection of tumour-specific cytolytic cells in EMT6 immunized mice bearing EMT6 multicellular tumour spheroids. Spheroid associated (SAC) and peritoneal (PC) effector cells were specific to EMT6 as the target cell, not killing line 1, P815 or RIF-1. The natural killer (NK) cell sensitive target YAC-1 was also not lysed by SAC or PC, indicating undetectable levels of NK cells present within infiltrated spheroids. In contrast, high levels of cytolytic activity were present in SAC, PC and spleen cells against WEHI-164, a line sensitive to natural cytotoxic (NC) and macrophage mediated killing. The EMT6 specific activity was mediated by Thyl+, Lyt2+ cells. The anti-WEHI-164 effector cell population was Thyl-, Lyt2-. The WEHI-164 killer cells were present in SAC and PC from unimmunized mice while the EMT6 specific effector cells were not. After separation of SAC and PC by size using centrifugal elutriation, anti-EMT6 activity was present only in the lymphocyte fraction while anti-WEHI-164 activity was enriched in the macrophage fraction.     

Killer cell Ig-like receptors ligand-mismatched, alloreactive natural killer cells lyse primary solid tumors

BACKGROUND: Donor alloreactive natural killer (NK) cells have a potent antileukemic effect in haploidentical stem cell transplantation. Whether alloreactive NK cells are able to specifically kill fresh tumor cells from primary solid tumors was analyzed. METHODS: NK cells were purified from healthy donors for the expression of inhibitory killer cell immunoglobulin (Ig)-like receptors (KIRs), ex vivo expanded, and used as effector cells. Their cytotoxic effect on tumor cells freshly obtained from surgical specimens was assessed by means of a single-cell cytotoxic assay (SCCA). RESULTS: Tumor cells from 1 ovarian, 1 gastric, 3 colon, and 4 renal cell cancers were analyzed and found susceptible to alloreactive NK cell killing (>20% lysis at an effector cell to target cell [E:T] ratio of 10:1 for tumor cells not expressing at least 1 human lymphocyte antigen [HLA] class I KIR-ligand group). Remarkably, NK cells that recognized specific HLA-C group mismatches were able to kill HLA-C KIR ligand-mismatched tumor cells, whereas no lysis of target cells occurred with KIR ligand-matched tumor targets. CONCLUSIONS: Alloreactive NK-cell mediated antitumor effects might provide useful insights for designing new cell therapy approaches against solid tumors.     

Augmentation of tumor immunity against syngeneic tumors in mice by beta-carotene

The effect of beta-carotene on tumor immunity was examined with the use of a syngeneic murine tumor system. Oral administration of beta-carotene (120 micrograms/mouse/day) for 9 days from day 1 to the BALB/c mice inoculated sc with 10(7) syngeneic BALB/c Meth A fibrosarcoma cells (Meth A) led to a remarkable rejection against rechallenged Meth A implanted sc on day 10. The growth of Meth 1 fibrosarcoma (Meth 1), another syngeneic tumor of BALB/c origin, as a rechallenge tumor was unaffected by treatment with beta-carotene, thereby suggesting that beta-carotene may augment tumor rejection specific to tumor-specific antigens. Winn assay revealed that the suppressive effect on tumor growth of immune lymph node cells obtained from Meth A-inoculated beta-carotene-treated mice on day 12 was enhanced dose dependently. Primary effector cells responsible for the augmented rejection are Thy-1-positive, Lyt-1-negative, and Lyt-2-positive lymphocytes, presumably cytotoxic T-lymphocytes.     

Natural cytotoxicity of human blood lymphocytes and monocytes and their cytotoxic factors: effect of interferon on target cell susceptibility

The effect of interferon (IFN) on target cell susceptibility to human natural killer (NK) cells and monocytes was analyzed in direct cell-mediated and their cytotoxic factor-mediated cytotoxicity assays. Treatment of K562 cells with IFN resulted in a decrease in their sensitivity to lysis by nonadherent lymphocytes and Percoll-purified large granular lymphocytes (LGL) when tested in a 4-hour 51Cr release assay. In contrast, the treatment did not affect the target susceptibility to monocytes purified by adherence to autologous serum-coated plastic surfaces. In the target-binding assay with LGL or monocytes the number of conjugates was not altered after IFN treatment of K562. Lymphocytes and monocytes were induced to release soluble cytotoxic factors, termed "natural killer cytotoxic factors (NKCF) and monocyte cytotoxic factors (MCF)," respectively, when co-cultured with K562. Both NKCF and MCF lysed K562 in a 48-hour microcytotoxicity assay or in an 18-hour 51Cr release assay in the presence of dactinomycin. IFN-treated K562 reduced or completely lost their ability to stimulate the release of NKCF, whereas they triggered MCF secretion as effectively as did the untreated K562. When lymphocytes or monocytes were pretreated with IFN, they released NKCF or MCF with augmented lytic activity. In contrast to the sensitivity to NK cell-mediated lysis, IFN pretreatment of K562 induced no change in their susceptibility to NKCF and MCF. When IFN was added to NKCF and MCF assays, the cytotoxicity was enhanced. The addition of IFN to K562 that had been pretreated with NKCF or MCF and washed resulted in no increase in lysis. The capacity of K562 to absorb the lytic activity of NKCF and MCF was not altered by IFN. These results indicate that IFN treatment of target cells can be used to distinguish the two distinct types of blood mononuclear cells with natural cytotoxicity, NK cells and monocytes, and that each effector cell type is stimulated to release cytotoxic factors by the different target determinant after the initial effector-target cell binding.     

Antitumor activity of a Streptococcus pyogenes preparation (OK-432). II. Analysis of the cytotoxic lymphocytes induced by OK-432 injection into tumor-bearing F344 rats

In the present study, the antigenic phenotype and target cell specificity of the cytotoxic lymphocytes observed in F344 rats following the ip inoculation of a syngeneic MADB106 mammary carcinoma and the single injection of OK-432 were examined. When the cytotoxicity of peritoneal exudate cells (PECs) was measured in a 4-hour 51Cr release assay, appreciable cytotoxicity against MADB106 tumor cells was evident by day 7-14 following OK-432 injection. With the use of an antibody (R1-3B3) and complement depletion of cytotoxic PECs, the MADB106 killer cells appeared to consist of both R1-3B3- (non-T) and R1-3B3+ (T) cells, with most of the anti-MADB106 killing residing in the R1-3B3- cell population. The R1-3B3- killer cells were further defined as: a) phenotypically asialoGM1+, b) present in athymic nude rats, and c) accompanied by some augmentation of YAC-1 killing [the prototype rat natural killer (NK) target], suggesting that some of these R1-3B3- killer cells were typical NK cells. However, it was also observed that most of the R1-3B3- cells that killed MADB106 tumor cells were: 1) phenotypically or functionally different from either cytotoxic T-cells or typical NK cells; 2) observed only in MADB106 tumor-bearing rats challenged with OK-432; 3) not present on day 1-2 following OK-432 injection, the time when YAC-1 killing was maximally augmented; and 4) present in high numbers in a secondary response following reinoculation of the MADB106 tumor cells into cured rats. The in vivo relevance and possible derivation of these various cytotoxic lymphocyte populations in the syngeneic tumor-bearing hosts are discussed.     

Killing of normal cells by activated mouse natural killer cells: evidence for two patterns of genetic regulation of lysis

Normal primary mouse thymocytes and peritoneal cells are sensitive for lysis by mouse spleen cells activated by in vivo infection with lymphocytic choriomeningitis virus (LCMV). With both thymocytes and peritoneal cells as targets, the active killer cell seemed to be an NK cell, as judged by high activity in the mouse mutant nude and low activity in the mouse mutant beige. Thymocytes from 1- to 3-week-old mice were found to be most NK-sensitive, while peritoneal cells showed highest sensitivity for lysis when harvested from older (6- to 8-week-old) animals. In a direct comparison the genetic regulation of NK activity against thymocytes and peritoneal cells was analyzed. The same strain origin of both target cells and effector cells in either system was used. With thymocytes as target cells, spleen cells from LCMV-infected C3H/St mice invariably showed the highest activity closely followed by cells from the BALB/c strain, while the SWR/J and the A/J strains both were considerably less reactive. The same pattern of high- or low-reactive strains was seen regardless of the genotype of the thymocyte target donor, and the same pattern of cytotoxicity was also seen against the YAC-I lymphoma target. An entirely different genetic regulation was active in the lysis of peritoneal cells, where a unique pattern of reactivity was seen for each effector cell genotype, dependent on the strain origin of the peritoneal target cell. In line with previous findings, syngeneic combinations of effector-target cells yielded little or no reactivity, while various allogeneic combinations showed considerable levels of activity. Also, primary cultures of embryonic fibroblasts were used as target cells. The genetics of the fibroblast target system was more similar to that seen in the thymocyte-YAC-I assay since no evidence for a preferential killing of allogeneic target cells was seen.     

Inhibition of syngeneic fibrosarcoma growth by lymphocytes sensitized on tumor-cell monolayers in the presence of the thymic humoral factor.

The present experiments were performed to investigate the possibility of inhibiting tumor growth in vivo with syngeneic lymphocytes sensitized in vitro on monolayers of the tumor under test, and to study the effect of a thymic humoral factor (THF) in this sensitization process. Monolayers of fibrosarcoma cells were used to sensitize spleen cells from syngeneic donors against the tumor. Such sensitized lymphocytes manifested cytotoxic activity against cells fo the fibrosarcoma in a microassay measuring tumor-cell detachment. However, when the sensitized lymphocytes were mixed with the fibrosarcoma cells and injected into syngeneic mice, enhanced tumor growth was observed in vivo. Addition of thymic humoral factor to the cultures during sensitization resulted in increased cytotoxic activity by the lymphocytes in vitro and a reduction in the tumor enhancement caused by these cells when injected in vivo. Enhanced tumor growth occured when activated lymphocytes of allogeneic as well as syngeneic origin were injected together with the fibrosarcoma cells. Enhancement, which was already apparent when the spleen cells had been sensitized for 24 h, could be circumvented by separate administration of lymphocytes and tumor cells. Syngeneic lymphocytes injected systemically after sensitization for 5 days exerted anti-tumor reactivity against the fibrosarcoma grafted in the foot-pad of syngeneic mice. Tumor growth was further inhibited by systemic injection of lymphocytes which had been sensitized in the presence of the thymic humoral factor.     

Natural cytotoxicity of human blood monocytes and natural killer cells and their cytotoxic factors: discriminating effects of actinomycin D

The effect of actinomycin D on target susceptibility to human blood natural killer (NK) cells and monocytes was analysed in direct cell-mediated and their cytotoxic factor-mediated cytotoxicity assays. Treatment of K562 cells with actinomycin D reduced their susceptibility to lysis by non-adherent lymphocytes and Percoll-purified large granular lymphocytes (LGL) in a 4-hr 51Cr-release assay, without affecting their sensitivity to monocytes purified by adherence to autologous serum-coated plastic surfaces. The drug treatment caused no shift in the kinetics of cytotoxicity. In the target binding assay LGL formed fewer conjugates with actinomycin-D-treated K562 cells than with untreated ones, while the binding of monocytes to targets was not reduced by the drug treatment of K562 cells. The cold target competition assay revealed that actinomycin-D-treated cold K562 cells showed less successful inhibition than untreated cold K562 cells. Lymphocytes and monocytes could be induced to release soluble cytotoxic factors, termed natural killer cytotoxic factors (NKCF) and monocyte cytotoxic factors (MCF), respectively, when co-cultured with K562 cells. Both cytotoxic factors lysed NK-sensitive target cells in a 48-hr assay. Actinomycin-D-treated K562 cells reduced or abolished the ability to stimulate the release of NKCF from lymphocytes, whereas they induced MCF secretion from monocytes as effectively as untreated ones. On the other hand, actinomycin D treatment of K562 cells enhanced their susceptibility to NKCF and MCF. This actinomycin-D-induced augmentation of target sensitivity to the cytotoxic factors was restricted to NK-sensitive target cells (K562 and Molt-4). NK-resistant target cells (Raji, YAC-I, EL4 and T blasts) were not lysed by NKCF and MCF even after they were treated with actinomycin D. The capacity of K562 cells to bind NKCF and MCF was not altered by actinomycin D. Treatment of the adherent cell population with OKMI or Leu-MI plus complement abrogated both cell-mediated cytotoxicity and MCF production, while Leu-IIb plus complement was ineffective. These results suggest that the effect of actinomycin-D treatment can be used to distinguish the two distinct types of blood mononuclear cells with natural cytotoxicity, NK cells and monocytes, and that each effector type recognizes different plasma membrane moieties of NK target cells, although the cytotoxic factors released from each effector cell similarly bind to and lyse the target cells.     

In vitro destruction of tumor cells by macrophages from mice treated with Corynebacterium granulosum.

Peritoneal macrophages from C3Hf/Bu mice treated with killed Corynebacterium granulosum bacteria were tested for their effect on in vitro growth of syngeneic fibrosarcoma cells, tumorigenic mouse L-P59 cells, human malignant melanoma cells, allogeneic fibroblasts, erythrocytes, and epithelial kidney cells. Only the cell cultures having neoplastic properties were destroyed by stimulated macrophages; the rate of tumor cell destruction was greater as the ratio of effector to target cells was increased. Neither irradiation nor trypsinization of macrophage monolayers altered the cytotoxicity of stimulated macrophages. The results indicated that C. granulosum activated macrophages to destroy tumor cells in an immunologically nonspecific manner but had no cytotoxic effect on normal allogeneic cells.     

Metastatic potential of murine fibrosarcoma cells is influenced by cell surface laminin

In order to examine the role of cell surface laminin in tumor metastasis we have utilized four well-characterized murine fibrosarcoma cell lines. Two of these lines were highly metastatic when injected into syngeneic mice while the remaining two lines were significantly less metastatic. Using indirect immunofluorescence techniques, we detected cell surface laminin on the cell surface of both highly metastatic cell lines but not on the low-metastatic cell lines. Although the low-metastatic cell lines did not possess endogeneous cell surface laminin, they had the ability to specifically bind exogenous laminin to their surface in a time- and concentration-dependent manner, indicating the presence of laminin receptors on these cells. Incubation of the low-metastatic cells with exogenous laminin prior to injection into syngeneic animals significantly increased their metastatic potential. No such increase was observed when the highly metastatic lines were preincubated with exogenous laminin. On the basis of these results, we conclude that in this fibrosarcoma model, metastatic potential is influenced by cell surface laminin and that the presence of unbound laminin receptors on the cell surface is not alone sufficient to promote metastasis of these cells.