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.     

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.     

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.     

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.     

Activated macrophages mediate interferon-independent inhibition of herpes simplex virus

Activated macrophages exhibit extrinsic antiviral activity (inhibition of virus replication in other cells) which may involve mechanisms similar to macrophage antitumor activity or macrophage-mediated immunosuppression. Peritoneal macrophages elicited in mice by Corynebacterium parvum vaccine suppressed the growth of herpes simplex virus (HSV) in infected cells by an interferon-independent mechanism. This was demonstrated by expression of activity against HSV-infected xenogeneic (Vero) cells. Culture supernatant fluids also did not mediate antiviral activity, and did not contain detectable levels of interferon (< 3 IU/ml). Moreover, antiviral activity was not affected by the presence of anti-mouse interferon IgG. Antiviral activity was expressed at 12–16 hr after infection, at the end of the first cycle of virus replication. Cell contact was required for optimal activity. No enhanced adsorption or phagocytosis of HSV by C. parvum macrophages could be detected nor was macrophage cytotoxicity responsible for the activity. Cytotoxicity (⁵¹Cr release) by macrophages for virus infected cells was low (< 6% specific cytotoxicity), and was not significantly higher with C. parvum macrophages than with resident macrophage controls. Although C. parvum macrophages were not cytotoxic at the macrophage-host cell ratio employed, they did significantly inhibit uptake of [³H]leucine by the host Vero cells. This suggests that inhibition of host cell metabolism by the macrophage, similar to macrophage immunosuppression, may be responsible for the antiviral activity in this system.     

Systemic administration of Corynebacterium parvum during sensitization to tumor alloantigen-modified response to rechallenge

Summary Intravenous administration of Corynebacterium parvum (C. parvum) to mice during a primary immune response against tumor alloantigens impairs their ability to generate memory cell-mediated cytotoxicity (CMC) in response to an intraperitoneal rechallenge with the same tumor alloantigens. Decreased CMC was observed in spleen and mesenteric lymph nodes, whereas CMC of lymphoid populations from the peritoneal cavity was merely delayed, reaching comparable levels to those found in control animals by day 5. Serum levels of cytotoxic antibody were unaffected, indicating that C. parvum administered during a primary immune response has selective effects on the cytotoxic memory response.     

Cell-mediated immune response to syngeneic UV induced tumors: I. The presence of tumor associated macrophages and their possible role in the in Vitro generation of cytotoxic lymphocytes

A primary in vitro sensitization system employing a chromium release assay was utilized to investigate reactivity of murine spleen cells toward syngeneic ultraviolet (uv) light induced fibrosarcomas. These tumors are immunologically rejected in vivo when implanted into normal syngeneic mice but grow progressivly when implanted into syngeneic mice that had previously been irradiated with subcarcinogenic levels of uv light. Following appropriate sensitization, spleen cells from both normal and uv irradiated mice are capable of developing cytotoxic lymphocytes in vitro against the uv induced tumors. It was subsequently discovered that in situ uv induced tumors all contained macrophages of host origin that became demonstrable only after enzymatic dissociation of the tumor tissue. These macrophages were immunologically active in vitro as their presence in the stimulator cell population was necessary to achieve an optimum anti-tumor cytotoxic response following in vitro sensitization. Anti-tumor reactivity generated by mixing spleen cells and tumor cells in the absence of tumor derived macrophages could be greatly enhanced by the addition of normal syngeneic peritoneal macrophages. When in vitro anti-tumor reactivity of spleen cells from normal and uv treated mice was compared under these conditions we again found no significant difference in the magnitude of the responses. In addition, the cytotoxic cells generated in response to uv induced tumors appeared to be highly cross reactive with respect to their killing potential. Cross reactive killing was observed between all uv induced tumors tested as well as with a syngeneic benz[a]pyrene (BP) induced fibrosarcoma. No cytotoxicity was observed against normal syngeneic PEC's even through these cells were shown to be susceptible to lysis by anti-H-2^k effector cells. It was concluded that: (a) A significant number of host-derived macrophages are present in uv tumor tissue. (b) These macrophages are important for the in vitro generation of tumor specific cytotoxicity. (c) Spleen cells from uv treated mice are capable of recognizing and responding against uv tumor associated antigens in vitro. Cytotoxic effector cells generated in response to uv induced tumors appear to have specificity for tumor associated antigens (TAA) present on all uv tumors tested as well as a syngeneic BP induced tumor. The relationship between in vivo and in vitro reactivity against uv tumors is discussed.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

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.     

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.     

Prostaglandin synthesis in spleen cell cultures of mice injected with Corynebacterium parvum.

Spleen cells of C57BL/6 mice produced high amounts of PGE in vitro when tested 5 to 10 days after injection of heat-killed C. parvum organisms. Little or no PGE was produced by spleen cells from untreated mice or from mice injected with a strain of coryneform bacteria that does not stimulate the lymphoreticular system of mice. Significant release of PGE from spleen cells of C. parvum injected mice could be detected as early as 30 min after initiating the cultures and maximal levels were usually seen after 48 hr. Treatment by indomethacin completely abolished this PGE production. Removal of the adherent population from the spleen cell suspension resulted in markedly decreased levels of PGE, but PGE release of the remaining population was never completely abolished. These data suggest that the cells responsible for most of the PGE synthesis in this system were adherent cells, presumably macrophages. The levels of PGE produced in spleen cells of C. parvum-treated mice were further increased by in vitro addition of C. parvum. This effect could also be observed after addition of zymosan particles indicating that it was not an immunologically specific effect. The reported data suggest that prostaglandins may represent important mediator molecules of the described immunostimulatory and immunosuppressive effects of C. parvum.     

Regulation of the In Vitro Secondary Cell-Mediated Cytotoxic Response against Syngeneic FBL-3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.     

Selective protection of murine thymic helper T cells from glucocorticosteroid inhibition by macrophage-derived mediators

We investigated the in vitro effects of dexamethasone (DEX) on the functional capacities of virgin murine T cells cultured in the absence and presence of adjuvant-stimulated macrophages or factors derived from them. Immunologically mature thymocytes, isolated on the basis of their inability to bind peanut agglutinin (PNA^? thymocytes), were used as virgin T cells. Treatment of PNA^? thymocytes with DEX for 24 hr in vitro eliminated their subsequent capacity to function as helper cells for primary humoral responses, to proliferate when stimulated by plant mitogens or allogeneic cells, or to generate T-cell-mediated cytotoxic responses. However, when PNA^? thymocytes were pretreated with DEX in combination with either adjuvant-activated macrophages or their culture supernatants, which contained Interleukin 1, the capacity of the T cells to subsequently express helper activity was preserved. The macrophage products, however, did not prevent DEX from inhibiting the capacities of PNA^? thymocytes to proliferate in response to plant mitogens or alloantigens or to generate cytotoxic effector cells; thus, protection was selective. The data indicate that, prior to activation, helper T cells are distinguished by their capacity to become steroid resistant in response to macrophage products. Although T-cell proliferative and cytotoxic responses have been reported to be protected from DEX inhibition by Interleukin 2, our results suggest that macrophages prevent steroid effects on virgin helper T cells by Interleukin-1-dependent mechanisms that do not involve Interleukin 2. While we have not delineated the biochemical pathways of protection, we show that the acquisition of DEX resistance by helper T cells cannot be attributed to the polyclonal induction of helper activity by macrophage factors.     

Immunoadjuvant activity of pyran copolymer: I. Evidence for direct stimulation of T-lymphocytes and macrophages

A single injection of pyran copolymer has been shown to greatly increase the number of hemolytic plaque forming cells to sheep erythrocytes (sRBC). Pyran given from 1 day before to 2 days after sRBC inoculation increased both specific activity and plaques/spleen, suggesting that macrophage activation was probably not responsible for the enhancement seen. In addition, pyran given 1 day prior to the primary injection of sRBC was found to increase the secondary response to SRBC given alone. As similar experiments using thymectomized irradiated bone marrow reconstituted mice showed no increase in specific activity following pyran administration, it was unlikely that pyran was acting directly on B cells. Furthermore, experiments measuring the antibody response to Escherichia coli lipopolysaccharide, a thymic independent antigen, pyran did not increase the response to this antigen. In contrast to the above, pyran delayed and depressed cell mediated cytotoxicity to the allogeneic DBA/2 P815 mastocytoma. However, no difference in the titers of cytotoxic antibody against mastocytoma cells was seen between pyran-treated and normal animals. Pyran was mitogenic for spleen cells in vitro. However, following the administration of pyran in vivo, mitogen induced blastogenesis in vitro to PHA and LPS was inhibited and this inhibition was determined to be macrophage-dependent. These results are consistent with a model in which the immunoregulatory effects of pyran act through macrophages and T-lymphocytes.     

The requirement of Ia-positive accessory cells for the induction of tumor-specific cytotoxic T lymphocytes in vitro

The mechanism for the induction of cytotoxic T cells specific for tumor-associated antigens was studied by using fractionated responder T cells, tumor cells, and accessory cells in vitro. The tumor-specific cytotoxic T cells were induced by culturing immunized spleen cells with the tumor cells in vitro for 5 days. Nylon-column-purified T cells alone did not induce cytotoxic T cells upon culture with tumor cells, but the addition of normal spleen cells as accessory cells did successfully induce the cytotoxic T cells, suggesting that the presence of accessory cells is required for the activation of tumor-specific cytotoxic T cells in vitro. The accessory function was associated with spleen cell populations adhering to a plastic dish, a Sephadex G-10 column or a nylon wool column, and was sensitive to anti-Ia serum and C treatment, but was resistant to anti-Ig serum or anti-Thy 1 serum and C treatment, suggesting that the accessory cells are Ia-positive macrophages. Not only syngeneic but also allogeneic macrophages had the accessory function and the allogeneic macrophages were also Ia positive. These results suggest that Ia-positive macrophages play a crucial role in the induction of tumor-specific cytotoxic T cells in vitro. The possible role of Ia-positive accessory cells in the induction of tumor-specific cytotoxic T cells is discussed from the standpoint of cellular interactions.     

Positive correlation between the levels of natural killer cells and the in vivo resistance to syngeneic tumor transplants as influenced by various routes of administration of Corynebacterium parvum bacteria.

Depending on the route of administration, heat-killed Corynebacterium parvum (C. parvum) bacteria caused an increase or decrease of natural killer (NK)-cell activity in mice. We used a syngeneic tumor with known susceptibility to NK lysis in vitro. The tumor was administered to mice whose NK levels had been increased or decreased by previous inoculations of C. parvum bacteria. A positive correlation between changes in NK-cell activity as measured in vitro and changes in tumor resistance as measured in vivo was observed. Additional evidence was provided in support of the view that NK cells may play an important role in resistance to tumor growth. The route of administration of C. parvum was considered important for protection against tumor growth.     

Adjuvant-induced nonspecific supressor cells: in vitro and in vivo studies

The in vitro mitogen responses of spleen cells from mice injected ip with the nonantigenic adjuvant, Al(OH)₃, are markedly depressed. This depressed reactivity was found to be mediated by a population of nylon wool adherent, Fc-receptor-bearing suppressor cells. Suppressor cells were detected only in the spleens of the adjuvant-treated mice, as the response of lymph node cells to mitogenic stimulation in vitro was found comparable to that of normal controls. Moreover, elevated levels of suppressive activity could be detected in sera of Al(OH)₃-treated mice during the first week after adjuvant administration, which, however, did not correlate with either the long-lasting presence of suppressor cells or the in vivo normal immune response of the adjuvant-treated animals. Studies designed to test the effect of suppressor cells on the generation of splenic PFC in vivo revealed that both the direct and indirect PFC responses against SRBC inoculated iv were enhanced rather than suppressed, as compared to those of the normal controls. Furthermore, the level of cytotoxic lymphocytes generated in spleens of Al(OH)₃-treated mice immunized with allogeneic tumor cells was equal to or higher than that of the normal controls. In view of the present results, we feel that the concept that splenic, nonspecific suppressor cells (macrophages) are immunosuppressive in vivo as well as the in vivo relevance of in vitro findings should be carefully reevaluated.     

Biological effects of the adjuvant Corynebacterium parvum. II. Evidence for macrophage-T-cell interaction

The mechanism underlying the markedly reduced PHA responsiveness of spleen and peripheral blood lymphocytes from Corynebacterium parvum-treated mice is due to inhibition of the responsive T-lymphocytes by C. parvum-activated macrophages. Inhibition is a result of a qualitative rather than quantitative change in the macrophage population and GVH-activated macrophages behave similarly. Corynebacterium parvum-activated macrophages need to be viable and will inhibit normal lymphocytes. This inhibitory effect appears to be mediated through cell-cell contact.     

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.     

Granzyme A Is Expressed in Mouse Lungs during Mycobacterium tuberculosis Infection but Does Not Contribute to Protection In Vivo

Granzyme A, a serine protease expressed in the granules of cytotoxic T and Natural Killer cells, is involved in the generation of pro-inflammatory cytokines by macrophages. Granzyme A has been described to induce in macrophages in vitro the activation of pro-inflammatory pathways that impair intracellular mycobacterial replication. In the present study, we explored the physiological relevance of Granzyme A in the control of pulmonary Mycobacterium tuberculosis infection in vivo. Our results show that, even though Granzyme A is expressed by cytotoxic cells from mouse lungs during pulmonary infection, its deficiency in knockout mice does not have an effect in the control of M. tuberculosis infection. In addition our findings indicate that absence of Granzyme A does not affect the protection conferred by the live-attenuated M. tuberculosis vaccine MTBVAC. Altogether, our findings are in apparent contradiction with previously published in vitro results and suggest that Granzyme A does not have a crucial role in vivo in the protective response to tuberculosis.