Oxidation of glucose by mouse peritoneal macrophages: a comparison of suspensions and monolayers

Macrophages, when maintained in vitro, take up glucose from the medium and oxidize it to CO2. The rate of oxidation of glucose varies considerably, depending on the physical state of the cell preparation. Cells in suspension oxidize glucose at a level six-fold that of cells in monolayers. The differences cannot be attributed to change in the rats of transport of glucose. On the other hand, an increase in intracellular glycogen (about three-fold) and free glucose plus glucose-6-P (many-fold) was found in the cells prepared as monolayers. During subsequent incubation with glucose-14C, this could be the cause of an isotope dilution effect and could explain the lower production of 14CO2 by the adherent cells. Since oxidation of glucose-1-14C to 14CO2 is used by many investigators to indicate the functional state of macrophages, we suggest close attention be paid to the system used, i.e., monolayers vs. suspensions.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

Oxygen consumption of mammalian myocardial cells in culture: measurements in beating cells attached to the substrate of the culture dish

A Lucite attachment which permitted the measurement of oxygen consumption in cells in culture without manipulating the cells was constructed. The attachment fit over commercially available dishes for cell culture and had an oxygen electrode built into it. Oxygen uptake of cells in culture was thus measured. Cells were attached to the substrate of the culture dish during the measurements and could be observed in an inverted phase microscope. Cells did not show any morphological changes, e.g., cell shapes or beating rate in case of myocardial cells, before and after the measurements of oxygen consumption. Using this method the rate of oxygen consumption was determined in rat myocardial and heart non-muscle cells in culture and also in HeLa and L6 cell lines. Myocardial cells in culture had an approximately four times higher rate of oxygen uptake compared with heart non-muscle, HeLa, and L6 cells. The oxygen uptake of beating myocardial cells was higher by about 50% compared with quiescent myocardial cells.     

Mitogenic activation of B cells in vitro: the properties of adherent accessory cells as revealed by partition analysis

The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that "promoted" contact ("Rock and Roll" experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation.     

Cytostatic activity of in vitro generated macrophages: evidence for a prostaglandin-independent reversible cytostatic mechanism

Culture of spleen cells for 5 days has previously been shown to result in the generation of strongly adherent cells from nonadherent precursors. In the current report it is shown that the majority (85-95%) of adherent cells are Mac-1+, FcR+, Thy 1.2- macrophages. Expression of effector activity by these macrophages requires exposure to activating signals. Coculture of the macrophages with Con A-stimulated spleen cells results in the expression of cytostatic activity against lymphocytic and monocytic tumor cell lines. Significant cytostatic activity is apparent within 6 hr after addition of the activating cells. Culture supernates of Con A-stimulated spleen cells (CAS-CM) are not effective in inducing cytostatic activity in the adherent macrophage population either alone or in the presence of additional Con A. However, stimulation of the culture generated macrophages with LPS in the presence of CAS-CM does induce cytostatic activity. The effector cell must be metabolically active in order to effect cytostasis insofar as heat fixation of the culture generated macrophage population eliminates effector activity. Proliferation of the tumor cells is significantly reduced after a 4-hr incubation period with the activated macrophages and is reduced two- to threefold after an 8- to 12-hr incubation period. The cytostatic effect is rapidly reversible. Proliferative activity of the tumor cells returned to control level within 12-24 hr after removal from activated macrophages. Cell cycle analysis indicated that the target cells were not arrested in a single stage of cell cycle, although an increase in frequency of cells in G1-phase was observed. Fluorescence analysis of bromodeoxyuridine (BrdU) incorporation rate demonstrated that the rate of DNA synthesis was reduced in all of the cells in the target population and that the mean rate of BrdU incorporation of the inhibited cells was three- to fivefold lower than control cells. RNA and protein synthesis were not affected to the same degree as DNA synthesis. The cytostatic effect was not mediated by prostaglandins or thymidine insofar as addition of indomethacin and 2-deoxycytidine did not prevent the cytostatic activity of the macrophages. The supernates of activated macrophages contained little inhibitory activity especially when indomethacin was included in the culture medium (19% inhibition of tumor cell proliferation by 1:1 dilution of supernate). The activity that was present could be eliminated by dialysis against fresh culture medium using Spectropor membranes with a 1000-Da molecular cutoff.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chicken thrombocytes in culture: lymphocyte-conditioned medium delays apoptosis.

Chicken thrombocytes are nucleated cells, analogs to mammalian platelets. These cells are involved in hemostasis, phagocytosis and secretion of specific products. Most of the properties of avian thrombocytes have been established in experiments that employed recently isolated blood cells. Attempts to cultivate these cells for a long period of time under optimal culture conditions for peripheral blood cells were unsuccessful; thrombocytes died after 24 h of cultivation unlike macrophages cocultured with them. Here we investigate the reasons and type of thrombocyte death in culture. Thrombocytes were separated from peripheral blood of roosters and cultured for 48 h. The influence of different culture conditions on thrombocyte viability was studied. Cells were cultured as adherent cell monolayers or under agitation (preventing adherence), in the presence or lack of lymphocytes or their soluble factors, and various concentrations of fetal bovine serum. After 24 h in standard culture thrombocytes displayed cytoplasm and chromatin condensation, DNA cleaved into oligonucleosomal fragments and unaltered mitochondria. These results strongly suggest that thrombocytes suffer an apoptotic cell death in culture. Apoptosis could be delayed by culturing thrombocytes in the presence of lymphocytes or their soluble factors.     

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.     

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.     

Functional maturation of murine B lymphocyte precursors. II. Analysis of cells required from the bone marrow microenvironment

The development of mature B cells in cultures of early B cell precursors depends on the presence of a confluent adherent bone marrow (aBM) cell layer. Adherent and sIgM+ cell-depleted bone marrow (BM) from untreated or 5-fluorouracil-pretreated donors or day 12 fetal liver cells were used as precursor cell populations. When adherent cells from thymus or highly enriched BM-derived macrophages were co-cultured with precursor cells, mature B cells were not developed. Similarly, aBM cell layers generated in the presence of hydrocortisone and horse serum were unable to support aBM cell-dependent precursor differentiation, even though cortisone was removed before the addition of precursor cells. In contrast, this type of microenvironment promoted the differentiation of precursor of myeloid cell lineages. Repeated treatment of established aBM cell populations with a monoclonal anti-macrophage antibody (31.3, known to recognize a surface marker on a subset of BM macrophages) and complement abolished the capacity of otherwise functional aBM cells to sustain the development of B cell precursors. Macrophage-depleted aBM cells regained their function after supplementation with highly enriched BM-derived macrophages grown in vitro. Limiting dilution analysis of aBM cells in microcultures containing saturating numbers of early B cell progenitors also suggests the participation of more than one cell type in the BM cell population. In conclusion, differentiation of early B cell progenitors requires macrophages in addition to at least one additional cell type contained in the aBM cell population.     

Depletion of NK by cellular immunoadsorption.

The binding of human natural killer (NK) cells to their tumor cell targets was investigated by using monolayers of sensitive target cell lines. Monolayers of K562 and HSB, a myeloid and T cell line, respectively, were prepared on poly-L-lysine-coated plastic tissue culture dishes and briefly fixed with 0.2% formaldehyde. Freshly isolated peripheral blood lymphocytes (PBL) were incubated on the monolayers. Nonadherent PBL were then removed, after gentle agitation, by decanting and gently washing the monolayer. They were tested, along with unseparated controls, for NK activity in a short-term 51Cr release assay. PBL that were nonadherent to a tested monolayer had only 20 to 60% of the control cytotoxic activity. Our results suggest that NK recognition sites on the effector lymphocytes were able to interact with reciprocal determinants on the target cell monolayers, resulting in selective loss of NK effector cells from the PBL population. The specificity of the NK effector-target interaction was investigated by testing the ability of each monolayer to remove activity against both targets. These data imply heterogeneity with regard to recognition structure within the NK effector population as well as among the target cells.     

Effect of rabbit anti-asialo GM1 treatment in vivo or with anti-asialo GM1 plus complement in vitro on cytotoxic T cell activities

The susceptibility of cytotoxic effector lymphocytes and their induction to in vivo or in vitro treatment with rabbit anti-neutral glycolipid ganglio-N-tetraosylceramide (anti-ASGM1) antiserum was investigated. Intravenous injection of anti-ASGM1 antiserum eliminated measurable natural killer (NK) cell activity in spleen cells of mice infected for 5 days with Vaccinia virus, or for 8 days with lymphocytic choriomeningitis virus (LCMV) if injected 24 hr prior to testing. In addition, this treatment lowered measurable virus-specific cytotoxic T cell activity by 60 to 95%. Virus-specific cytotoxic T cell and NK cell activity generated during a primary infection in vivo was also sensitive to treatment in vitro with anti-ASGM1 antiserum (1/300 to 1/600 dilution) plus rabbit complement at a dilution of 1/15 (20 to 50% cell death, more than 30-fold decrease of cytotoxic activity); in vitro treatment with rabbit complement alone often enhanced NK and cytotoxic T cell activity slightly. In vivo treatment with anti-ASGM1 before primary immunization decreased generation of primary CTL only if high doses of anti-ASGM1 antiserum were injected twice. Antiviral T cells generated during secondary stimulation in vitro and alloreactive cytotoxic T cells from a mixed lymphocyte culture were resistant to treatment in vitro with anti-ASGM1 plus complement at the end of the culture period. Treatment in vitro of in vivo-primed responder spleen cells with anti-ASGM1 plus complement before their addition to a secondary restimulation culture resulted in complete inhibition of a secondary antiviral cytotoxic T cell response. In vivo treatment with anti-ASGM1 24 hr before their spleen cells were harvested and restimulated in vitro significantly reduced the virus-specific T cell activity of mice that had been immunized with virus several weeks previously. A cloned T cell line exclusively exerting NK-like activity was resistant, and two cloned virus-specific cytotoxic T cell lines were susceptible to treatment with anti-ASGM1 plus complement in vitro. These results caution the general use of rabbit anti-ASGM1 as a marker to distinguish NK from CTL cells; they indicate a possible relationship between NK and CTL cells and suggest that in vitro culture of lymphocytes may alter or select the cell surface expression or availability of the ASGM1 marker(s).     

Plasma from cirrhotic patients induces inotropic changes on cultured rat heart cells

Cultured monolayers of newborn rat heart cells were perfused with plasma from patients with alcoholic liver cirrhosis. The results showed that cirrhotic plasma, when compared to control plasma, depressed contractility of beating cardiac cells. The in-vitro deleterious effects of plasma from cirrhotic subjects on cell cultures were neither related to hemodynamic parameters nor to the levels of plasmatic catecholamines. This study suggests the presence in the plasma of patients with alcoholic liver cirrhosis, of humoral factor(s) which cause depressive effects on rat heart cells in culture.     

T cell recognition in the mixed lymphocyte response. I. Non-T, radiation-resistant splenic adherent cells are the predominant stimulators in the murine mixed lymphocyte reaction.

The ability of subpopulations of murine spleen cells to stimulate a mixed lymphocyte response (MLR) was studied. It was found that T cells (nylon-nonadherent spleen cells) and B cells [G-10 passed and treated with rabbit anti-mouse brain serum (RAMB) and complement (C)] were poor stimulators of an MLR. In contrast, whole spleen cells or B cells plus adherent cells (RAMB +C-treated spleen cells) produced good stimulation. However, a non-T, radiation-resistant splenic adherent cell (SAC) population was up to 20 to 50 times more efficient as a stimulator of an MLR on a per cell basis than an unseparated spleen population. These SAC were shown to express Ia determinants encoded by genes in I-A and I-E/C. These results suggest that Ia+ SAC may be the predominant stimulating cells in spleen cell populations, and the preferential target for T cell recognition in cell interaction events.     

Generation of EPR-detectable nitrosyl-iron complexes in tumor target cells cocultured with activated macrophages.

After immunostimulation, murine macrophages oxidize L-arginine into nitric oxide (NO) which acts as an effector molecule. In this study, we attempted to establish whether activated macrophage-derived NO forms paramagnetic complexes in tumor target cells which do not express by themselves the L-arginine:NO pathway. Accordingly, murine L1210 leukemia cells were cocultivated with activated peritoneal macrophages from Bacillus-Calmette-Guérin-infected mice, or activated in vitro with interferon-gamma. In control experiments, macrophages were prevented from producing nitrogen oxides by incubation with NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine:NO pathway. After coculture, L1210 cells were removed from adherent macrophage monolayers and analyzed by electron paramagnetic resonance at 77 K. In the L1210 cells cultured with activated macrophages, we detected a signal typical of nitrosyl-iron-sulfur complexes, with g values of 2.041 and 2.015. This signal was not present when L1210 cells were either cultured alone or cocultured with activated macrophages in the presence of NG-monomethyl-L-arginine. Mitochondria from activated macrophage-injured L1210 cells also exhibited the signal with g values of 2.041 and 2.015. These results show that when tumor target cells undergo cell-to-cell contact with activated macrophages during culture, the macrophages promote target cell nitrosylation in compartments like mitochondria.     

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: I. Study of proliferative response

Culture of normal CBA lymphocytes on monolayers of syngeneic thyroid epithelial cells leads to significant thymidine incorporation. The specificity of this model was demonstrated by depletion of the CBA lymphocytes binding to syngeneic thyroid cells and the increase of thymidine uptake after secondary exposure on syngeneic thyroid monolayers. Removal of B cells (by treatment with anti-Ig serum plus complement) or of adherent cells does not modify the proliferative response whereas T-cell depletion strongly diminishes the response. Thus T cells are stimulated to undergo DNA synthesis and are sensitized when exposed to syngeneic thyroid epitelial cells. The nature of the antigens recognized by T cells (native autoantigen, enzyme, or virus-modified autoantigen) is not yet determined. Whether such autoreactive T cells play a role in the onset of experimental autoimmune thyroiditis as regulatory T-cells or cytotoxic effector cells is discussed.     

Evaluation of intrauterine immune suppression during pregnancy in a species with epitheliochorial placentation

Cells capable of suppressing the immune response of PBL to the mitogen PHA are associated with the epitheliochorial placenta during normal first pregnancy in the pig. Because placentation in the pig is noninvasive, these suppressor cells are not associated with decidua. Cells with similar activity are also found between the implantation sites and in the uterus of pseudopregnant pigs, suggesting that fetal trophoblast is not essential for recruitment of intrauterine suppressor cells. Cell separation studies demonstrate that two independent populations of suppressor cells are present in the pig uterus on day 28 of gestation, as well as a population of PHA-responsive cells. The inability of unseparated porcine uterine cells to respond to PHA, plus the reconstitution of suppression in remixing experiments, demonstrate directly that a functional, putative T effector cell population is blocked within the uterus during normal mammalian pregnancy.     

The macrophage adherence phenomenon: Its relationship to prostaglandin E2 and superoxide anion production and changes in transmembrane potential

Mononuclear phagocytes are undoubtedly the sine qua non of chronic inflammatory reactions. This is demonstrated by their unique ability to function as phagocytic, secretory, or effector cells during the course of an immune event. Although macrophages can perform a variety of immune tasks, their ability to function appropriately is dependent upon the mode of elicitation, the stimulus under investigation, the source of the macrophages (peritoneal, alveolar, etc.), and whether the macrophages are monolayers or in suspension. We have examined the relationship between adherent and non-adherent elicited peritoneal macrophages in terms of prostaglandin E₂ (PGE₂) and superoxide anion (O₂) production; in addition, we hae studied these elicited macrophages in suspension for their ability to undergo transmembrane potential changes in response to several stimuli. Non-adherent, elicited peritoneal macrophages demonstrated an increase in basal PGE₂ production, and were refractory to particulate stimulus. After monolayer formation, basal PGE₂ levels dropped and the cell could respond to both soluble and particulate stimuli. Only adherent macrophages could respond to a specific challenge and synthesize O₂. Both O₂ production and depolarization of the transmembrane potential were suppressed in cell in suspension. Furthermore, both exogenous PGE₂ and supernatant from macrophages in suspension could modulate O₂ production by PMA challenged macrophages monolayers. These studies indicate that PGE₂ may modulate macrophage function and dictate activity as macrophages go from the non-adherent to adherent state.     

The relationship of calcium and parathyroid hormone in their effect on heart cells

Parathyroid hormone (PTH), the plasma concentration of which is raised in uremia, has been suggested as one of the agents responsible for the myocardial changes commonly seen in uremia. The effect of intact [1–84] PTH on rat heart cells grown in tissue culture has been studied. Addition of the hormone to the media significantly stimulated beating rate. The stimulation was directly proportional to the amount of PTH in the medium. Excessively high concentration of PTH caused immediate cessation of the beating, which was reversed by the addition of calcium to the medium. The extent of stimulation by PTH was inversely proportional to the calcium concentrations in the medium. Isoproterenol and phenylephrine at excessively high concentrations in the medium did not mimic the PTH effect either alone or together with PTH. When beating ceased due to verapamil the effect was not reversed by the addition of calcium to the medium.Calcium added to the myocytes seen after beating ceased reversed the effect and the cells started to beat again. Cells kept for a longer period in the arrested state were not revived by the addition of calcium.     

Automated surface area measurement of cultured cardiac myocytes

An automated method for rapidly measuring surface area of individual cardiac myocytes was used as an index of myocyte growth. Hearts from 2- to 4-day-old rats were digested by overnight incubation in cold trypsin solution. Enriched suspensions of myocytes were plated at 2×10⁵ cells/well in 12-well-culture plates. Cells were grown in M199 supplemented with 1%, 10% serum or 10% serum plus 10^–7 M norepinephrine. On days 1–4 after plating, cells were fixed in Bouin's Solution and stained with Weigert's Iron Hematoxylin and Biebrich Scarlet-Acid Fuchsin. An inverted microscope, video camera and monitor were coupled to a video image processor (Image Technology Corp.). The enhanced image of stained heart cells was digitized, and perimeter, length, width and area of each selected cell were calculated. One hundred randomly selected cells were measured in each of eight wells from each treatment-day group. Areas of individual myocytes varied widely in culture dishes and the distribition was skewed toward larger cells. The standard deviation increased in proportion to an increase in mean cell area. A logarithmic transformation of the data normalized the data and yielded a more homogeneous variance. The geometric mean area of heart cells supplemented with 1% serum increased only slightly, but significantly, during four days in culture. Geometric mean area of cells supplemented with 10% serum increased nearly four-fold. Supplementing cells with norepinephrine (10^–7 M) in addition to 10% serum did not induce a further increase in cell size. This technique has the potential to rapidly and objectively monitor heart cell growth following pharmacological or toxicological treatments.     

Mechanism(s) of in vitro macrophage activation with Nocardia rubra cell wall skeleton: the effects on macrophage activating factor production by lymphocytes

Summary BALB/c mouse peritoneal macrophages prepared from WPC which had been treated with N. CWS demonstrated potent cytostatic activity against syngeneic Meth A fibrosarcoma cells. The maximum cytostatic activity developed in the macrophages when WPC were incubated with 25 g/ml N. CWS for 3 days. NAPC from BALB/c mice given an i. p. injection with 100 g N. CWS 7 days previously (N. CWS-NAPC) or supernatants from N. CWS-NAPC also activated peritoneal macrophages in vitro. However, when peritoneal macrophages were incubated with N. CWS in the absence of NAPC, or when T cells were depleted from WPC by treatment with anti-Thy 1.2 antibody and complement, N. CWS failed to enhance the cytostatic activity of the macrophages. Furthermore, thioglycollate-elicited peritoneal macrophages from C3H/HeN mice increased their cytolytic properties by incubation with supernatant fluids from N. CWS-treated spleen cells. These findings suggest that in vitro macrophage activation with N. CWS depends on MAF secreted from T lymphocytes. Abbreviations used: N. CWS, Nocardia rubra cell-wall skeleton; BRM, biological response modifier; MAF, macrophage activating factor; IL-1, interleukin 1; IL-2, interleukin 2; IFN-, interferon gamma; PCCM, peritoneal cell culture medium; SCCM, spleen cell culture medium; TCM, tumor culture medium; HI-FCS, heat-inactivated fetal calf serum; Con A, concanavalin A; PC, peritoneal cells; PEC, peritoneal exudate cells; WPC, whole peritoneal cells; APC, adherent peritoneal cells; TGC-APC, thioglycollateelicited adherent peritoneal cells; NAPC, nonadherent peritoneal cells; SN, supernatants; NK cells, natural killer cells; LAK cells, lymphokine activated killer cells E:T ratio, effector: target cell ratio; WSA, water soluble adjuvant; LPS, lipopolysaccharide; MDP, N-acetyl-muramyl-L-alanyl-D-isoglutamine