Effects of immune lymphocyte products and serum antibody on the multiplication of Toxoplasma in murine peritoneal macrophages.

In vitro studies on cell-mediated immunity against Toxoplasma infection were carried out by estimating the ability of antigenically stimulated lymphocytes. Splenic lymphocytes from normal mice and from hyper-immunized mice were cultured in the presence or absence of Toxoplasma lysate antigen. The cell-free supernatant fluids from the lymphocyte cultures were assassed for their ability to alter the functional capacities of normal macrophages. When glycogen-induced peritoneal macrophages were incubated with the culture supernatant from immune lymphocytes reacting with specific angigen, the intracellular multiplication of the organisms was inhibited remarkably. In contrast, the addition of antitoxoplasma antibody to culture medium had no effect on the enhancement of phagocytic activity of culture macrophages. However, when extra-cellular organisms were exposed to fresh or heat-inactivated immune serum just before infection of the monolayers, the intracellular multiplication of Toxoplasmas was inhibited significantly by either activated or normal macrophages.     

In vitro and in vivo studies on the mechanism of experimental autoimmune thyroiditis in guinea pigs

Thyroid explants of inbred strain 13 guinea pigs were grown in a semisynthetic medium containing 0.3 IU of thyroid-stimulating hormone. The monolayer retained the capacity in vitro to form thyroglobulin. Sensitized lymphocytes from animals with autoimmune thyroiditis could specifically lyse these thyroid target cells in vitro in the presence of an appropriate amount of specific antigen. This cytotoxicity was not observed in thyroid epithelial cells which had been incubated (a) with normal lymphocytes or (b) with purified macrophages either from normal animals or from animals with autoimmune thyroiditis. When thyroid cells were incubated with hyperimmune antithyroglobulin serum, cytolysis did not occur, whether or not complement was added. The cytopathic effect of sensitized lymphocytes was further demonstrated to be caused by a soluble cellular product, termed thyroid cytotoxic factor, or TCF, which was released from sensitized lymphocytes under the stimulation of specific antigen, thyroglobulin, and could exert a cytotoxic effect directly on the target cells. Direct cell-to-cell contact was not required in this type of cell-mediated cytolysis.     

Identification of the principal cell type yielding immune RNA capable of transferring tumor-specific cellular cytotoxicity

A search was made for the lymphoid cell type(s) which are the source of immune RNA (I-RNA) capable of transferring tumor-specific cell-mediated cytotoxicity (CMC). Hartley guinea pigs were immunized with syngeneic murine fibrosarcomas (BP-10 or BP-11) induced by 3,4-benzo(a)pyrene in C3H/HeJ mice, and the I-RNA was extracted individually from their spleens, lymph nodes, and peritoneal exudate (PE) cells. All three I-RNA preparations were able to convert normal C3H/HeJ mouse lymphocytes to effector cells significantly cytolytic to the specific syngeneic mouse tumor in vitro. Furthermore, lymphocytes and macrophages were purified from the spleens, lymph nodes, and PE cells of tumor-immunized guinea pigs. I-RNA was extracted from these purified cell populations and also from the pooled guinea pig lymphoid tissues. Normal C3H/HeJ lymphocytes were incubated with each type of I-RNA and tested in vitro for CMC against the specific tumor cells. Significant CMC against BP-10 targets was observed with mouse lymphocytes incubated with I-RNA extracted from pooled lymphoid tissues of BP-10 tumor-immunized guinea pigs. There was a reduced but still significant CMC when mouse lymphocytes were incubated with I-RNA extracted from purified guinea pig lymphocytes, whereas there was a markedly increased CMC when the I-RNA was extracted from purified guinea pig macrophages. As indicated by sucrose density gradient analysis, the lesser effectiveness of lymphocyte I-RNA was not due to RNA degradation resulting from lymphocyte purification or I-RNA extraction. Treatment of all types of I-RNA with RNase abrogated the transfer of CMC, whereas treatment of I-RNA with DNase or pronase did not. RNA extracted from the lymphoid tissues of guinea pigs immunized with complete Freund's adjuvant without tumor was ineffective. Mouse lymphocytes incubated with BP-10 macrophage I-RNA destroyed BP-10 but not BP-11 tumor cells, whereas lymphocytes incubated with BP-11 macrophage I-RNA killed BP-11 but not BP-10 tumor cells, thus indicating tumor specificity of the immunity transferred by macrophage I-RNA. Our results suggest that macrophages are the principal source of I-RNA capable of transferring tumor-specific CMC.     

Activated lymphocytes depress phagocytosis of latex particles by human monocyte-macrophages

In this study, monocyte-macrophages of normal human donors were cultured with and without lymphocytes and antigen in order to define the effect of antigen-stimulated lymphocytes on phagocytosis by macrophages. Phagocytosis was assessed by uptake of radio-labeled latex particles by macrophage monolayers. Although no effect was noted when purified macrophage monolayers were cultured in the presence of antigen, marked inhibition of phagocytosis was observed when macrophages were cultured in the presence of autochthonous antigen-stimulated lymphocytes. The degree of phagocytic depression correlated with the delayed cutaneous hypersensitivity response of the donor and with the concentration of antigen present in the system.     

Studies on the cellular site of action of macrophage RNA-antigen complexes.

Nonadherent spleen cell populations exposed in vitro to ribonucleic acid-rich preparations from mouse macrophages that had been incubated with human γ-globulin (RNA : HGG) were able to produce specific antibody, as measured by rosette-forming cells, in lethally irradiated (800 R), reconstituted, syngeneic mice. Exposure of the RNA to anti-HGG serum abrogated its ability to initiate antibody synthesis, as did monospecific anti-human γ chain and anti-human κ chain serum. Normal rabbit serum, anti-bovine albumin serum, anti-human μ chain or anti-human λ chain serum, when substituted for anti-HGG serum had no effect. Thus, the presence of both γ heavy chains and κ light chains of the antigen in the RNA moiety was indicated. Although both an adherent and a nonadherent cell were required by HGG to stimulate rosetteforming cells in irradiated mice, the need for the adherent cell was eliminated when RNA : HGG was substituted for HGG. In addition, anti-θ-treated bone marrow cells exposed to the RNA : HGG were capable of rosette-cell formation, suggesting that RNA attachment converted a T cell-dependent antigen to a T cell-independent antigen.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages

Alveolar macrophages obtained by lung lavage from rats were incubated with monoclonal mouse anti-DNP IgE and specific antigen (DNP-HSA) and were found to release a slow reacting substance (SRS), which was characterized by high performance liquid chromatography as leukotriene C4 (LTC)4. Alveolar macrophages incubated with 1 microM A23187 (calcium ionophore) released similar amounts of SRS (6.0 +/- 2.2 and 5.7 +/- 3.7 X 10(-10) mol of LTC4 per 5 X 10(6) alveolar macrophages, respectively). The optimal conditions and mechanism of LTC release by IgE and antigen were examined. LTC4 release was maximal when freshly retrieved alveolar macrophages were incubated for 20 min with 10 micrograms/ml IgE and then for 20 min with 100 ng/ml antigen or for 20 min with IgE and antigen that had been preincubated together for 30 min at room temperature. In addition, LTC4 release was maximal when cells were challenged with IgE and antigen in a protein-free balanced salt solution and when the cells were tumbled to prevent adherence. Dose response experiments revealed that macrophages released LTC4 when stimulated with as little as 10 ng IgE and 100 ng DNP-HSA. Alveolar macrophages did not release LTC when challenged with IgE or DNP-HSA alone. Activation of LTC4 release by IgE and antigen was rapid in onset (2.5 to 5 min), and washing to remove fluid phase IgE and antigen revealed that once activated, alveolar macrophages were capable of prolonged and continuous release of LTC4. Peritoneal lavage cells stimulated with IgE and antigen did not release SRS but could release SRS when incubated with A23187 (5.7 +/- 1.3 X 10(-10) mol LTC4/5 X 10(6) macrophages). A large variability existed between individual rats in the ability of their alveolar macrophages to be activated by IgE and antigen to release LTC4. DNP-HSA labeled with 125I was used to show formation of immune complexes of IgE and antigen when IgE and antigen were incubated together before macrophage challenge. IgE immune complexes containing as little as 2 ng of antigen elicited the release of LTC4 from alveolar macrophages. These data indicate that rat alveolar macrophages release primarily LTC4 when challenged with IgE immune complexes, and that the alveolar macrophage may differ in this respect from peritoneal macrophages that do not release detectable quantities of LTC4 when challenged under identical conditions.     

艾氏腹水癌细胞核糖核酸体外对C_(57)BL/6脾自然杀伤活性的影响

本文采用~(51)Cr释放法,研究了艾氏腹水癌细胞核糖核酸(EAC-RNA)体外对C_(57)BL/6小鼠脾自然杀伤细胞(NK)对YAC-1靶细胞杀伤活性的影响。结果表明,EAC-RNA能显著抑制NK活性。经RNase处理后,其抑制活性消失,DNase或Pronase的处理不改变其抑制活性。     

Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.     

The association of suppressor cells with mononuclear cell aggregates in spleens of tumor-bearing mice

Spleen cell suspensions from mice with progressive B-16 melanoma consistently contained significant numbers of aggregates of mononuclear cells (MN-Agg), when compared to spleen cell suspensions from normal mice or mice in the early stages of tumor growth. Histological, histochemical and immunological characterization of the cells involved in MN-Agg from tumor-bearing mice indicated that aggregates were composed of macrophages and T and B lymphocytes. The formation of MN-Agg was dependent upon the macrophage content of the spleens of tumor-bearing mice since the appearance of MN-Agg correlated temporally with an increase in the number of splenic macrophages demonstrable in tumor-bearing animals. An antigen nonspecific suppressor cell was identified in the spleens of mice 15 days following the appearance of palpable B-16 tumor, and the appearance of the suppressor cell population closely correlated with the appearance of MN-Agg. Additionally, fractionation of MN-Agg-containing cell suspensions demonstrated that fractions highly enriched in MN-Agg were concomitantly enriched for suppressor cells. The suppressor cell associated with MN-Agg was a T lymphocyte since suppressor activity of MN-Agg could be abolished by treatment of MN-Agg with a rabbit anti-mouse brain serum and complement. It is proposed that the generation of suppressor cells in mice with B-16 melanoma may require specific interaction between macrophages and lymphocytes which is manifested in the spleens of tumor-bearing mice by the formation of MN-Agg.     

The active E-rosette test: a sensitive in vitro correlate for human delayed-type hypersensitivity.

The "active" rosette test was adapted as an in vitro assay and correlated with human delayed cutaneous hypersensitivity (DCH) to two microbial antigens. Peripheral lymphocytes were purified from donors known to be responders or nonresponders to PPD-tuberculin or tularemia on the basis of prior DCH reactions. Skin test antigen, incubated with lymphocytes from antigen-sensitive donors, produced a significant increase (+2 S.D.) in the ability of the lymphocytes to form active rosette-forming cells (A-RFC) when compared to lymphocytes cultured without antigen. Skin test antigen incubated with lymphocytes from nonsensitive donors produced no increase in their A-RFC. The optimal dose of each antigen was approximately 100 ng/ml. The percentage of A-RFC rose to maximum levels between 3 and 4 hr after the addition of antigen to the lymphocytes incubated at 37 degrees C. The assay appears to be specific for the antigen to which the individual demonstrates DCH. This assay may provide a new in vitro method for investigating mechanisms of cell-mediated immunity and a rapid diagnostic test for sensitization to microbial antigens.     

Quantitation of lymphocyte response to antigen by flow cytofluorometry.

The response of human peripheral blood lymphocytes to antigenic stimulation has been studied in vitro using flow cytofluorometry and an acridine orange (AO) staining technique for cellular deoxyribonucleic acid and ribonucleic acid (RNA). Antigen-stimulated "pyroninophillic" immunoblasts, identified by an increase in cellular content of RNA (red fluorescence with AO), were quantitated in triplicate cultures incubated up to 7 days with and without bacterial antigen. These results were similar to 14C-thymidine incorporation into identical cultures incubated in parallel. Cytofluorometric analysis showed a peak in percentage of immunoblasts after 6 days in culture, while maximum thymidine incorporation was seen on day 7. Cells from patients with depressed immune response secondary to cancer showed lower than normal antigen response by cytofluorometry. Kinetic studies revealed both a lower percentage of immunoblasts when compared to normal and a lower average per cell RNA content of the stimulated cells. AO cytofluorometry is suggested as a convenient method of simultaneously assessing lymphocyte proliferative and nonproliferative response to antigen.     

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

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

Electrokinetic study of the reactions of peritoneal macrophages and eosinophils with IgG immune complexes

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of guinea pig IgG immune complexes on the electrophoretic mobility distributions of guinea pig resident peritoneal cells. The resident population of cells is composed of macrophages (approximately 75%) and eosinophils (approximately 25%). These cells were separated according to the well-established method of Boyum. Populations of resident macrophages, eosinophils, and the unfractionated samples were incubated with soluble immune complexes, antigen alone, or antibody alone. The mean mobility of the resident macrophages decreased approximately 60% when incubated in the presence of immune complexes, although no effect could be discerned in the presence of antigen or antibody alone. The width of the resulting macrophage mobility distribution was larger than that of the control distributions, with a broad shoulder on the high-mobility side, indicating a heterogeneous response of the macrophages to the immune complexes. Eosinophils react in two distinct fashions. One population of eosinophils is present near the control experiments. The second population reacts in a manner very similar to that of macrophages. This suggest that at least two populations of eosinophils are present in the unstimulated guinea pig peritoneal cavity. Results that are intermediate between these two cases are found when unfractionated samples are studied.     

Histamine-induced suppressor factor (HSF): effect on migration inhibitory factor (MIF) production and proliferation.

Histamine added in vitro to cultures of sensitized lymphocytes suppresses antigen-induced production of migration inhibitory factor (MIF) and proliferation by these cells. Recent studies have suggested that lymphocytes bearing histamine type-2 receptors play a regulatory role in these in vitro responses. The present studies were undertaken to determine if suppressor function by cells having histamine receptors was mediated through a soluble product. It was found that lymph node cells from nonimmune or immune strain 2 guinea pigs elaborate a nondialyzable factor into the culture supernatant when incubated with 10(-3) to 10(-5) M histamine (histamine-induced suppressor factor of HSF). HSF, when cocultured with sensitized lymphocytes, suppressed their MIF and proliferative responses to antigen. HSF was made by lymphocytes but not macrophages. Its production could be blocked by an H2 receptor antagonist (burimamide) but not an H1 receptor antagonist (chlorpheniramine). Furthermore, the inhibitory effect of HSF was reversible as lymphocytes washed free of the factor after 24 hr and recultured with fresh medium and antigen were able to produce MIF. Gel filtration by Sephadex G-100 chromatography indicated that HSF had an approximate m.w. of 23,000 to 40,000. These results suggest that the release of histamine at the sites of immediate hypersensitivity reactions, possibly by generating HSF activity, may play a regulatory role in the subsequent development of cellular-immune reactions at the same site.     

The polyvalent protease inhibitor, trasylol, inhibits DNA synthesis of mouse lymphocytes by an indirect mechanism.

By the use of incorporation of radiolabeled thymidine, uridine, and leucine into mouse lymphocytes, the inhibitory effect of the protease inhibitor, trasylol, on antigenor mitogen-induced lymphocyte triggering was studied in vitro. DNA synthesis, as well as RNA and protein syntheses, were effectively inhibited by 0.3–2.5 × 10^?7 mol of trasylol when responses were induced by homologous antigen, allogeneic cells, phytohemagglutinin, or endotoxic lipopolysaccharide of Escherichia coli. The inhibitory effect of trasylol was reversible. On the contrary, DNA synthesis by nonadherent spleen cells was hardly inhibited by the inhibitor when the cells were stimulated with a relatively large amount of concanavalin A. Antigen-induced DNA synthesis by non-adherent lymph node cells was enhanced by the culture supernatant of macrophages. This helping effect of macrophage supernatant was effectively inhibited either by soluble or insoluble trasylol. These results suggest that the inhibitory action of trasylol on lymphocyte triggering may operate indirectly to interfere with the helping action of macrophages on lymphocytes.     

Specific MIF release by rat lymphocytes following incubation with syngeneic anti-tumor “Immune” RNA

Other investigators have previously shown that normal nonimmune lymphoid cells, after incubation with “Immune” RNA, will release MIF when these cells are incubated with the specific antigen used to immunize the RNA donor. This conversion can be detected with the macrophage migration inhibition assay. These observations have been confirmed in a system involving the transfer of immune response to tumor associated antigens with syngeneic “Immune” RNA. Syngeneic “Immune” RNA was extracted from the spleens of Fischer 344/N rats bearing growing transplants of one or another of two syngeneic chemically induced sarcomas. Normal, nonimmune Fischer 344/N spleen cells were incubated with these RNA preparations. When these RNA-incubated spleen cells were exposed to solubilized antigens from that particular tumor used to immunize the RNA donor, MIF was released. RNAse treatment of the “Immune” RNA abrogated the response, while DNAse or pronase treatment did not.     

Suppression of Ag-induced release of EP (IL 1) by spleen cells of specifically desensitized donors: evidence for the role of a suppressor cell

Monocytes or macrophages may be induced to produce IL 1 by activators (e.g., lipopolysaccharide endotoxin) that act directly or by antigens/mitogens (e.g., Con A) that stimulate inducer lymphocytes to release a lymphokine that stimulates macrophages. Using guinea pigs (GP) rendered delayed hypersensitive to ovalbumin (OVA), we investigated the role of spleen cells from normal, sensitized, and specifically desensitized GP in suppressing release of IL 1, measured as endogenous pyrogen (EP), from peritoneal exudates of sensitized GP when incubated with OVA in vitro. Co-cultivation of all three sources of spleen cells with GP peritoneal exudate cells and OVA suppressed EP release as measured in the rabbit fever assay, the effect being most marked with cells from desensitized GP, intermediate with cells from sensitized GP, and least with normal cells. This suppressor activity of spleen cells on in vitro EP release was not explained by nonspecific absorption of EP by the added cells and did not affect EP release by a stimulus that activates macrophages directly (heat-killed staphylococci). It required both lymphocytes and macrophages for its effect, but unlike some other suppressor factors, it was not modified by indomethacin, an inhibitor of prostaglandin release. This appears to be the first reported evidence for cell-mediated suppression of lymphokine-mediated release of IL 1, an important modulator of the immune system through its combined role as a lymphocyte-activating factor and an inducer of fever (EP).     

Tumor-induced alteration in macrophage accessory cell activity on autoreactive T cells

Using a tumor-model system, differences in the accessory cell capabilities on autoreactive T cells of splenic macrophages from normal and tumor-bearing hosts (TBH) were assessed in the syngeneic mixed lymphocyte reaction. Tumor development caused a drop in autoreactivity. At 0 and 7 days of tumor growth, no drop in reactivity occurred when TBH macrophages were used as accessory cells and L3T4+ autoreactive T cells from normal mice were used as responder cells. However, by day 14, there was a 32% drop in reactivity, and by day 21 only 22% of the T cell reactivity remained when TBH macrophages were used as accessory cells. Alterations in macrophage Ia antigen during tumor growth were first investigated as the potential cause of reduced autoreactivity. Before tumor growth (day 0) 59% of the splenic macrophages were found to be Ia+. Day-7 TBH macrophages showed no difference in Ia antigen expression when compared to day 0 macrophages. However, by day 14, TBH macrophages showed a 9% decrease, and by day 21 they showed a 36% decrease in the number which were Ia+. Concomitant with the decrease in the number of Ia+ cells was a decrease in the density of Ia antigen expression on day-14 and -21 TBH macrophages. In day-14 and -21 TBH macrophages, two populations were seen that were Ia+. The first had a 10%-20% decrease in Ia antigen expression per cell while the second population had a greater than 50% drop in Ia antigen expression per cell. By titrating and mixing TBH macrophages with normal host macrophages, we assessed whether they could actively mediate suppression of autoreactive T cells. A titratable suppressive phenomenon was demonstrated using day-21 TBH macrophages. In contrast, day-7 and -14 TBH macrophages titrated with normal host macrophages had no effect on the syngeneic mixed lymphocyte reactivity. Lastly, we investigated whether the macrophage-mediated suppression was caused by increased prostaglandin secretion. Addition of indomethacin to cultures increased autoreactive T cell reactivity stimulated by normal or TBH macrophages (59% and 99% increase, respectively). Although indomethacin reduced suppression mediated by TBH macrophages, autoreactivity did not return to levels induced by untreated or indomethacin-treated cells from a normal host. Taken together, the data suggested that tumor growth modulates the function of macrophage accessory cells with autoreactive T cells in at least two ways: by decreasing Ia antigen expression and by increasing suppressor activity.     

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.