In vitro and in vivo release of cytostatic factors from Lactobacillus casei-elicited peritoneal macrophages after stimulation with tumor cells and immunostimulants

Summary The effect of tumor cells and immunostimulants on the release of cytostatic factors (CF) from Lactobacillus casei YIT 9018 (LC)-, Corynebacterium parvum (CP)- or peptone-elicited peritoneal macrophages (PM) was investigated in vitro and in vivo. Significant release of CF into the culture medium from PM elicited with LC was induced by seven of eight mitomycin C-pretreated tumor cell lines and not by normal spleen cells, while no CF was released extracellularly from peptone-elicited PM given the same stimulus. CF were released from LC-elicited PM (LCEPM) after stimulation with LC, bacille Calmette-Guérin, streptococcal preparation OK-432, fucoidan or lipopolysaccharide, and LC but not CP induced CF production in the peritoneal cavities of LC- or CP-primed mice. The release of CF from LCEPM after stimulation with mitomycin C-pretreated 3T12-3 cells was inhibited by D-mannose and not by L-fucose. L-Rhamnose and mannose 6-phosphate, but not D-mannose or L-fucose, caused the release of CF from the PM.It was suggested that the release of CF from activated PM is caused by stimulation by some tumor cells, sugars, or bacterial immunostimulants, D-Mannose and L-rhamnose on the surface of tumor cells or bacteria, respectively, may play an important role in the release of CF from activated macrophages.     

Suppressor T cells and suppressor macrophages induced by Corynebacterium parvum

Corynebacterium parvum, injected intravenously into C57B1/6 mice (H-2^b) previously alloimmunized with P815 (H-2^d) mastocytoma cells, generated splenic suppressor cells that inhibited the development of primary cytotoxic lymphocytes in vitro. These suppressor cells differed from those generated by intravenous C. parvum injection of naive C57B1/6 mice. The former suppressor cells were effectively induced by administration of 700 μg of C. parvum whereas the latter suppressor cells were dependent upon higher doses (1400 μg) of adjuvant for their activation. Furthermore, suppressor cells generated in alloimmunized mice could only suppress C57B1/6 anti-P815 in vitro cytotoxic responses whereas suppressor cells generated in naive mice could suppress C57B1/6 anti-CBA (H-2^k) responses as well. Suppressor cells were not H-2 restricted in their action. Fractionation of spleens from alloimmunized, C. parvum-treated mice revealed the presence of suppressor T cells and suppressor macrophages. We were unable, however, to determine which cell was responsible for “antigen specificity” of suppression since the fractionation procedures seemed to trigger both suppressor cell types prior to adding them to the primary culture.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

Lymphocyte and macrophage adenyl cyclase activity in animals with enhanced cell-mediated resistance to infection and tumors.

As cyclic AMP has been associated with the inhibition of lymphocyte cytotoxicity, studies were performed to investigate adenyl cyclase activity in lymphocytes and macrophages of Toxoplasma-infected mice in which the efferent limb of the cell-mediated immune response had previously been found to be activated. In peritoneal or splenic lymphocytes from $\text{Balb}\text{c}$ mice chronically infected with Toxoplasma in which growth of an isogeneic bladder tumor was found to be inhibited, adenyl cyclase activity was significantly less than in lymphocytes from uninfected control mice. Stimulation by prostaglandin E₁ or NaF in vitro led to higher levels of adenyl cyclase activity in lymphocytes from unifected animals than in cells from Toxoplasma-infected animals. Similar observations were made with peritoneal macrophages from Toxoplasma-infected and uninfected mice. Lower levels of adenyl cyclase activity were also found in lymphocytes from tumor-bearing mice than in lymphocytes from nontumor-bearing controls. These data suggest that production of cyclic AMP by lymphocytes is inhibited with activation of certain cell-mediated immune functions.     

Enhancing and suppressive effects of macrophages on T-lymphocyte stimulation in vitro.

The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [¹²⁵I]UdR incorporation as a measure of lymphocyte proliferation. [¹²⁵I]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers of peritoneal macrophages beyond 5–10%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9–10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [¹²⁵I]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.     

Suppression of lymphokine production: I. Macrophage-mediated inhibition of MIF production

Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum, whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.     

Cytotoxic factor production by kupffer cells elicited with Lactobacillus casei and Corynebacterium parvum

Summary The ability of Kupffer cells, spleen macrophages, pulmonary macrophages, and peritoneal macrophages (PM) to produce cytotoxic factor (CTF) was investigated in vitro. The production of CTF by Kupffer cells elicited with Corynebacterium parvum (CP) or Lactobacillus casei YIT9018 (LC9018) was higher than that of spleen, pulmonary macrophages, or PM. In addition, oxygen radical (OR) production by Kupffer cells or PM was measured. The production of OR by Kupffer cells or PM was significantly augmented by i.v. or i.p. injection of LC9018 or CP. No significant correlation was observed between the increase in OR production by Kupffer cells or PM and CTF production by Kupffer cells or PM elicited with either organism. It was suggested that activated Kupffer cells may be one important source of CTF production in serum and that the CTF-producing macrophages may be different from the OR-producing macrophages.     

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor

The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

Inhibition of memory cell-mediated cytotoxic response by systemic administration of Corynebacterium parvum.

Intravenous administration of Corynebacterium parvum to mice during a developing immune response to alloantigens resulted in the marked inhibition of the generation and expression of memory cell-mediated cytotoxic response in the spleen. The inhibition was observed following rechallenge in vivo or by in vitro culturing with the same alloantigen. The impairment in vitro was due, in part, to the generation of regulatory cells which were non-T phagocytic cells, probably macrophages activated by C. parvum administration. These suppressor macrophages appear to act by inhibiting proliferation and clonal expansion of memory cytotoxic cells.     

Leishmania major: Secreted antigens of Leishmania major promastigotes shift the immune response of the C57BL/6 mice toward Th2 in vitro

BALB/c mice are sensitive to Leishmaniamajor infection, while C57BL/6 mice are resistant and able to mount an effective immune response against the parasite. Since the secreted antigens of L. major suppress the proliferation of BALB/c mice lymphocytes in vitro, we analyzed their effects on the immune system of resistant C57BL/6 mice. Secreted antigens were semi-purified and two fractions with immunosuppressive activity were isolated. 15 μg/ml of fraction could suppress 60% of lymphocyte proliferation and prevent the stimulated lymphocytes entering from G1 phase into the S phase of the cell cycle. These fractions decreased the production of IFN-γ, increased IL-4 level in the lymphocyte culture and down-regulated the nitric oxide production by activated macrophages. These results may suggest that L. major parasite by secreting immunosuppressive factors could down-regulate the immune system of both sensitive and resistant mice for own survival advantage.     

Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr, whereupon either the inhibition of [¹²⁵I]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8–10% and 10%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4–6 and 6–8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.     

Separation of functionally distinct subpopulations of Corynebacterium parvum-activated macrophages with predominantly stimulatory or suppressive effect on the cell-mediated cytotoxic T cell response.

Peritoneal cells (PEC) from mice injected ip with Corynebacterium parvum (CP) showed greatly enhanced suppressive activity on the growth of syngeneic tumor cells and on the generation of alloreactive cytotoxic T lymphocytes (CTL) in vitro. On the other hand, CP-activated PEC exhibited increased immunostimulatory (accessory or A cell) activity as measured by the restoration of the CTL response of nonadherent spleen cells. After fractionation of the CP-activated PEC according to cell size by velocity sedimentation, the mutually antagonistic A cell and immunosuppressive activities were clearly separated and found to be associated with functionally distinct subpopulations of macrophages. Thus A cell function was detected in fractions rich in small and medium sized macrophages which were probably derived from recently arrived monocytes. Immunosuppressive (and anti-tumor) activity was associated with the largest macrophages which were almost devoid of A cell function and probably represented a highly activated and differentiated macrophage subpopulation.     

Mechanism for Macrophage Activation against Corynebacterium parvum—Participation of T Cells and Its Lymphokines

It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T-cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c- + / + mice was abrogated completely by the pre-treatment of spleen cells with anti-Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c-nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin-P (a PHA-induced lymphokine) made it possible for BALB/c-nu/nu mice to produce TNF, associated with an induction of Lyt-1⁺ cells and Lyt-2⁺ cells. However, treatment with the lymphokine did not augment the increases of Ia⁺ cells or liver and spleen weights. These results suggest that increasing the number of Ia⁺ cells is not sufficient to bring about TNF production; Ia⁺ cells must also be stimulated by T cells or T-cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia⁺ cells against C. parvum.     

Studies on the specificity of killing of intracellular pathogens by macrophages.

To determine whether macrophages can discriminate in an immunologically specific manner between the intracellular pathogens which they inhibit or kill, unelicited peritoneal macrophages from mice infected with either of two related but antigenically dissimilar protozoa were challenged with these protozoa in vitro. Experimental conditions were varied in an attempt to establish a state in vivo in which macrophage specificity might be demonstrated. No differences could be discerned between the ability of macrophages from three different strains of mice infected with the protozoa to kill Besnoitia and Toxoplasma. The effect of macrophages on Toxoplasma as compared with Besnoitia did not evolve or vary during development, expression, or decline of an immune response, i.e., with varying times after infection of mice as well as with varying times after treatment of mice with irradiated Toxoplasma. The route of infection could not be shown to confer specificity on macrophages, as subcutaneous and intraperitoneal inoculation of Toxoplasma did not lead to differential ability of macrophages to inhibit or kill the protozoa. The different strains of protozoa used for infection of mice did not affect the ability of peritoneal macrophages from Besnoitia- and Toxoplasma-infected mice to inhibit multiplication of or kill Besnoitia and Toxoplasma comparably in vitro. Peritoneal macrophages of mice treated with Corynebacterium parvum kill both organisms efficiently. These macrophages were employed to determine whether stimulation of macrophages by treatment of mice with a substance unrelated to the protozoa would produce activated macrophages. Uninfected mice and mice infected with either Besnoitia or Toxoplasma were challenged with varying doses of the protozoa in parallel with examination of macrophages from the same groups of mice in vitro to determine whether the presence of stimulated macrophages in the peritoneal cavity was necessary for protection against Toxoplasma and Besnoitia, and if so if their presence was sufficient for protection. Only mice with activated peritoneal macrophages were protected. However, protection was greater when the primary infection was with the same organisms used for challenge at a time when macrophages inhibited or killed both protozoa efficiently in vitro. The possible role of other effector cells, subpopulations of macrophages of different functional abilities in various sites, and antibody or other lymphocyte products acting in concert with macrophages as factors which may explain the differences observed between in vivo protection and in vitro capacity to inhibit or kill the protozoa are discussed.     

Suppression of Splenic Lymphocyte Proliferation by Eucommia ulmoides and Genipin

We investigated the modulation of innate and adaptive immune cell activation by Eucommia ulmoides Oliver extract (EUE) and its ingredient genipin. As an innate immunity indicator, the phagocytic activity of macrophages was determined by measuring engulfed, fluorescently labeled Escherichia coli. As a surrogate marker for the respective activation of cellular and humoral adaptive immunity, concanavalin A (Con A) and lipopolysaccharide (LPS) induction of primary splenocyte proliferation was assayed in in vitro and ex vivo systems. EUE and genipin suppressed the proliferation of primary splenic lymphocytes induced by Con A or LPS, but not macrophage phagocytosis. Oral administration of EUE and genipin to mice decreased splenic lymphocyte proliferation induced by Con A or LPS. These results revealed that E. ulmoides and genipin suppressed cellular and humoral adaptive immunity, and they suggest that E. ulmoides and genipin are promising candidates for immunosuppressive drugs that target diseases that involve excessive activation of adaptive immunity.     

Chemical characterization and radioprotective effect of polysaccharide from <Emphasis Type="Italic">Monostroma angicava</Emphasis> (Chlorophyta)

Polysaccharide from the green alga Monostroma angicava was extracted with boiling water and was purified by ion-exchange and size-exclusion column chromatography. The radioprotective effect of the polysaccharide was investigated in mice. The results show that polysaccharide from M. angicava has a different chemical composition to other Chlorophyta having a high rhamnose – containing sulfated polysaccharide. The sulfate ester content was estimated to be 21.8%. When the polysaccharide was applied to BALB/c mice following whole-body X-ray irradiation the counts of leukocytes, thrombocytes and erythrocytes recovered more rapidly in the polysaccharide treated mice after irradiation. In the irradiated mice, the polysaccharide significantly increased the spleen index, natural killer cytostatic activity and the transformation response of splenic lymphocytes. The present observations suggest that polysaccharide from M. angicava led to leukocytogensis and hematopoetic activation in mice after irradiation and that the biological response might be caused by immune activation.