Activation of natural resistance against lung metastasis of an adenocarcinoma in T-cell depressed spontaneously hypertensive rats by infection with Listeria monocytogenes

Summary We report here our study of the role of natural host defense mechanisms mediated by macrophages and natural killer (NK) cells in an experimental model of spontaneous pulmonary metastases of a mammary adenocarcinoma SST-2 in spontaneously hypertensive rats (SHR) with congenital T-cell depression. To activate macrophages and NK cells, Listeria monocytogenes (LM) was injected IV into SHR which had received a transplantation of SST-2. To assess the antimetastatic responses induced by LM, the number of lung nodules and the lung weight in SHR were evaluated 30 days after tumor inoculation. The growth of lung metastases, though not of primary tumors, was significantly reduced if 10⁷ LM were injected IV into SHR 2, 10 and 20 days after the SC transplantation of 5×10⁴ or 5×10⁵ SST-2. An inhibitory effect of LM on pulmonary metastases was also observed in tumor-excised rats, in which the number of lung metastases and the lung weight were enhanced as compared with those in tumor-bearing rats which had not undergone surgery. Peritoneal resident cells which were harvested from rats injected with LM showed a significant augmentation of tumoricidal activity against SST-2 cells as measured by in vitro cytotoxicity. Similarly, the NK activity of spleen cells of SHR injected with LM increased significantly when compared with untreated SHR. These data suggest that the inhibition of metastatic growth, though not of pirmary tumor growth, was accomplished by the, possibly T-cell independent, activation of macrophages and NK cells.     

Effect of interleukin-18 on metastasis of mouse osteosarcoma cells

The effect of interleukin-18 (IL-18) on metastasis of highly metastatic LM8 mouse osteosarcoma cells was investigated using nude mice treated with anti-asialo GM1 serum to exclude anti-tumor actions of IL-18 through activation of T and natural killer cells. Injection of LM8 cells which do not express IL-18 receptor β into a tail vain resulted in the formation of pulmonary and hepatic metastatic foci. Daily injection of mice with IL-18 starting the fifth day from the cell injection had no significant effect on the number of metastatic foci, while five daily injections of IL-18 before and after the cell injection resulted in marked decreases. Culture of LM8 cells with IL-18 for 5 days before the injection into mice produced no significant effect on the number of pulmonary and hepatic metastatic foci. In contrast, pretreatment of mice with IL-18 for 5 days before the cell injection markedly decreased metastatic foci. The retention of LM8 cells in the lung 24 h after their injection was also reduced by the pretreatment of mice with IL-18. Serum obtained from mice pretreated with IL-18 for 5 days suppressed mobility of LM8 cells but IL-18 itself did not. These results suggest that IL-18 inhibits metastasis of LM8 cells partly by inducing a factor(s) in the host which suppresses cell mobility.     

Comparative evaluation of stress-induced depression of normal killer activity in old and young animals and its elimination by interferon

The data are presented on the comparative evaluation of a poststress decrease in the activity of normal killer cells, determined in the test of the release of 41Cr from target cells YAC-1 in young and old CBA mice after 6-hour immobilization. The use of mouse leukocyte interferon and poly I . poly C acid has been shown to be highly effective for the restoration of the activity of normal killer cells in young animals after its stress-induced suppression, while in old animals these immunomodulators have proved to be considerably less effective in restoring the activity of natural killer cells at the period following the stress. The probable role of disturbances, affecting the functional properties of the normal killer cell population in old animals, in the mechanism of the poststress decrease of natural cell-mediated immunity is discussed.     

Inhibitory effect of metastasis by combined administration with interleukin-2 and sizofiran,a single glucan — immunohistochemical study —

Innovations in methods of combined administration with other BRM or chemotherapeutic drugs have been discussed. We have reported [1] that combined administration with recombinant interleukin-2 (rIL-2) and sizofiran (SPG) is effective in prolonging survival time of C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The immunomechanisms of the combined administration were clarified investigating the intraperitoneal cell population in the primary tumor site, especially the tumor infiltrating lymphocyte (TIL) quantitatively. In the present study, to clarify the antitumor effects of combined administration with rIL-2 and SPG on the metastatic sites, the immnunomechanisms of the suppressive effects of combined administration on the metastasis were studied in EL-4 lymphoma cells intraperitoneally transplanted to mice. Inasmuch as EL-4 lymphoma shows rapid hepatosplenic metastasis, we studied the metastatic foci in the liver and the spleen semiquantitatively investigating the histopathological and immunohistochemical findings of the metastatic foci, especially the TIL. The metastatic foci were stained by hematoxylin-eosin (HE) and monoclonal antibodies (L3T4, Lyt2, asialo GM1, Mac-1, and Ia). The combined administration resulted in: 1) fewer infiltrating tumor cells, 2) more lymphocytic infiltration, and 3) more antitumor effector cells (cytotoxic T cells: Lyt2 and natural killer cells: asialo GM1), macrophages (Mac-1), helper T cells (L3T4), and cells with MHC-class-II antigen (Ia) than did administration of rIL-2 alone or SPG alone, or no administration of these two at all. Combined administration with rIL-2 and SPG appears to activate antitumor-immune response at the metastatic site more effectively than when either agent is administered alone.     

Treatment of mice with anti-asialo-GM1 antibody or poly-I:C: effects on metastasis dissociable from modulation of macrophage antitumor activity

Treatment of C57BL/6J mice with poly-I:C or antibodies to asialo-GM1 enhances and depresses respectively natural killer (NK) cell activity while inversely altering lung metastasis, suggesting a critical role for these cells in controlling tumor formation. We assessed the effect of these treatments on antitumor activity mediated by macrophage (M phi) populations likely to be important in lung metastasis. Alveolar and lung interstitial M phi were asialo-GM1 positive (98%) and were sensitive to in vitro treatment with the antibody plus complement; however, treatment of mice with antibodies to asialo-GM1 failed to alter their tumoricidal activity in vitro. Few blood monocytes (4%) or spleen M phi (2%) were asialo-GM1 positive and their antitumor activity was similarly unaffected. In contrast, however, this same in vivo treatment resulted in a 14-fold increase in lung metastasis. Intraperitoneal injection of poly-I:C greatly reduced metastasis formation but also failed to significantly affect in vitro cytolytic activity of the M phi populations. These findings suggest that the major metastasis altering effects of these agents result from modulation of NK rather than M phi function.     

Inhibition of experimental metastasis with indomethacin: role of macrophages and natural killer cells

The metastatic capacity of murine mammary tumor line 410.4 is greatly increased by treatment of the host with asialo-GM1 antiserum (5-fold), 2-chloroadenosine (4-fold) or k-carrageenan (2.5-fold). This suggests that both NK cells and macrophages contribute to control of metastatic dissemination. The metastatic potential of these cells is associated with the synthesis of high levels of prostaglandin E2 (PGE2) (1). When line 410.4 cells are cultured in vitro in the presence of the prostaglandin synthesis inhibitor indomethacin (INDO) 1 microM) their subsequent lung colonization ability (experimental metastasis) is reduced by 50-90% as compared to solvent-treated cells. The inhibitory effect of INDO is highly dependent on the presence of asGM1 positive cells, and is compromised to a lesser extent by treatments directed towards macrophages. The INDO-mediated inhibition is neither due to differential arrest of tumor cells in the lung nor does it appear to be due to shifts in the replication cycle.     

人黑色素瘤细胞裸鼠肺转移模型的建立

目的建立整合素αvβ3高表达的黑色素瘤细胞肺转移模型。方法通过将不同数目的M21细胞经尾静脉接种裸鼠,适时处死后计数肺表面癌结节。通过实时定量PCR和明胶酶谱的方法比较肺转移灶细胞与亲代M21细胞差异。结果M21细胞1×10^6、2×10^6、5×10^6尾静脉接种裸鼠,均能形成肺转移灶,癌结节数目均值分别为：84±8、70±6、88±12,三组之间没有明显差异,阴性对照组未形成转移灶。M21肺转移灶细胞与亲代细胞相比,增殖增快,MMP-2活性增高,整合素αv和β3mRNA表达水平明显增高。结论M21细胞1×10^6经尾静脉接种裸鼠50d内即可100%成瘤。M21肺转移灶细胞具有更快的增殖能力,整合素αvβ3和MMP-2表达水平明显增高。本实验建立了稳定的肺转移模型,为黑色素瘤和整合素αvβ3的研究提供重要的动物模型。     

Different metastatic modes of malignant melanoma implanted in the ear of young and old mice

Summary An attempt has been made to determine (a) whether aging plays an important role in resistance against metastasis and (b) whether dithiothreitol, an effective in vitro mitogenic potentiator of splenic cells of young and old mice, can modulate the occurrence of pulmonary metastasis. B16-F10 melanoma cells were injected into the outer ear of young and old female C57BL/6 mice; and the growth of the primary tumor, the palpable size of the cervical lymph node, and the number of lung metastases were then determined at various intervals. The ear was amputated when the primary tumor reached 4 mm in mean diameter. The following results were obtained. (a) The growth rate of the primary tumor in young mice is comparable to that in old mice. (b) Enlargement of the cervical lymph node occurs earlier in old than in young mice. (c) Old mice are more vulnerable to pulmonary metastases, but small metastasized pulmonary colonies are more prominent in old than in young mice. (d) Dithiothreitol (100 g) injected every 2 days after the inoculation of tumor cells is effective in reducing the incidence of pulmonary metastases in old mice.     

NK and NKT cell functions in immunosenescence

Immunosenescence is defined as the state of dysregulated immune function that contributes to the increased susceptibility to infection, cancer and autoimmune diseases observed in old organisms, including humans. However, dysregulations in the immune functions are normally counterbalanced by continuous adaptation of the body to the deteriorations that occur over time. These adaptive changes are likely to occur in healthy human centenarians. Both innate (natural) and adaptive (acquired) immune responses decline with advancing age. Natural killer (NK) and natural killer T (NKT) cells represent the best model to describe innate and adaptive immune response in aging. NK and NKT cell cytotoxicity decreases in aging as well as interferon-gamma (IFN-gamma) production by both activated cell types. Their innate and acquired immune responses are preserved in very old age. However, NKT cells bearing T-cell receptor (TCR) gammadelta also display an increased cytotoxicity and IFN-gamma production in very old age. This fact suggests that NKT cells bearing TCRgammadelta are more involved in maintaining innate and adaptive immune response in aging leading to successful aging. The role played by the neuroendocrine-immune network and by nutritional factors, such as zinc, in maintaining NK and NKT cell functions in aging is discussed.     

Induction of tumoricidal macrophages from bone marrow cells of normal mice or mice bearing a colony-stimulating-factor-producing tumor

Summary Nonadherent cells of the bone marrow of C3H/HeN mice were incubated for 3 days with the culture supernatant of an L-929 cell line containing macrophage-colony-stimulating factor. Approximately, 70% of the cells became phagocytic, adherent to plastic dishes and positive for nonspecific esterase staining. The adherent cells exhibited a weak tumoricidal activity against MM48 syngeneic mammary carcinoma cells, and the cytotoxicity was strongly augmented by the addition of bacterial lipopolysaccharide to the cytotoxicity assay. The cytotoxicity induced by lipopolysaccharide was also shown to be mediated by Thy1.2^– and asialo-GM1⁺ cells, and was abrogated by the addition of carrageenan. Macrophage-colony-stimulating-factor-producing (D66) and nonproducing (A23) variants were separated from the MM48 tumor line in in vitro culture following limiting dilution. There was no difference between these two variants in either the in vitro growth rate or the susceptibility to macrophage-mediated cytotoxicity. C3H/HeN mice inoculated i.p. with D66 survived longer than did those inoculated i.p. with A23. C3H/HeN mice bearing D66 or A23 as an ascitic form were given i.p. injections of Nocardia rubra cell wall skeleton (N-CWS). N-CWS significantly prolonged the survival period of mice bearing D66, whereas it exhibited no apparent antitumor effect on mice bearing A23. The increase in the cell number of D66 in the peritoneal cavity was significantly retarded, compared with that of A23. In contrast, the number of peritoneal macrophages increased more in D66-bearing mice than in A23-bearing mice. The increase in the peritoneal macrophage number was further augmented by an i.p. injection of N-CWS. Peritoneal macrophages of D66-bearing mice exhibited apparent tumoricidal activity against MM48 tumor cells in the presence of lipopolysaccharide, and the cytotoxicity was significantly augmented by i.p. injection of N-CWS. On the other hand, the responsiveness of peritoneal macrophages to lipopolysaccharide was found to be poor in A23-bearing mice and the tumoricidal activity was only weakly augmented by N-CWS. These results strongly suggest that M-CSF plays an important role not only in the maturation of macrophage progenitors but also in the induction and the accumulation of activated macrophages. Abbreviations used: M-CSF, macrophage-colony-stimulating factor; NABMC, nonadherent bone marrow cells; CM, conditioned medium; NK, natural killer; N-CWS, Nocardia rubra cell-wall skeleton     

Induction of nonspecific killer cells by delayed-type hypersensitivity against soluble protein antigens in murine peritoneal cavities

Summary We induced nonspecific killer cells in the local site of delayed-type hypersensitivity against keyhole limpet hemocyanin or ovalbumin. Delayed-type hypersensitivity was induced in the peritoneal cavities of mice, and peritoneal exudate cells (PEC) were collected. These PEC were found to have killer activity toward SP2 and YAC-1 cells (target cells susceptible to natural killer cells) by 4-h ⁵¹Cr-release assays. The induction of killer activity in PEC was observed in parallel with the eliciting of delayed-type hypersensitivity in the peritoneal cavity, in which the killer activity was maximum 24–48 h after the antigen challenge, but was not induced in nu/nu mice and was induced in an antigen-specific way. These killer cells did not adhere to nylon wool and had Thy1 and asialo-GM1 antigens on their surfaces. Their precursor cells were also asialo-GM1-positive. These findings indicate that the killer cells probably belong to the NK cell lineage. Results of tumor challenge experiments showed that these killer cells had an antitumor effect in vivo as well as in vitro.     

Delineating the relationship between immune system aging and myogenesis in muscle repair

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y‐O) or old to old (O‐O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL‐10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y‐O chimeras). Improved immune responses in Y‐O chimeras were associated with greater satellite cell proliferation compared with O‐O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age‐related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.     

In vitro augmentation of rat natural killer (NK) cell activity

In vitro augmentation of rat natural killer (NK) cell activity was produced by 2 types of treatment. Increased activity occurred "spontaneously" when spleen cells were cultured alone at 37 degrees C. This augmentation was dependent on the presence of adherent, phagocytic cells, presumably macrophages, and was independent of LPS of FCS. Normally low levels of NK activity, present in macrophage-depleted cultured cells, could also be boosted in vitro by incubation with Corynebacterium parvum. This augmentation appeared to be independent of both B cells and macrophages and may be due to stimulation of rat NK cells themselves. Both forms of augmentation were associated with the production of interferon, were found in rats of all ages and strains tested, and should provide an excellent in vitro system for detailed studies of activation of rat NK cells.     

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.     

肺癌转移瘤动物模型的建立

目的通过尾静脉注射,建立一种符合临床特征的肺腺癌转移瘤动物模型,为下一步的肺腺癌转移机制的研究提供可靠的实验造模方法。方法取对数生长期的A549细胞,11只SPF级、4~6周龄BALB/c裸鼠,分别以1×106个细胞/只注射入裸鼠尾静脉。接种后每天观察小鼠状态。分别于接种肿瘤细胞后第4、5、6、7周随机处死2只,余3只小鼠处于濒死状态时处死。解剖小鼠,观察肺部有无转移、转移结节的数目及全身其他器官的转移情况,并做病理取材,HE染色观察。结果注射过程中小鼠均存活。未处死的3只分别于第11、13、14周出现恶液质。第4周肺部未见转移结节;第5周出现镜下肺部转移结节;第6周肉眼可见肺部转移结节;第7周转移结节数增多;第11周出现纵隔淋巴结转移。第11、13、14周出现肺部结构大量破坏,弥漫性的肿瘤细胞浸润,出现淋巴结浸润,病理证实为腺癌。结论通过尾静脉注射A549细胞可以成功建立人肺腺癌转移瘤模型。