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.     

Macrophage migration stimulation factor, migration inhibition factor and the role of suppressor cells in their production.

Guinea pig lymph node lymphocytes and human peripheral blood lymphocytes when stimulated by specific antigen or mitogen will release factors that affect in vitro macrophage migration. Migration inhibition factor production appears to be under the control of suppressor cells which are T lymphocytes. When suppressor cells are generated by stimulation with Con A for 4 days, migration stimulation factor (M.St.F.) activity is found. In other situations where M.St.F. is found this is thought to be due to increased suppressor cell activity. For example, young adults produce this lymphokine when stimulated with Con A, whereas aged individuals produce MIF. Concanavalin A appears to be the mitogen of choice for M.St.F. production, and phytohemagglutinin for MIF production. The release of this putative factor M.St.F. from suppressor T cells helps to explain some of the difficulties that have existed in studies of macrophage migration inhibition.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Regulation of bone marrow lymphocyte production: IV. Cells mediating the stimulation of marrow lymphocyte production by sheep red blood cells: Studies in anti-IgM-suppressed mice,athymic mice,and silica-treated mice

Some cellular requirements have been examined for the stimulation of lymphocyte production in mouse bone marrow by injected sheep red blood cells (SRBC). The increased genesis of marrow lymphocytes after a single dose of SRBC assayed radioautographically after [³H]thymidine labeling was unimpaired in the marrow of mice treated with anti-IgM antibodies from birth to eliminate B lymphocytes, and in congenitally athymic mice lacking T lymphocytes. However, pretreatment of mice with silica to depress macrophage function completely abolished the SRBC effect both on the total lymphocyte production and on the number of B and null small lymphocytes in the marrow. Comparative studies were performed on the thymus and spleen. The results demonstrate that the stimulation of marrow lymphocyte production by SRBC is mediated by a silica-sensitive mechanism, does not require B or T lymphocytes, and is independent of the humoral immune response. Thus, extrinsic agents may amplify the production of primary B cells and other lymphocytes in the bone marrow by an antigen-nonspecific mechanism, putatively mediated by macrophages.     

Modulation of the immunosuppressive effects of splenic macrophages in Fischer rats bearing adenocarcinoma 13762

Summary The nature of spleen cells in Fischer rats bearing a large size (>1 cm diameter) mammary adenocarcinoma 13762A (MAC) which block the immunostimulating capacities of MTP² (a synthetic immunomodulator) and suppress proliferation in vitro of splenic T and B lymphocytes by their respective mitogens was investigated. Splenic macrophages were recognized as the suppressor cells by (a) restoration of mitogenic responses by depletion of macrophages from spleen cell suspensions and (b) continued suppressor activity in spleen cell suspensions of tumor bearers devoid of viable T lymphocytes. Macrophage contact with T lymphocytes was required for the inhibition of T lymphocyte proliferation by concanavalin A as shown by (a) the absence of suppressor activity in supernatants derived from cultured suppressor macrophages, (b) lowering of the suppressor activity of intact macrophages after treatment with neuraminidase, (c) lowering of the suppressor activity of macrophages by addition of red cells to spleen cultures of tumor bearers indicating red cell interference with macrophage-T cell interaction and (d) lack of inhibiting action of suppressor macrophages on allogenic T lymphocyte proliferation showing macrophage T cell recognition for suppression.Animals bearing a large size tumor exhibited spleen hypertrophy and an increase in macrophage:lymphocyte ratio and a decrease in red cell:lymphocyte ratio. Splenic macrophages did not appear to be implicated in blocking antitumor immunity induction since (a) suppressor macrophages were absent in spleens during the inductive phase of the immune response and (b) MAC implanted in allogenic Wistar rats grew to about 2 cm diameter, induced splenic suppressor macrophages but the tumor was later rejected by the animals. Collectively the results suggest that suppressor macrophages are the result of increasing tumor volume rather than its cause.This study was supported by a grant from the National Cancer Institute of Canada Abbreviations used: Con A, Concanavalin A; LPS, lipopolysaccharide; PHA, phytohemagglutinin; MTP, maltose tetrapalmitate; MAC, mammary adenocarcinoma 13762; RPMI, Roswell Park Memorial Institute; TBR, tumor bearing rat; RBC, red blood cell     

Interleukin 2 enhancement of lymphokine secretion by T lymphocytes: analysis of established clones and primary limiting dilution microcultures

The effect of exogenous interleukin 2 (IL 2) on lymphokine production by T lymphocytes was examined in two systems: the secretion of macrophage-activating factor (MAF) and interferon (IFN) by cloned long-term T cell lines, and a limiting dilution system for estimating the frequency of precursors of MAF-secreting cells in normal spleen. An IL 2-containing, MAF- and IFN-free supernatant from the EL-4 thymoma (EL-4 SN) significantly enhanced release of MAF and IFN by mitogen- or antigen-stimulated, cytolytic or noncytolytic T lymphocyte clones directed against alloantigens or Moloney leukemia virus-associated antigens. Highly purified IL 2 produced equivalent enhancement as EL-4 SN in cultures of alloreactive clones stimulated with concanavalin A. Kinetics experiments showed that EL-4 SN increased both the rate and duration of MAF release by T cell clones. EL-4 SN also increased MAF production when added during restimulation of limiting dilution cultures of positively selected Lyt-2+ and Lyt-2- C57BL/6 splenic T lymphocytes activated against DBA/2 alloantigens. This enhancement resulted in a threefold increase in the apparent precursor frequency of MAF-secreting cells among Lyt-2+ lymphocytes, but did not affect the frequency among Lyt-2- cells. Additional analysis indicated that average MAF production in cultures of Lyt-2-+ cells was sixfold lower than in cultures of Lyt-2- cells, and hence that EL-4 SN allowed detection of a significant proportion of Lyt-2+ cell cultures secreting low levels of MAF. Under these improved conditions, the MAF assay detected the majority of responding Lyt-2+ and Lyt-2- lymphocytes.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses. I. Tumor-specific Lyt-1+2- T cells are required for producing MAF able to generate cytolytic as well as cytostatic macrophages

In the present study we investigated some of the cellular mechanisms for the generation of macrophage-activating factor(s) (MAF) in immune responses to tumor antigens. C3H/HeN mice were immunized to syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma by intradermal inoculation of viable tumor cells, followed by the surgical resection of the tumor. Spleen and lymph node cells from these tumor-immune mice were stimulated in vitro with the corresponding tumor cells, and supernatant from such a culture was tested for an ability to activate macrophages to exert their cytostatic and cytolytic activities as detected on tumor cells unrelated to immunizing tumors. Peritoneal adherent cells as a macrophage source, which were preincubated with supernatant from co-culture of tumor-unimmunized normal spleen and lymph node cells plus tumor cells, failed to exhibit any significant antitumor effect on unrelated X5563 tumor cells, whereas the addition of supernatant from cultures containing immune lymphocytes to adherent cells resulted in appreciably potent cytostatic and cytolytic effects on X5563 tumor cells, indicating the generation of MAF in culture supernatant. The activation of tumor-immune spleen and lymph node cells for MAF generation was tumor-specific, because anti-MH134- and anti-MCH-1-A1-immune lymphocytes produced MAF by the stimulation with the respective but not with the other alternative tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 but not with anti-Lyt-2.1 antibody plus complement before culturing. These results indicate that the tumor-specific Lyt-1+2- T cell subset has a crucial role in generating MAF by which an adherent cell population as a source of macrophages acquires the potential for inducing a cytolytic as well as a cytostatic effect on tumor cells.     

Suppressive effect of macrophages on interferon-gamma production by human peripheral lymphocytes stimulated with Mycoplasma pneumoniae

Production of interferon (IFN)-gamma was investigated in human peripheral lymphocytes stimulated with Mycoplasma pneumoniae. Lymphocytes obtained from non-immune individuals produced no IFN. IFN-gamma was produced by T cells obtained from immune individuals, and the helper/inducer T cells produced two- to sixfold higher titer of IFN-gamma than the suppressor/cytotoxic T cells. The addition of macrophages in T cell cultures suppressed the production of IFN-gamma; this differs from the previous result wherein the addition of macrophages enhanced the production of IFN-gamma, when stimulated with mumps virus or measles virus.     

Composition of splenic T lymphocytes in allophenic mice.

Guinea pig peritoneal macrophages were activated in vitro by culturing with MAF (macrophage activating factor)-containing fractions from stimulated lymphocytes. These macrophage preparations demonstrate a 60% increase in the production of prostaglandins of the E series (PGE) when compared with macrophages cultured with fractions from unstimulated lymphocytes. PGE accumulation in macrophage cultures is maximal after 24 hr with MAF; tumor cytotoxicity is also maximal at this time. The final PGE concentration in cultures of activated macrophages averaged 3 × 10^?8M.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

Changes in lymphocyte subsets and macrophage functions from high, short-term dietary ethanol in C57/BL6 mice

Chronic administration of a diet containing 7% ethanol (36% of total calories) for 8 days to male C57/BL6 mice resulted in significant changes in functioning of macrophages. Peritoneal exudate macrophages from the ethanol-fed mice released more tumor cell cytotoxic materials upon culturing in vitro than cells from controls. However, peritoneal exudate cells continued to respond to exogenous beta carotene in vitro to produce additional cytotoxic materials. Phagocytosis of sheep red blood cells in vitro was suppressed in cells from ethanol treated mice. The number of splenic lymphocytes of various subsets was significantly changed by the ethanol exposure. Total T cells and T suppressor cells were lower, with a significant decrease in B cells containing IgM on their surface. The percentage of spleen cells showing markers for macrophage functions and their activation were significantly reduced. It is concluded that short-term chronic consumption of dietary ethanol, which was sufficient to produce physical dependence, results in significant alterations in lymphocyte subtypes and suppression of some macrophage functions.     

Reversal of macrophage-augmented MLR reactivity by tumor-induced splenic suppressor T cells and their soluble factor(s)

Augmenting concentrations of macrophages or their supernatants failed to reverse T-cell hyporeactivity in tumor-bearing mice (TBM). Serial passaging over nylon wool columns depleted TBM spleen cells of a mildly adherent tumor-induced suppressor cell and restored mixed lymphocyte reaction (MLR) reactivity to the purified TBM T-cell population. The tumor-induced suppressor cell was extensively plated to remove macrophages and characterized as a T cell by its anti-Thy 1 serum sensitivity. This suppressor T cell, when added to normal T cells, abrogated all enhancing effects caused by addition of macrophages. Suppressor T-cell inhibition was non-contact dependent, since suppressor T-cell supernatants inhibited MLR activity in T cells treated with enhancing concentrations of macrophage supernatants. Thus it appears that tumor-induced T-cell debilitation is a reversible phenomenon, mediated not by macrophages but by soluble factor(s) from a nonphagocytic, mildly adherent, suppressor T cell.     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Stimulation of mouse migration inhibitory factor (MIF) production form MSV-immune lymphocytes by soluble tumor-associated antigen: requirement for histocompatible macrophages.

Spleen cells from C57BL/6 mice immunized with murine sarcoma virus (MSV) are capable of producing migration inhibition factor (MIF) in response to stimulation with a specific tumor-associated antigen prepared by solubilization with 3 M KCL. We have previously demonstrated that this response is T cell-dependent. Further investigations into the effector cells involved in the production of MIF have revealed that spleen cells from mice immunized with MSV cannot produce MIF when stimulated with tumor extract if the population has been previously depleted of macrophages. However, the response can be restored by adding nonimmune syngeneic macrophages but not by allogeneic macrophages. The inability of allogeneic macrophages to provide this function was not due to their increased suppressor activity since in mixing experiments they did not interfere with the ability of immune spleen cells to produce MIF. Furthermore, they were not defective since they could supply this "cooperative function" to appropriate F1 mice. The results indicate that macrophages are required for stimulation of MIF by soluble tumor antigens and that for efficient interaction the macrophages and lymphocytes must share some genetic similarities.     

Activation of the production of cytotoxic factors by mouse spleen cells exposed to the combined action of lipopolysaccharide and muramyl dipeptide in vitro

Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) stimulated murine splenocytes in vitro to produce cytotoxic factors (CF) that killed target cells L-929. This effect was synergic at LPS dose of 10 ng/ml and MDP dose of 10 micrograms/ml. CF production started 2 hours after spleen cell activation and was maximum in 6 hours. CF were produced by macrophages as well as by lymphocytes stimulated by LPS, MDP or their combination. However, synergic effect of immunomodulators was registered only if nonfractionated spleen cells were stimulated during 24 hours. Lymphocytes depleted on T cells did not lack the ability to generate CF upon activation. In addition, LPS and MDP activated synergically the production of interleukin-I by spleen cells in vitro.     

Polyclonal activation of murine B lymphocytes by immune complexes

Murine splenic B lymphocytes are stimulated by homologous immune complexes to proliferate and secrete polyclonal antibody. The use of antibody from whole serum or monoclonal antibodies to form complexes resulted in the stimulation of mouse B lymphocytes. The ratio of antibody to antigen appears to be critical for the generation of the polyclonal antibody response. Because antigen and antibody are added independently at culture initiation, the exact nature of the complex is unknown, but optimal polyclonal antibody formation occurs in slight antigen excess. Immune complex-induced polyclonal antibody production requires the presence of both macrophages and T cells, whereas B cell proliferation requires only macrophages. The role of the macrophage appears to be to cleave a low m.w. (17,000) fragment from the complex, which is responsible for lymphocyte activation.     

Molecular mechanisms involved in T cell activation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-induced proliferation of cloned cytotoxic T lymphocytes

The role that extracellular calcium plays in activating resting cloned cytotoxic T lymphocytes (CTL) to proliferate and to produce lymphokines was examined. In these cells, stimulation with interleukin 2 (IL-2) induced a proliferative response without a concomitant production of macrophage-activating factor (MAF), whereas stimulation with antigen or lectin (in the absence of IL-2) induced MAF production but not proliferation. In the case of IL-2-induced proliferation, extracellular calcium was required to initiate proliferation as well as to prevent cellular arrest later in the G2 + M phase of the cell cycle. In MAF production extracellular calcium was required both to activate the phosphatidylinositol signal-transducing mechanism and to mobilize intracellular calcium in antigen- or lectin-stimulated cytotoxic T lymphocytes. Further, extracellular calcium was required for only 8 of the 18 hr of stimulation time which was needed to achieve maximal MAF production, indicating that both calcium-dependent and -independent events exist in the signal pathway. Additional experiments with calcium ionophores and activators of protein kinase C indicated that although both intracellular calcium mobilization and de novo protein phosphorylation are involved in MAF production, an optimal increase in the level of intracellular calcium by itself is insufficient to induce the production of this lymphokine.     

Regulation of immunoglobulin production in human peripheral bood leukocytes: cellular interactions.

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This system was shown to be totally T lymphocyte dependent as purified B lymphocytes (less than or equal to 1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period. T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished. T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes campable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populations in the mouse.     

Production of specific macrophage activating factor by lymphocytes from tumor-bearing mice.

Lymphocytes from C57BL mice bearing a syngeneic UV-induced fibrosarcoma (UV-112) produced macrophage activating fatcor (MAF) when cultured with UV-112 cells in vitro. This MAF rendered normal C57BL macrophages cytotoxic in vitro to UV-112 cells. MAF production and lymphocyte-mediated cytotoxicity were detected in the early stages of tumor growth, but were absent in mice bearing large tumors. This eclipsed reatcivity was specific for the growing tumor. Lymphocytes from mice bearing a large UV-112 tumor were still able to produce MAF in response to B16 melanoma to which they had been preimmunized. In all instances, the MAF produced was specific in that it rendered syngeneic macrophages cytotoxic against only the tumor used for immunization.     

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.