Immune interferon. II. Different cellular site for the production of murine macrophage migration inhibitory factor and interferon.

The production of macrophage migration inhibitory factor (MIF) and immune interferon (IF) by concanavalin A (Con A)-stimulated cultures of thymus, lymph node and spleen cells was investigated. It was found that all cultures produced MIF activity, whereas only spleen cells produced marked IF activity. The capacity to produce IF was found to be correlated with the macrophage content of a cell preparation as evidenced by staining for esterase-positive cells. Furthermore, column-purified spleen T cells produced MIF but no IF. Migration inhibition caused by residual mitogen could be ruled out. On the other hand, when macrophages grown from bone marrow cells were pre-exposed to supernatants of mitogen-stimulated lymphocytes, IF activity was released into freshly added medium while no significant MIF activity was found. IF was also found in supernatants of macrophage cultures after exposure to conventional inducers in vitro (polyinosinic-polycytidylic acid, Corynebacterium parvum) or in vivo (C. parvum), whereas no MIF was detected. An anti-Type I IF serum neutralized IF in supernatants from Con A-stimulated spleen cells but did not affect MIF in the same supernatants. This indicates that IF and MIF activity are associated with different molecules. It is, therefore, concluded that under the described conditions, IF and MIF are produced by different cells. T cells are the prime producers of MIF while IF is released by macrophages following induction by lymphokines.     

In vitro production and cellular origin of murine type II interferon.

Antigen-specific type II interferon was produced in vitro by harvesting supernatants of spleen cell cultures from Swiss-Webster mice sensitized with Mycobacterium bovis strain BCG and challenged with old tuberculin. Treatment of C3H mouse spleen cell cultures with appropriate anti-Ia, anti-IgG, anti-Thy-1 or anti-Ly-2,3 sera resulted in a significant decrease in production of type II interferon. Removal of nylon wool adherent cells or cells with histamine receptors by column chromatography similarly caused reduced production of type II interferon. Recombination of spleen cell cultures treated with anti-Ia and anti-Thy-1 sera or of cells treated with anti-IgG and anti-Thy-1 resulted in restored production of type II interferon. Interferon production was also restored by combination of cells passed through histamine columns with anti-Ia treated cells, or those passed through nylon wool columns with anti-Thy-1 treated cells. Anti-Ly-1 serum treatment had no effect on interferon production. Removal of plastic-adherent cells or cells that had phagocytosed carbonyl iron also decreased interferon production, suggesting that macrophages were also involved in type II interferon production. Recombination of non-adherent spleen cells with anti-Ia and anti-Thy-1 sera treated spleen cells, however, did not restore interferon production, suggesting that other cells in addition to macrophages are depleted by the adherence procedure. These findings indicate that type II interferon is produced by suppressor or cytotoxic (Ly-2,3+) T lymphocytes in co-operation with one or two additional cell types: (i) B lymphocytes, and (ii) macrophages.     

Induction of interferon in murine bone marrow-derived macrophage cultures by 10-carboxymethyl-9-acridanone

10-Carboxymethyl-9-acridanone (CMA) induced high titers of interferon (IFN) in murine leukocyte cultures. Thymocytes, lymph node, spleen and peritoneal exudate cells responded to CMA with IFN production. Pure macrophages derived from the bone marrow were the most efficient producers of CMA-induced IFN. The yields of IFN-α β in the macrophage supernatants depended on the concentration of the inducer and titers up to 3000 IU/ml were measured after exposure to the optimal dose (500 μg/ml). CMA was found to be the first low molecular weight compound that induced in vitro titers of IFN nearly as high as obtained after exposure to Newcastle disease virus, which is one of the most potent interferon inducers.     

Tumour-Host Interactions in Vivo

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

Inhibition of contact sensitivity by macrophages

Lymphoid cells of mice injected with picrylsulphonic acid and then painted with picryl chloride produce a specific T suppressor factor (TSF) in vitro. This factor arms peritoneal exudate cells, which then produce a nonspecific factor which inhibits the transfer of contact sensitivity by immune cells incubated in it. An adherent, theta-negative cell, which is presumably a macrophage, is responsible. This justifies the use of the term macrophage suppressor factor. As a separate phenomenon, passive transfer cells lose their activity when incubated on high density monolayers of normal peritoneal exudate cells. However, this is not associated with the production of a supernatant factor. The inhibition of transfer when immune cells are incubated with specific TSF is unaffected by nylon wool filtration (which removes macrophages). This suggests that TSF is able to depress the passive transfer of contact sensitivity by a macrophage-independent process.     

Antibody-dependent and -independent cytotoxicity of human mononuclear phagocytes: defective stimulation of tumoricidal activity in milk macrophages.

Human peripheral blood monocytes, milk macrophages and peritoneal exudate macrophages were purified by adherence. antibody-dependent cellular cytotoxicity (ADCC) was measured using the murine TLX9 lymphoma pre-labelled with 3H-thymidine. Direct, antibody-independent tumoricidal activity was measured against the murine TU5 line pre-labelled with 3H-thymidine. All mononuclear phagocyte populations tested were similarly effective in mediating ADCC against TLX9 cells. In the absence of deliberate stimulation blood monocytes and peritoneal macrophages had appreciable spontaneous cytotoxicity against the susceptible TU5 line. In contrast, four out of 10 milk macrophage preparations lacked detectable spontaneous killing activity on this target. In vitro exposure to partially purified fibroblast interferon (IFN) or to lymphokine supernatants from PHA stimulated lymphocytes augmented the direct tumoricidal activity of blood monocytes and peritoneal exudate macrophages. Milk macrophages were completely unresponsive to IFN and lymphokines. therefore the capacity to mediate antibody-dependent and -independent cytotoxicity against tumour cells can be dissociated to some extent in human mononuclear phagocyte populations from diverse anatomical sites.     

Polyclonal immunoglobulin synthesis induced by a macrophage factor acting via T cells.

New Zealand White rabbits were killed 7 days after immunization with 1 mg of alum precipitated, keyhole limpet hemocyanin (KLH) into each hind footpad and 3 days after intraperitoneal injection of thioglycollate. When supernatants from cultures of purified, elicited macrophages were added to Mishell-Dutton cultures of primed popliteal lymphocytes, they induced synthesis of both general immunoglobulins and antibody specific for KLH. The active factor(s), polyclonal lymphocyte activator (PLA), appears to be a glycoprotein with a molecular weight between 150,000 and 200,000 daltons. Absorption with high concentrations of thymocytes but not bone marrow cells removed polyclonal stimulatory activity from peritoneal macrophage supernatants which contained PLA. Purified lymph-node B cells were stimulated by PLA only in the presence of T cells. In addition, supernatants from PLA activated, washed T cells were effective at inducing polyclonal B-cell activation. Thus, PLA appears to act indirectly on B cells by stimulating T cells to produce a soluble factor which induces polyclonal B-cell activation.     

Studies of the producer cell of herpes simplex virus-induced interferon in mouse spleen cell cultures

The producer cell of type I interferon was studied in spleen cell cultures of C57BL/6 mice stimulated by inactivated Herpes Simplex Virus (HSV). Interferon production was not abolished by pretreatment of the spleen cells by anti-theta serum plus complement. The producer cell of interferon was not removed by plastic adherence and was not destroyed by the addition of silica. It was present in spleens of 3 day old C57BL/6 mice and in spleens of nu/nu mice. It was not inactivated by treatment of nu/nu spleen cells by anti-theta serum plus complement. HSV-induced interferon production was abolished by passage of the spleen cells through nylon wool columns and by irradiation (1000 R) of the spleen cells. Collectively these data suggest that in murine spleen cell cultures type I interferon is produced by B cells. However, our data do not allow to rule out that the interferon producing cell may be an immature macrophage or an immature T cell.     

Interleukin-4-triggered, STAT6-dependent production of a factor that induces mouse mast cell apoptosis

IL-4 can suppress mast cell development from mouse spleen, bone marrow and peritoneal cells by an indirect process that is dependent on the presence of macrophages. Mast cells undergo apoptosis when exposed to supernatants collected from cultures of IL-4-stimulated peritoneal cells due to the IL-4-induced production of an apoptosis-inducing factor in the cultures. This effect of IL-4 is shown to be dependent on STAT6 signaling, because IL-4 and IL-13 do not suppress mast cell development from the spleen and peritoneal cells of STAT6-/- mice. Moreover, supernatants from cultures of IL-4- and IL-13-stimulated peritoneal cells of STAT6-/- mice do not exhibit apoptosis-inducing activity. We confirm, by using deficient mice, neutralizing antibodies and recombinant cytokines, that IL-4-induced apoptosis is not related to the well-known apoptosis-inducing factors Fas, Fas ligand, TNF-alpha, TRAIL, TGF-beta or perforin. These results demonstrate a novel mechanism whereby IL-4 and IL-13 can suppress mast cell development by inducing the production of an apoptosis-inducing factor from macrophages.     

Heavy-metal mitogenesis: Zn++ and Hg++ induce cellular cytotoxicity and interferon production in murine T lymphocytes

Splenic and lymph node lymphocytes from Balb/c mice were activated in vitro by the heavy-metal cations, Zn++ and Hg++, as noted by the several-fold increases in 3H-thymidine incorporation at 144 h of culture. Optimal mitogenic concentrations of Zn++ and Hg++ were 200 microM and 10 microM, respectively. Data from experiments using T or B splenic lymphocytes enriched by cell passage over nylon wool columns, through use of athymic Nu/Nu mouse spleen cells, or by cell lysis with monoclonal anti-Thy-1 and antibody plus complement, indicated than Zn++ and Hg++ are mitogens for T cells. Removal of macrophages from spleen cells by treatment with carbonyl iron, followed by cell passage through nylon wool, eliminated the lymphocyte responses to Zn++ and to Hg++. Moreover, addition of macrophage-depleted lymphocytes to monolayers of resident peritoneal macrophages restored the lymphocyte responses to these mitogens. Both Zn++ and Hg++ activated splenic lymphocytes to display lectin-dependent cytotoxicity and to produce acid-labile interferon.     

Modulation of macrophage fibronectin secretion by LPS

The secretion of fibronectin (Fn) by rat peritoneal macrophages was found to be down-regulated by LPS. A sensitive ELISA was used to quantitate both substrate-attached and supernatant Fn. Thioglycollate-elicited peritoneal exudate cells were observed to release a considerable amount of Fn during the adherence procedure for macrophage purification. After this procedure, macrophage Fn levels peaked within 2 hr and then declined steadily to baseline levels by 96 hr. Fn release by exudate cells during adherence purification was less affected by cycloheximide treatment than was subsequent Fn secretion by purified macrophages. Macrophages elicited with thioglycollate and P. acnes displayed enhanced Fn secretory activity when compared with resident unstimulated cells. Exposure to lipopolysaccharide (LPS) suppressed the levels of immunoreactive Fn in supernatants of elicited cells. This inhibition was shown to be dose-dependent and most significant after 24 hr of incubation. The inclusion of polymyxin B in the culture medium did not reverse the LPS-induced inhibition of Fn production but did prevent LPS stimulation of interleukin-1 secretion in the macrophage cultures. These observations demonstrate that Fn secretion by macrophages is regulated according to the functional state of the cell.     

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

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

Alveolar macrophages. VI. Regulation of alveolar macrophage-mediated suppression of lymphocyte proliferation by a putative T cell.

Alveolar macrophages (AM) from normal rats suppressed antigen- or mitogen-stimulated blastogenic responses in cultures of splenic or lymph node lymphocytes, high levels of suppression often being observed when added AM comprised as few 0.6% of the total cells in culture. The efficiency of AM-mediated suppression of spleen cell blastogenesis declined with the age of the spleen cell donors, was severely curtailed by pretreatment of donors with low levels of cyclophosphamide, and was depleted by adult thymectomy coupled with thoracic duct drainage. The suppressive activity of AM was most obvious at high cell density, was unaffected by the presence of indomethacin in the cultures, or by prior X-irradiation of the spleen cells. Fractionation of spleen cells by velocity sedimentation yielded cell populations of greatly varying sensitivities to AM-mediated suppression, from small splenocytes (sedimentation velocity 1.1-2.8 mm/h) which were almost totally refractory to AM-suppression when assayed in isolation from the remainder of the spleen cell population, to larger cells (sedimentation velocity greater than 3.,5 mm/h) exhibiting high levels of sensitivity. Fractionation of spleen cells by glass wool adherence indicated decreased sensitivity to AM-suppression in the effluent population. Examination of the suppressive activity of individual subpopulations of AM separated by velocity sedimentation indicated that the larger macrophages were the most active in vitro. Suppressive activity of this nature was not seen with unstimulated peritoneal macrophages, but was observed when 'activated' peritoneal exudate cells were tested. These data are discussed in terms of a two-cell model for suppression of blastogenesis, the ultimate effector cell being a macrophage, the activity of which is controlled by a long-lived, recirculating lymphocyte, which we have provisionally designated as a T lymphocyte.     

Study of the biological activities of toxic shock syndrome toxin-1: II. Induction of the proliferative response and the interleukin 2 production by T cells from human peripheral blood mononuclear cells stimulated with the toxin

Toxic shock syndrome toxin-1 (TSST-1) is an exotoxin produced by Staphylococcus aureus isolated from patients with toxic shock syndrome. We investigated the proliferative response of human lymphocytes and their interleukin 2 (IL-2) production after stimulation with TSST-1 in vitro. Human cord blood mononuclear cells (HCBM) and human peripheral blood mononuclear cells (HPBM) could proliferate with TSST-1 stimulation. T cell-depleted HPBM showed only a marginal response to this toxin. A IL-2-like factor with a molecular weight of 15-18 kD was obtained from the supernatants of TSST-1-stimulated HPBM cultures. The factor was absorbed by CTLL-2 cells but not by T cell-depleted murine spleen cells, indicating that it is IL-2. HPBM are very sensitive to TSST-1: a low concentration of TSST-1 (0.01 ng/ml in 36 h stimulation) and a short period of stimulation (8 h at 10 ng/ml of the toxin) were fully effective for HPBM to produce substantial amounts of IL-2. Removal of T cells abrogated the TSST-1-induced IL-2 production by HPBM. Reconstituted cell cultures of nylon wool column-passed T cells and macrophages produced IL-2 by TSST-1 stimulation and, furthermore, the accessory activity of the macrophages could be partially replaced by a macrophage-derived factor containing interleukin 1. These findings indicate that T cells require macrophages or IL-1 for TSST-1-induced production of IL-2. The roles of lymphokines, including IL-2, in the development of this illness are discussed.     

Macrophages as a source of tumoricidal activity (tumor-necrotizing factor).

Macrophage-enriched peritoneal exudate cells from mice infected with Mycobacterium bovis BCG, macrophage-like tumor cells (PU 5-1.8), and peritoneal macrophages propagated in vitro with macrophage growth factor released tumoricidal activity into the culture medium within 2 to 3 h after stimulation with nanogram quantities of bacterial lipopolysaccharide. The cytotoxic activities from each of the macrophage culture supernatants eluted from diethylaminoethyl-Sephacel columns at a sodium chloride concentration of 200 mM exhibited a molecular weight of 50,000 to 60,000 as estimated by gel filtration, were stable at 56 degrees C for 30 min, and were active at a pH range of 6 to 10. A rabbit antiserum directed against serum-derived cytotoxic activity (tumor-necrotizing factor) from BCG-infected and lipopolysaccharide-challenged mice inhibited all of the cytotoxic activities generated in vitro. This suggests that the macrophage-derived cytotoxins are identical with serum-derived cytotoxic factor, which further implies that the macrophage is the cellular source of tumor-necrotizing factor.     

Detection of different macrophage-activating factor and interferon activities in supernatants of chicken lymphocyte cultures

Chicken spleen lymphocytes were cultivated under various conditions in order to produce and characterise functionally avian lymphokines. Biological properties of lymphokine-containing culture supernatants were evaluated with regard to their antiviral activity and their effects on cultured bone marrow-derived chicken macrophages, including induction of cytostatic activity, enhancement of phagocytic activity towards vital Candida albicans, and giant cell formation. Optimal doses of concanavalin A (ConA) for stimulation of lymphocytes varied between 2.5 and 40 mug/ml, depending on the cell concentration and presence or absence of serum. Lymphokine production occurred even without exogenous mitogen stimulation when cells were cultured at sufficiently high concentrations (1 to 2 x 10(7) cells/ml). Cytostasis-inducing capacity of lymphokine preparations against lymphoblastoid MDCC-RP1 cells was always combined with the presence of antiviral activity. Experimental results suggested that these two activities had to be attributed to at least two distinct lymphokines, i.e. macrophage-activating factor and interferon (IFN). ConA stimulation of lymphocytes from a single donor appeared to be the appropriate signal for production of IFN-gamma. Endogenous stimulation in mixed lymphocyte cultures more appropriately triggered production of IFN-alpha or IFN-ss, although production of IFN-gamma was not completely suppressed. Phagocytic activity of macrophages could also be increased by a cyto-kine present in conditioned media from confluent cultures of chicken embryo fibroblasts, chicken kidney cells, or bone marrow-derived chicken macrophages. In lymphokine preparations, this mediator may-even be a cofactor necessary for induction of multinucleated giant cell formation of cultured macrophages.     

Synergism between tumor necrosis factor-alpha and interferon-gamma on macrophage activation for the killing of intracellular Trypanosoma cruzi through a nitric oxide-dependent mechanism.

Intracellular replication of the protozoan parasite Trypanosoma cruzi inside macrophages is essential for the production of the disease and the development of the parasite. Two CD4+ T cell lines, A10 and A28, were established from T. cruzi-infected BALB/c mice which specifically proliferated to parasite antigens. The trypanocidal activity of BALB/c macrophages was induced upon culture with the A10, but not with the A28 T cell line. The cell-free supernatant from this A10 line, as well as from immune spleen cells stimulated with specific antigen or concanavalin A, but not from the A28 T cell line also activated the trypanocidal activity of peritoneal macrophages or of the J774 macrophage-like cell line. when the lymphokine content of the supernatants from both cell lines was analyzed, it was found that the A10 T cell line secreted interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin 2, whereas the A28 line did not secrete IFN-gamma upon stimulation. Furthermore, the trypanocidal-inducing ability of A10 supernatant was completely abrogated by neutralizing anti-IFN-gamma antibodies and partially abrogated by neutralizing anti-TNF-alpha antibodies. When recombinant cytokines were added to J774 cells, IFN-gamma was able to induce significant trypanocidal activity whereas TNF-alpha was almost ineffective. However, TNF-alpha or lipopolysaccharide (LPS) showed a synergistic effect with IFN-gamma on macrophage activation. IFN-gamma triggered nitric oxide (NO) synthesis by J774 cells whereas TNF-alpha was almost ineffective. TNF-alpha and LPS were also synergistic with IFN-gamma in the NO production. Both the NO production and the trypanocidal activity in J774 cells induced by T cell supernatants or lymphokine combinations were inhibited by N-monomethyl-L-arginine, a competitive inhibitor of NO synthase activity. A good correlation between the levels of NO production and trypanocidal activity induced by different lymphokine preparations was found. Those results suggest that IFN-gamma and TNF-alpha, secreted by T. cruzi-immune T cells, are involved in the activation of the trypanocidal activity of mouse macrophages through an NO-dependent mechanism.     

Feedback suppression of staphylococcal enterotoxin-stimulated T-lymphocyte proliferation by macrophages through inductive nitric oxide synthesis.

Staphylococcal enterotoxin A (SEA)- or SEB-stimulated T-lymphocyte proliferation was suppressed by the addition of high numbers of murine peritoneal macrophages or rat peritoneal or alveolar macrophages, whereas lower numbers of murine peritoneal macrophages enhanced the T-lymphocyte response. Suppression was associated with the increase of accumulation of nitrite, a product of nitric oxide, in the culture supernatants. This macrophage-mediated suppression was totally reversed by the addition of NG-monomethyl-L-arginine, a homolog of L-arginine, indicating that macrophage-mediated suppression of T-lymphocyte proliferation was mediated through the nitric oxide-synthesizing pathway activity. Macrophages in large numbers spontaneously produced nitric oxide in culture supernatant fluids. By the addition of autologous or allogeneic spleen cells but not thymocytes to SEA- or SEB-stimulated macrophage culture, nitric oxide production was greatly increased. When T lymphocytes in spleen cells were killed by antibody before addition to macrophage culture, nitric oxide production was diminished to the basal level. These results suggest that in addition to the action to support the process of T-lymphocyte activation by SEA or SEB, macrophages display a feedback regulatory action on the SEA- or SEB-stimulated T-cell proliferative response by releasing nitric oxide through interaction between macrophages and activated T lymphocytes.     

Induction of Helper T Cells by Antigen Specific Factor from Macrophages

An immunostimulatory antigen specific factor (ASF) was found to be secreted by antigen-pulsed macrophages. Macrophages obtained from peritoneal exudate of C57BL/6 mice were pulsed with horse spleen ferritin (HSF). The PM10 ultrafiltration membrane-retained supernatant from the cultures of these macrophages was able to generate helper T cells when introduced into cultures of nylon wool purified T lymphocytes from spleens of C57BL/6 mice. The generation of helper T cells was measured by the cooperation between ASF-induced T cells and splenic B cells in the presence of trinitrophenyl (TNP)-HSF, and subsequently by the anti-TNP plaque forming cell (PFC) assay using TNP-coated sheep red blood cells. The number of PFC obtained from these cultures was significantly higher than the control (T cell cultures without ASF). Background levels of PFC were obtained when the T-B cooperation cultures were challenged with other haptenated antigens (e.g. TNP-BSA) instead of TNP-HSF. In addition, ASF from allogeneic macrophages was unable to facilitate helper cell induction. These results indicated that helper T cell induction by ASF is antigen specific as well as genetically restricted. When small amounts of ASF were injected into syngeneic mice without any adjuvant, specific helper T cells could be obtained from the spleens of these animals which showed that ASF is also active in vivo.     

Alveolar macrophages. V. Comparative studies on the antigen presentation activity of guinea-pig and rat alveolar macrophages.

The addition of syngeneic alveolar macrophages (AM) to mitogen-stimulated lymphocyte cultures from the rat and the guinea-pig resulted in markedly dissimilar effects upon in vitro blastogenesis, guinea-pig AM stimulating the response and rat AM exhibiting strong suppressive activity. The capacity of guinea-pig and rat AM to initiate antigen-specific blastogenesis was also examined. Ovalbumin-immune lymph node cells from guinea-pigs were passed through nylon wool columns to deplete macrophages. This process abolished their capacity to respond to the antigen via blastogenesis. The addition of ovalbumin-pulsed AM to these cultures restored their blastogenic responsiveness, and did so with considerably greater efficiency than was observed employing peritoneal macrophages from the same animals. Identical manoeuvres in the rat again yielded opposite results; the addition of rat AM to syngeneic antigen-stimulated lymphoid cell cultures consistently suppressed blastogenesis, an effect not seen employing peritoneal macrophages from the same species.