The effect of yoghurt on the cytotoxic and phagocytic activity of macrophages in tumour‐bearing mice

In a previous paper, it was demonstrated that feeding yoghurt was able to inhibit the growth of an intestinal tumour induced chemically with 1,2‐dimethylhydrazine (DMH). This effect was due to the increase in IgA‐producing cells and a diminution of the inflammatory immune response. In this paper the phagocytic and cytotoxic capacity of macrophages both involved and not involved in the target organ are studied. The study was aimed at determining whether in the intestinal tumour inhibition demonstrated previously the systemic immune response was also increased. The cytotoxic capacity and ß‐glucuronidase enzyme levels of the peritoneal macrophages were analyzed together with the cytolytic effect of the serum on tumour cells and the phagocytic activity of the macrophages infiltrating the intestinal mucosa. Groups of mice were split into three experimental groups. One group was treated with DMH. The others were treated with DMH, and their diets were supplemented with yoghurt for 7 or 10 consecutive days, during 24 weeks. It was demonstrated that feeding yoghurt for 7 or 10 days increased cytotoxic and ß‐glucuronidase levels in peritoneal macrophages, and also the cytolytic capacity of serum, reaching values significantly higher than those in the DMH control. Enhancement of the phagocytic activity of the macrophages associated with the large intestine was also observed. This increase in the macrophage activity involved in the systemic and mucosal immune responses could also be responsible for the tumour inhibition observed in the group of mice fed with yoghurt. The presence in the serum of lytic factors (cytokines) which were released by immune cells activated by feeding yoghurt may also have had a role in tumour inhibition.     

Tumour-Host Interactions in Vivo

We have earlier shown that coculture of macrophages and cells from a methylcholanthiene-induced sarcoma (MCI M-AA) in vitro resulted in macrophage activation and production of type II (gamma) interferon. When ascites fluid from the MCIM-AA sarcoma (shown previously to activate macrophages in vitro) was added to spleen cell populations from semisyngeneic C₃D₂ mice in vitro. NK activity was markedly enhanced. After intraperitoneal injection of MCI M-AA cells or ascites fluid, 4- to 12-fold increased NK cell activity with a peak at 3–5 days could be measured in the spleen cell population and in the non-adherent peritoneal exudate cell population. The mice injected with tumour cells or ascites fluid developed a pronounced splenomegaly, and maximum spleen size coincided with peak NK activity. Injection of tumour cells or tumour ascites fluid resulted in marked changes in the T-cell, B-cell, macrophage, and 'null' cell content of ihe peritoneal exudate.     

Relationships among differentiated T-cell subpopulations. V. T-cell dependent mechanism of xenograft rejection and its radioresistant nature.

Rat tumours grew progressively in nu/nu mice but not in nu/+ mice, normal AKR mice or sublethally irradiated AKR mice after subcutaneous inoculation. Suppression of tumour growth appeared to depend on radioresistant cytotoxic T cells which were detected in in vivo neutralization tests (Winn's test) with spleen cells of mice immunized with rat lymphocytes or tumour cells 5 days previously. Radioresistant cytotoxic activity was detected by 51Cr-release test in glass-non-adherent peritoneal exudate cells 5 days after intraperitoneal immunization with rat lymphocytes. These results suggested that immune lymphocytes might differentiate to mature cytotoxic cells at the site of direct graft rejection.     

Regulation of NK cell function in vivo by the dose of tumour transplanted in the peritoneum

Antigen dose is known to regulate T cell activation and anergy. Similarly, dose of antigen also regulates NK cell lytic potential and phenotype development. Resident peritoneal cells of rat contain a small population of NK and NKT cells. Inoculation of AK-5 tumour cells intraperitoneally modulate the cytotoxic function of NK and NKT cells present in the peritoneal exudate cells (PEC) in a dose dependent manner. Low dose of tumour causes activation of NK and NKT cell cytotoxic function and enhanced NK and NKT cell population in PEC, whereas, high doses of tumour cause inactivation of NK and NKT cell cytotoxic function and depletion of the two sub-populations in the peritoneum. Different doses of tumour inoculation in the peritoneal cavity did not suppress the cytotoxic function of NK cells from spleen suggesting that a direct interaction between NK cells and tumour cells is required for the suppression of NK cell cytotoxic function. Tumour inoculation induced secretion of IL-2, IL-12, IFN-gamma and TNF-alpha by tumour infiltrating mononuclear cells (TIM) in ascitic fluid as well as in serum. The levels of IL-2, IL-12, IFN-gamma and TNF-alpha secretion were higher in animals, which rejected tumours as compared with the animals that failed to reject the tumours. Injection of anti IL-12 and anti IFN-gamma antibody reduced the survival rate of tumour injected animals, however, anti IL-2 antibody had no effect on the survival of animals. Following incubation with AK-5 tumour cells, activated NK cells upregulated perform expression, whereas, there was upregulation of CD95 expression in inactivated NK cells.     

Activation of non-specific cytotoxic cells in Listeria-susceptible and -resistant mouse strains.

An increase in macrophage tumouricidal activity in the spleen was demonstrated following intravenous infection of genetically resistant C57BL/10 mice with Listeria monocytogenes, but not after infection of BALB/c mice. However, tumouricidal macrophages appeared in the peritoneal cavity of both strains after infection, while NK cell activity was generally higher in BALB/c mice. The activation of tumouricidal macrophages and NK cells in the C57BL/B10 mice was T independent and, at least in the peritoneal cavity, radioresistant (600 rads). The relevance of these results to the genetic control of resistance to Listeria is discussed.     

Natural antitumor defense system of the murine liver

Murine nonparenchymal liver cells from various genetic strains isolated by collagenase digestion and differential sedimentation contain both lymphocytes and macrophages. Nonparenchymal liver cells as well as spleen cells, mononuclear blood cells, and peritoneal exudate cells from C3HeB/FeJ mice were tested for natural cytotoxicity against YAC-1 (sensitive to NK cells) and P815 (resistant to NK cells) tumor cell lines. Resident peritoneal exudate cells exerted no cytotoxicity against either tumor cell, whereas spleen and mononuclear blood cells lysed only YAC-1. In contrast, nonparenchymal liver cells lysed both YAC-1(4 h) and P815 (18 h) tumor cells. Treatment of nonparenchymal liver cells with anti-asialo GM1 and complement abolished the antitumor activity against both tumor cell lines but not the phagocytic activity. Nonadherent nonparenchymal liver cells exerted greater cytotoxicity against YAC-1 tumor cells but little cytotoxicity against P815 tumor cells when compared with unfractionated cells. Adherent nonparenchymal liver cells (macrophages) from untreated mice exerted no antitumor activity against either tumor cell. In contrast, adherent nonparenchymal liver cells from Corynebacerium parvum treated mice were directly cytotoxic to P815 tumor cells. Spleen cells that are normally not cytotoxic to P815 tumor cells (18 h) became cytotoxic when mixed with adherent nonparenchymal liver cells from untreated mice. These results indicate that the tumoricidal effector cell in nonparenchymal liver cells from untreated mice appears to be the NK cell. Apparently, murine liver macrophages from untreated mice do not have tumoricidal activity per se but can "activate" NK cells to kill tumor cells normally resistant to NK cells.     

Comparison of natural killer cells induced by Kunjin virus and Corynebacterium parvum with those occurring naturally in nude mice.

Natural killer (NK) cells are rapidly elicited in the spleen and peritoneal cavity of mice inoculated intravenously or intraperitoneally with live Kunjin virus, and more slowly in the peritoneal cavity of mice inoculated intraperitoneally with Formalin-inactivated Corynebacterium parvum. NK cells induced by either agent display cytotoxicity for a similar spectrum of syngeneic, allogeneic, and xenogeneic cultured cell lines. By contrast, the cells occurring naturally in the spleen of congenitally athymic (nude) mice show substantially lower NK activity and are cytotoxic for a more restricted range of target cell lines. The distinction suggests that there may be more than one type of NK cell or that activation enhances the cytotoxicity and perhaps broadens the range of target specificity of endogenous NK cells.     

Use of a Macrophage Cytotoxicity System to Show Macrophage Activation by Listeria monocytogenes Cell Wall Fraction

Experiments were conducted to determine whether a partially purified listeria cell wall fraction could stimulate macrophages to high levels of activation. To detect activation of macrophages, a macrophage-mediated cytotoxicity system were established. The data demonstrate that listeria cell wall components are capable of activating thioglycollate-induced adherent peritoneal exudate cells to be cytotoxic for 51Cr-labelled target tumour cells, and that the listeria fraction is as effective as bacterial lipopolysaccharide in inducing cytotoxicity. The listeria fraction can also induce peritoneal exudate cells from congenitally thymusless nude mice to become cytotoxic, suggesting that mature T cells are not required. Furthermore, thioglycollate-induced adherent peritoneal exudate cells from mice hyperimmunized to live Listeria organisms are already stimulated to be cytotoxic for tumour cells, and do not need to be activated in vitro. Additional data are presented which characterize the system. These data demonstrate that a critical concentration of adherent peritoneal cells is required for in vitro activation. Moreover, only peritoneal cells induced with aged batches of thioglycollate, and not uniduced peritoneal cells or those induced with fresh thioglycollate or with protease peptone can be activated in vitro to kill tumour cells. Evidence is presented which suggests that the cytotoxic cell is a macrophage.     

A Macrophage Factor Enhancing the Systemic Anti-Tumour Effect of T Lymphocytes

Spleen cells sensitized to tumour cells have an anti-tumour effect on injected syngeneiclymphosarcoma cells in mice. This study shows that this anti-tumour effect can be enhanced by induced peritoneal macrophages and by macrophage-like tumour cells (macrophages). Addition of macrophages to the intraperitoneally injected sensitized spleen cells stimulated the anti-tumour effect. This was observed both with intraperitoneally injected tumour cells and with subcutaneously transplanted tumour cells. The anti tumour effect is the result of a cooperation between T cells and macrophages.In vitro incubation of immune T-cells with macrophages or macrophage-like cells enhancedthe in vivo anti-tumour activity of the sensitized T-lymphocytes. Neither the presence of antigen nor the proliferation of the immune T-cells were a prerequisite to enhance this anti-tumour effect. Our experiments suggest that a macrophage factor is responsible for the enhancement of the anti-tumour effect.Based on the results of this paper and other studies we propose the following sequence ofevents to explain the anti-tumour effect of injected sensitized T-lymphocytes and mac-rophages: injected macrophages enhance the anti-tumour effect of sensitized lymphocytes. These stimulated lymphocytes migrate to the tumour located elsewhere and recognize the tumour antigens. Subsequently, the lymphocytes render (host) macrophages in the tumour cytotoxic to tumour cells.     

Comparison of cytotoxic and microbicidal function of bronchoalveolar and peritoneal macrophages.

Studies were carried out with mice to explore in vitro the effector function(s) of macrophages from two different anatomical compartments (peritoneal cavity and lungs). The cytotoxic capacity of macrophages was measured by determining their cytostatic and cytocidal effects on EL-4 tumour target cells, and the microbicidal capacity of macrophages was measured by determining their ability to kill or inhibit the intracellular protozoan, Toxoplasma gondii. Neither peritoneal macrophages (PM) nor bronchoalveolar macrophages (BAM) from normal mice were ever microbicidal or cytotoxic. Intravenous treatment with Corynebacterium parvum greatly enhanced (activated) both effector functions of PM but did not activate BAM. Chronic infection with Toxoplasma activated PM throughout the period of observation (greater than 140 days), but the presence of activated BAM was transient and appeared to coincide with the occurrence of an inflammatory response in the lungs.     

Mechanisms of in vivo generation of cytotoxic activity against syngeneic tumours. I. Local differentiation of mature cytotoxic T lymphocytes in the rejection of tumours.

Mature cytotoxic T lymphocytes (CTL) were detected in the peritoneal cavity of syngeneic mice immunized intraperitoneally (i.p.) with mitomycin C (MC)-treated EL-4 or X5563 cells, but were not found in their spleens or lymph nodes. Mature CTL appeared among PE cells after transfer of spleen cells from those immune mice, along with MC-treated tumour cells, to the peritoneal cavity of syngeneic mice. These results lead us to the hypothesis that immature CTL primed in the spleen and lymph nodes may migrate to the site of tumour inoculation and differentiate into mature CTL after antigenic or non-specific stimulation at that site. Inability of primed CTL to differentiate to mature CTL in the spleen might be explained by the effect of splenic suppressor cells, since mature CTL became detectable in the spleen of immune mice by treatment with cyclophosphamide.     

Growth inhibition of tumour cells by a liposome-encapsulated, mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate.

In vivo growth of syngeneic tumour cells in the peritoneal cavity was strongly inhibited by intraperitoneal injection of a liposome-encapsulated, mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate (TTM), derived from a non-pathogenic, acid-fast bacterium. Gordona aurantiaca. Peritoneal macrophages from mice after this treatment lysed tumour cells in vitro at a low effector/target ratio, and their culture supernatant inhibited tumour cell growth. The supernatant inhibited growth of not only tumour necrosis factor (TNF)-sensitive tumour cells, but also TNF-insensitive tumour cells. This inhibitory activity was enhanced by addition of lipopolysaccharide (LPS) to the culture medium of the macrophages. The macrophages released more superoxide (O2-), TNF and interleukin-1 (IL-1) on LPS triggering, and the releases of these compounds were further increased by addition of recombinant interferon-gamma (IFN-gamma) to the medium. Moreover, splenic T cells of TTM liposome-primed mice were found to produce eight times more IFN-gamma upon stimulation with LPS. These results indicated that priming with TTM liposomes resulted in strong activation of macrophages, which lysed tumour cells directly and also inhibited tumour cell growth by released factors.     

Detection of either rapidly cytolytic macrophages or NK cells in "activated" peritoneal exudates depends on the method of analysis and the target cell type

The nature of the cytotoxic cells present in the peritoneal cavity of rats treated with Bacille Calmette-Guérin (BCG) or Corynebacterium parvum was investigated using a 6 hr chromium release assay and a quantitative method of analysis based on consideration of target-cell killing as an enzyme-substrate reaction. When the results of cell-fractionation experiments were evaluated in terms of recovery of total lytic units and when appropriate target cells (such as sarcoma Mc7) were used, the simultaneous presence of both cytotoxic macrophages and NK cells in peritoneal exudates could be readily demonstrated. With certain other target cells different results were obtained. Thus, with normal thymocytes, normal hepatocytes, or myeloma P3NSI as targets, NK cells were preferentially detected, whereas with leukaemias L5178Y, P815, and EL4 as targets, cytotoxic macrophages were preferentially detected. These findings resolve the previously conflicting reports concerning the nature of cytotoxic cells in activated peritoneal exudates.     

Effects of Antithymocyte and Antimacrophage Sera on the Survival of Mice Infected with Listeria monocytogenes

Antisera prepared in rabbits against purified mouse thymocytes (antithymocyte serum; ATS) and peritoneal macrophages (antimacrophage serum; AMS) were injected intraperitoneally into Balb/c mice infected with the bacterium Listeria monocytogenes. When administered near the initiation of infection, the ATS significantly decreased the survival time of the animals and increased the mortality rate. When ATS was administered 6 days after a sublethal dose of L. monocytogenes had been inoculated, an overt disease did not evolve. ATS that significantly potentiated primary listeriosis also had high cytotoxicity titers for thymocytes and lymphoid cells from the peritoneal cavity. Although cytotoxic activity against peritoneal macrophages could be demonstrated in lower dilutions of the ATS, this activity did not appear to correlate with the effects of the sera on listeriosis. The injection of AMS did not enhance the infectious process. In some trials more deaths occurred among mice receiving normal rabbit serum than those receiving AMS. All of the AMS had cytotoxic titers against peritoneal macrophages, and the sera were usually inactive against thymocytes and peritoneal lymphoid cells. Listeria was isolated from fatally infected mice with nearly equal success in all of the serum-treated groups, and the serum treatments did not appear to alter the pattern of gross lesions. The afferent limb of the immune response was markedly affected by the presence of antibodies to lymphocytes. However, antibodies reacting with macrophages did not demonstrably enhance the Listeria process, which depends upon cellular immunity as the principal means of acquired host defense.     

Low dose streptozotocin causes stimulation of the immune system and of anti-islet cytotoxicity in mice.

Multiple low doses of streptozotocin are known to induce immune-mediated insulin deficient diabetes and depression of immune reactivity. We show here that immune depression by streptozotocin is not general but that some parts of the immune system are stimulated. Spleen cells from streptozotocin-treated mice showed enhanced cytotoxicity against syngeneic islet cells and various tumour cells including insulinoma cells. Several cell types served as effector cells, including macrophages, asialo GM1+ and Lyt-2+ lymphocytes. The increased cytotoxic activity towards islet cells was mostly due to macrophages and to non-asialo GM1+ and non-Lyt-2+ lymphocytes. A higher activation state of macrophages in low dose streptozotocin-treated mice was demonstrated by measurements of superoxide anion release. We conclude that multiple low doses of streptozotocin stimulate 'natural cytotoxicity', i.e. the non-MHC restricted cytotoxic activity of macrophages, T cells and natural killer lymphocytes.     

The cellular cancer resistance of the SR/CR mouse

The SR/CR mouse phenotype, first described in 1999 in BALB/c and later bred into C57BL/6 mice, is resistant to cancer formation following high doses of cancer cells administered intraperitoneally. The tumor cell targeting and destruction mechanisms have not been identified. By fluorescence‐activated cell sorting analysis, the immune response of SR/CR mice after intraperitoneal injection of cancer cells was investigated and compared with parent strain mice. A massive influx of leukocytes into the peritoneal cavity was found. A large fraction of these leukocytes were polymorphonuclear granulocytes, macrophages and natural killer cells. A relative decrease in influx of B‐cells compared with controls was demonstrated. Increased proportions of leukocytes belonging to the innate immune system were also demonstrated in splenocytes of SR/CR mice. Cytospins of peritoneal fluid from SR/CR mice after cancer cell injection showed formations of immune cells morphologically resembling polymorphonuclear granulocytes and macrophages adjoining the cancer cells. The results point to the potential involvement of innate immune cells in cancer immunology. Our data support migration of polymorphonuclear granulocytes, macrophages and NK cells into the peritoneum of the SR/CR mouse in response to intraperitoneal injection of S180 cancer cells. The cell composition of spleens of SR/CR mice reflected the differential regulation of the innate immune cells in peritoneal exudates. Both peritoneal exudates and the spleens of SR/CR mice contained decreased proportions of B‐cells compared with BALB/c and C57BL/6 mice. We reproduce important aspects of previous published data and further extend them by showing differentially regulated populations of splenocytes including B‐lymphocytes in SR/CR mice compared with parent strain controls. Importantly, this differentially regulated immune response of SR/CR mice could not be found in response to challenge with the lymphoma cell line EL‐4.     

Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice: natural killer cell activity in cultured spleen leukocytes concomitant with T-cell-dependent immune interferon production

The characteristics and specificities of spleen and peritoneal cytotoxic cells generated during lymphocytic choriomeningitis virus (LCMV) infection of C3H/St mice were examined. Activated natural killer (NK) cell activity was identified in fresh leukocyte populations from the 2nd to 8th days postinfection, whereas virus-specific cytotoxic T-cell activity was detected from the 6th to 14th days. When leukocytes were cultured overnight at 37 degrees C before assay, T-cell activity was still observed, but nonspecific activated NK cell-like cytotoxicity was only detected on the 6th and to a lesser degree the 8th day postinfection. Overnight culture of leukocytes taken earlier in the infection eliminated their NK cell activity. Similar activities were seen with spleen cell, plastic-adherent peritoneal cell, and nonadherent peritoneal cell populations. The virus-specific cytotoxicity observed with adherent peritoneal cells was due to contamination with cytotoxic T cells, as shown by H-2-restricted cytotoxicity and sensitivity to anti-theta antibody and complement. The nonspecific cultured day 6 effector cell from either the spleen or peritoneum displayed killing specificities and other physical properties identical to those of activated NK cells, but had sensitivities to anti-theta antibody and complement intermediate between activated day 3 NK cells and cytotoxic T cells. Culture stable NK-like cells were not found in athymic nude mice, suggesting a T-cell-dependent mechanism. Whereas LCMV spleen homogenates contained 10-fold-higher levels of interferon at day 2 than at day 6 postinfection, substantially more (nearly 20-fold) interferon was made in cultures of day 6 cells than day 2 cells. Spleen interferon was predominantly type I, whereas the culture interferon was predominantly type II, as shown by acid lability studies. Significant levels of interferon were produced by nylon-wool-passed day 6 spleen cells, and virtually all interferon production was eliminated by treatment of either day 2 or day 6 cells with antibody to theta antigen and complement, suggesting that T cells produced the interferon in vitro. Furthermore, athymic nude mice had no culture-stable NK cells 6 days postinfection, and spleen cells from them failed to produce significant levels of interferon in vitro. Addition of interferon (type I, fibroblast) to cultured C3H spleen cells affect the already elevated levels of cytotoxicity in day 6 cultures, suggesting that the NK cells in the day 6 culture were already activated. Our results suggest that T cells responding to LCMV infection secrete interferon type II which causes the continued activation of NK cells in culture. The resulting population of activated NK cells therefore appears to be relatively stable in culture and to express more theta antigen because of this T-cell dependence. Although one could mistakenly or allospecific cytotoxic T cells or cytotoxic macrophages, more careful examination shows that they are most likely activated NK cells...     

A novel rat monoclonal antibody reactive with murine tumoricidal Kupffer cells and activated peritoneal macrophages from BCG-infected mice

A rat monoclonal antibody, termed 1A2.10D and raised against mouse BCG-activated peritoneal-macrophages, was used to detect antigenic determinant(s) on mouse tumoricidal activated peritoneal and liver macrophages from BCG-infected mice by indirect immunofluorescence flow cytometry and immunoblot analysis. This antibody was of the rat IgG2b subclass and not cytolytic to BCG-activated macrophages in the presence of rabbit complement. The antigen(s) was expressed on tumoricidal activated peritoneal and liver macrophages from BCG-infected mice and some macrophage cell lines. Its expression could not be detected on resident peritoneal cells, peritoneal exudate cells containing non- or low tumoricidal macrophages, thymocytes, resting bone marrow cells, normal spleen cells, various established mouse tumour cells or one macrophage cell line. Immunoblot analysis showed the antibody to bind to one major protein of 56,000 MW, and to be expressed on the peritoneal and liver macrophages from BCG-infected mice. Using this antibody and a fluorescence-activated cell sorter, selection was made of antibody-positive or -negative macrophages from BCG-infected mice to examine their tumour killing activity. The antibody-positive macrophages clearly showed higher tumour killing activity than the negative macrophages.     

Avian cellular immune effector mechanisms--a review

Considerable new knowledge has accumulated within the last few years on the immune functions of birds, and data show that the avian cellular immune effector mechanisms are quite similar to those of mammals. Cellular immunity in the chicken, the most widely studied avian species, is primarily mediated by T cells, macrophages and NK cells. The avian T lymphocytes express unique surface antigens that distinguish these cells from other cells. The T cells respond vigorously in vitro to T cell-specific mitogens and can perform cytotoxic and mixed lymphocyte reactions against allogeneic or tumour cell targets. The in vivo responses mediated by T cells include allograft and tumour cell rejection, graft-versus-host reaction and delayed type hypersensitivity reactions. T cells also serve as helper cells in enabling B Cells to produce antibody against thymus-dependent antigens. Avian T cells also perform immune regulatory functions and sub-populations of T suppressor cells can cause profound blockage of antibody production by B cells. Certain T cell functions require cooperation from other immune cells, particularly major histo-compatibility complex-compatible macrophages. Avian macrophages resemble their mammalian counterparts in being adherent and phagocytic, and they serve as accessory cells in a multitude of immune reactions as well as effector cells in their own right. Macrophages when co-cultivated in vitro with tumour target cells may non-specifically arrest proliferation of tumour cells or lyse tumour cells. They may also engage in specific anti-alloantigen cytotoxicity following hyperimmunisation. Under certain circumstances, macrophages can also be potent suppressors of immune reactivity. Birds appear to have a well developed NK cell system. NK cells of chickens share general characteristics with mammalian NK cells and lack the surface markers of T cells, B cells, or macrophages. Recent evidence indicates that the NK cell system plays a role in defence against Marek's disease. NK-like cells have also been shown to mediate ADCC in chickens and ducks. The immunoregulatory factors that may mediate avian immune effector cell functions are currently being actively studied.