Direct augmentation of cytolytic activity of tumor-derived macrophages and macrophage cell lines by muramyl dipeptide.

Macrophages derived from MSV-induced tumors and several macrophage cell lines showed direct cytolytic activity in an 18-hr ⁵¹Cr release assay against tumor target cells. The cytolytic activity of these macrophages was augmented by the addition of muramyl dipeptide (MDP) to the cytotoxicity assay, an effect similar to that observed with bacterial lipopolysaccharide. The stimulation of macrophage-mediated cytotoxicity by MDP appeared to be under genetic control since macrophages from BALB/c mice were augmented with MDP while those from C57BL/6 animals were not. MDP appears to act directly on the macrophage without the participation of any other cell type, since MDP increased the activity of the macrophage cell lines.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

Evidence for the involvement of cytolytic macrophages in rejection of SV40-induced tumors

The potential role of cytolytic macrophages in in vivo resistance to tumors induced by simian virus 40 (SV40) was evaluated in two experimental systems. First, a cell line produced by sequential in vivo passage of SV40-transformed fibroblasts through syngeneic C3H/HeJ mice was found to develop both increased neoplastic character and resistance to macrophage-mediated lysis, suggesting in vivo selection pressure against the macrophage-sensitive phenotype. In the second approach, SV40-transformed cells from C3H.OL mice, a strain that fails to produce SV40-specific cytolytic T lymphocytes (CTL), were cloned, and the cloned cells were tested for susceptibility to macrophage cytolysis in vitro. Two clones SV-COL-E8 and SV-COL-F5, which represent the extremes of macrophage susceptibility and resistance, respectively, were tested for progressive growth in syngeneic C3H.OL recipients. Progression in vivo was found to correlate with resistance to macrophage cytolysis in vitro. Other in vitro measures of the neoplastic phenotype, cell division rate and anchorage-independent growth, did not predict the relative abilities of clones E8 and F5 to form tumors. Likewise, the cells were indistinguishable in their sensitivity to cytolysis by allogeneic CTL and by natural killer cells. Finally, the presence of activated macrophages in the peritoneum of mice rejecting a challenge of syngeneic SV40-transformed cells was confirmed in both CTL responder and nonresponder strains. These studies suggest that cytolytic macrophages are indeed generated during rejection of SV40-induced mouse tumors and that, in the absence of an effective anti-SV40 CTL response, resistance of the transformed cell to macrophage-mediated cytolysis can be a determining factor in in vivo tumor growth.     

Regulation by transforming growth factor-beta 1 of expression and function of the receptor for IFN-gamma on mouse macrophages.

Transforming growth factor-beta (TGF-beta) is known phenomenologically as a negative regulator of several functions of mouse bone marrow macrophages. The studies reported here extend this list by showing that TGF-beta can suppress cytolytic activity of mouse bone marrow culture-derived macrophages that already have become activated by IFN-gamma and LPS for tumor cell killing, as well as confirm that this cytokine can interfere with the induction of activation. Suppression was caused by a shift in the dose response curve for IFN-gamma rather than absolute inhibition; the 50% effective dose for IFN-gamma was increased approximately fourfold by treatment with TGF-beta. TGF-beta also decreased the absolute number of IFN-gamma R on the surfaces of pretreated macrophages by approximately 30 to 35%, without altering the affinity with which IFN-gamma bound. The increased concentration of IFN-gamma needed to produce the higher level of receptor occupancy explained the observed shift in the IFN-gamma dose response curve. These results suggest that TGF-beta mediates its negative regulatory effects on macrophage activation by interfering with coupling of the IFN-gamma R to the pathways that induce and maintain macrophage activation for tumor cell killing. Such effects are consistent with the view that TGF-beta is a negative regulator of macrophage activation for tumor cell killing. Because of this fact, neoplastic cells that secrete this cytokine may have a distinct survival advantage.     

Maleylated bovine serum albumin triggers cytolytic function in selected populations of primed murine macrophages

The complex algal polysaccharide fucoidan has been reported as serving as a second signal for activation of macrophages primed in vivo by BCG. To assess the potential utility of this observation in analyzing biochemical mechanisms involved in macrophage activation, we examined the triggering effects of maleylated bovine serum albumin (maleylated-BSA), a defined molecule that clears via similar mechanisms. Cytolysis of P815 mastocytoma targets was triggered by maleylated-BSA, in a dose-dependent manner, in murine peritoneal macrophages primed in vivo by BCG. Unlike bacterial LPS, which triggered cytolysis when used to pretreat the macrophages, maleylated-BSA was only effective if present throughout the period of macrophage-target cytolytic interaction. Maleylated-BSA alone did not lyse the P815 targets and did not affect the binding of such targets by macrophages. Maleylated-BSA was equally effective in triggering cytolysis in BCG-primed macrophages from C3H/HeJ or C3H/HeN mice. Macrophages primed in vitro with IFN-gamma, however, could not be triggered by maleylated-BSA, even though these macrophages bound maleylated-BSA comparably to the BCG-primed macrophages. When responsive macrophages were fully activated in vitro by IFN-gamma and LPS and then allowed to decay to the primed state, maleylated-BSA was then as effective as LPS in triggering cytolysis. Taken together, the results indicate that maleylated-BSA can trigger cytolysis in certain populations of primed macrophages but not in others.     

Imbalanced accumulation of ribosomal RNA in macrophages activated in vivo or in vitro to a cytolytic stage

Previous studies have shown that peritoneal murine macrophages activated in vivo and in vitro to a tumoricidal stage have a depressed rate of RNA synthesis. In attempting to clarify the differences in RNA metabolism between noncytotoxic and tumoricidal macrophages, we have studied the relative accumulation of various species of RNA in macrophages activated in vivo and in vitro with the use of agarose gel electrophoresis. Macrophages activated in vitro to a cytotoxic stage with supernatants containing lymphokines (LK) and traces of lipopolysaccharide (LPS) have an imbalanced accumulation of mature ribosomal RNA (rRNA), with a decreased accumulation of 28S rRNA compared to 18S rRNA. In contrast, macrophages primed in vitro with LK free of detectable endotoxins that exhibit suppressive rather than tumoricidal activity do not manifest a decreased 28S:18S rRNA ratio. The conclusion that the decreased 28S:18S rRNA ratio was associated with the activation of macrophages to a cytolytic stage was supported by the finding that cytotoxic macrophages activated in vivo by i.p. injection of Propionibacterium acnes (formerly designated C. parvum) also demonstrated a decreased accumulation of 28S comparable with that observed in in vitro-activated macrophages. Moreover, activated macrophages that lost their cytolytic activity upon prolonged in vitro culture had an augmented accumulation of 28S rRNA. These results provide the first direct evidence that the expression of cytolytic activity is associated with modulation of a specific class of RNA. The unbalanced accumulation of rRNA appears to be a late molecular event in the activation process occurring during the transition from primed to cytotoxic macrophages, because inflammatory and primed macrophages had normal rRNA accumulation. A model of macrophage activation accounting for these results is proposed.     

Appearance of cytolytic granules upon induction of cytolytic activity in CTL-hybrids.

Examining a CTL-line (B6.1) with specific cytolytic activity, we have confirmed the ability of CTL-lines to produce poly C9-like, tubular complexes of two sizes (polyperforin 1 and 2). Isolated cytoplasmic granules from this cell line can induce hemolysis and contain the lysosomal marker arylsulfatase. Electron microscopial studies show that granules contain precursor molecules which are assembled into polyperforin upon addition of Ca2+. Antibodies raised against granules predominantly react with a 27-kd protein. We compared the B6.1 cell with a hybrid, PC60, derived from a cross between B6.1 and a non-cytolytic rat-thymoma. This hybrid line exhibits inducible CTL-activity. In PC60 with induced cytolytic activity, polyperforins 1 and 2, cytolytic granules and granule-associated proteins are detected. Non-cytolytic PC60 cells lack cytolytic granules and polyperforin 1, but contain polyperforin 2. These results suggest that cytolytic granules appear in parallel with the expression of cytotoxicity during T-lymphocyte differentiation.     

Modulation of macrophage function by gamma-irradiation. Acquisition of the primed cell intermediate stage of the macrophage tumoricidal activation pathway

Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. Although this synergistic response of normal macrophages to sequential incubation with activation signals has been well established, characterization of the intermediate stages in this pathway has been difficult, due in large measure to the instability of the intermediate cell phenotypes. We have developed a model system for examination of macrophage-mediated tumor cell lysis, with the use of the murine macrophage tumor cell line RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both interferon-gamma (IFN-gamma, the priming signal) and bacterial lipopolysaccharide (LPS, the triggering signal) in the development of tumor cytolytic activity. In this system, the priming effects of IFN-gamma decay rapidly after withdrawal of this mediator and the cells become unresponsive to LPS triggering. We have recently observed that gamma-irradiation of the RAW 264.7 cells also results in development of a primed activation state for tumor cell killing. The effects of gamma-radiation on the RAW 264.7 cell line are strikingly similar to those resulting from incubation with IFN-gamma, with the exception that the irradiation-induced primed cell intermediate is stable and responsive to LPS triggering for at least 24 hr. Treatment with gamma-radiation also results in increased cell surface expression of major histocompatibility complex-encoded class I antigens; however, class II antigen expression is not induced. Irradiation-induced development of the primed phenotype is not solely the result of cytostatic effects as treatment of the cells with a radiomimetic drug, mitomycin C, results in decreases in [3H]thymidine incorporation that are similar to those observed after irradiation, without concomitant development of cytolytic potential. In addition, priming by gamma-radiation does not appear to be mediated by the release of soluble autoregulatory factors. This alternate pathway for induction of the primed macrophage activation state should serve as a useful tool for identification of molecules important to the functional potential of primed cells, and for elucidation of the biochemical mechanisms of the priming event in tumoricidal activation.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. III. Inhibition by cytochalasins, colchicine, and vinblastine.

The effect of cytochalasin A and B, colchicine and vinblastine on tumor cell killing by macrophages activated in vitro with lymphocyte mediators was examined. Both cytochalasins reversibly inhibited the killing of tumor cells by activated macrophages. Kinetic studies with cytochalasin B suggested that this drug exerts its effect on an early step of the cytotoxic process. Additional studies revealed that the drug inhibited the binding of tumor cells by activated macrophages.Colchicine inhibited both the binding and the killing of tumor cells by activated macrophages, whereas its structural analogue, lumicolchicine, had no effect on either macrophage function.Vinblastine also inhibited the binding and killing of tumor cells. However, this drug no longer inhibited tumor cell binding at low concentrations (<10^?6M) that still inhibited tumor cell killing. Further, vinblastine inhibited tumor cell killing when added late to an ongoing cytolytic reaction.These results suggest that the cytochalasins, colchicine and vinblastine inhibit macrophage mediated cytotoxicity by preventing intimate contact between the effector macrophages and their targets. In addition, vinblastine also appears to inhibit a later step of the cytolytic process, possibly the secretion of a cytotoxic macrophage product.     

Phenotypic characteristics associated with the acquisition of HSV-specific CD8 T-lymphocyte-mediated cytolytic function in vitro.

Class I MHC-restricted, HSV-1-specific CD8(+) cytolytic T lymphocyte (CTL) function is rarely detected in lymphocytes isolated directly from the lymph node draining the site of infection. However, culture in vitro for 24 to 72 h in the absence of exogenous antigen results in the development of easily detectable levels of HSV-1-specific CTL effectors. The inability to detect virus-specific CTL in HSV-1-infected mice is not well understood. However, since the in vitro culture of HSV-1-immune lymphocytes results in the transition to CTL function, studies of the changes occurring to the CD8(+) T cell subpopulation may provide important insights into the development of virus-specific CTL. Therefore, the phenotypic changes taking place in the CD8(+) population of T cells from draining popliteal lymph nodes of HSV-1-infected C57BL/6 (B6) mice were investigated, focusing on changes in the expression of cell surface markers associated with T lymphocyte activation. The results demonstrate an increase in the percentage of CD8(+) T cells expressing the activation markers CD44 and CD25 in parallel with the acquisition of HSV-specific CTL effector function. Cytolytic function was found exclusively within the CD8(+) CD44(hi) CD25(hi) fraction of cells in culture, but, surprisingly, was not detectable in CD8(+) CD44(hi) CD25(lo) T cells. This suggested that the acquisition of high levels of the high-affinity IL-2 receptor was closely linked to cytolytic function and may define an important developmental stage in the transition from noncytolytic to cytolytic effector cell. In support of this, CD8(+) CD25(hi) T cells isolated from the regional lymph node exhibited direct ex vivo cytolytic function, indicating that cytolytic effector cells were present in the lymph node, but must emigrate rapidly after attaining this level of differentiation.     

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.     

Evidence for granulocyte-mediated macrophage activation after C. parvum immunization

It has been previously demonstrated that at the peak of the peritoneal response to Corynebacterium parvum (Day 4), cytolytic macrophages can be characterized by the presence of intracellular bacteria. In the present study, the role of neutrophils in the activation of peritoneal macrophages by C. parvum was investigated. Inflammatory neutrophils isolated 5 hr after ip administration of C. parvum were transferred to normal, syngeneic mice and the peritoneal macrophages of recipients harvested 4 days later were tested for cytoxicity against HeLa cells. Neutrophils isolated from mice 5 hr after C. parvum immunization were effective in inducing cytolytic macrophages. Less than 100-fold as much bacteria was needed to induce comparable levels of cytotoxic activity when introduced inside granulocytes. Neutrophils obtained from mice 48 hr after C. parvum injection or mononuclear cells were not good macrophage activators. Viable neutrophils were not required as freeze-thawed cells were able to activate macrophages in recipient mice. The intracellular distribution of C. parvum changed dramatically with time. Initially almost all bacteria were found within neutrophils. By 24 hr, many macrophages contained either bacteria or granulocytes which had ingested C. parvum. Pyridine extracts of C. parvum, which do not activate peritoneal macrophages when injected directly into mice, did not induce neutrophils capable of activating macrophages. The residue of pyridine-extracted C. parvum did induce neutrophils that could activate macrophages when transferred. The results suggest that processing of the bacteria by inflammatory granulocytes may be an obligatory step in macrophage activation by this agent. The peak response occurred earlier than T-cell immunity is usually observed and it is suggested that direct activation of macrophages via ingestion of neutrophils may represent the earliest stage of macrophage activation by C. parvum.     

Augmentation of macrophage antibody-dependent killing of tumor targets by microtubule inhibitors

Antibody-dependent cellular cytotoxicity (ADCC) to tumor targets was studied using murine resident peritoneal macrophages and a macrophage cell line RAW264.10A, both having low inherent cytolytic activity. The target was ¹²⁵I-labeled pre-B lymphoma 18-8. Pretreatment of both macrophage populations with 0.5 – 2 μM concentrations of the microtubule-stabilizing drug taxol greatly increased their antibody-dependent cytotoxicity with no stimulation of nonspecific killing. Taxol present only during the 18-hr cytolytic assays had no effect on target killing. Optimal killing activity was obtained by treating macrophages 2 days with taxol, similar to previously described cytokine stimulation of ADCC. This concentration completely blocked growth of RAW264 cells. Other microtubule inhibitors, lidocaine and colchicine, also augmented peritoneal and cell line macrophage ADCC at cytostatic concentrations. In contrast, the microfilament-disrupting agent, cytochalasin B, caused little or no stimulation of ADCC. These results show that microtubule reformation is not necessary for the development of cytotoxicity. Since microtubule inhibitors block lysosomal discharge, they may stimulate macrophage ADCC by causing accumulation of toxic molecules involved in cytotoxicity.     

Kinetics and mechanisms of macrophage activation by Corynebacterium anaerobium

Kinetics and mechanisms of macrophage activation by heat-killed Corynebacterium anaerobium (CA) in mice were investigated. The carbon clearance test revealed that the function of the reticuloendothelial system rose markedly on the 4th day after a single intravenous injection of CA and continued in a highly enhanced state until the 14th day. This activity declined gradually and dropped to a normal level around the 21st day. On the other hand, both lysosomal enzymes, beta-glucuronidase and acic phosphatase, of peritoneal macrophages decreased after the CA injection and then recovered, taking an almost inverse course to the function of the reticuloendothelial system. These results might be attributable to possible extracellular secretion of the lysosomal enzymes in accordance with macrophage activation by CA. A remarkable cytotoxicity of peritoneal macrophages, examined in vitro against L 929 cells, was detected on the 4th day following intraperitoneal administration of CA. It was maintained up to the 14th day and then declined rapidly. The mechanisms of macrophage activation by CA were also examined in vitro. CA-homogenate, heat-killed CA disrupted with an ultrasonicator, directly activated thioglycollate-induced macrophages. The macrophages were aslo activated by simultaneous treatment with both CA-homogenate and CA-sensitized spleen cells. Furthermore, the supernatant obtained from the culture of CA-sensitized spleen cells with CA-homogenate was capable of inducing activation of the macrophages. Conversely, the culture supernatant of spleen cells from CA-immunized athymic nude mice with CA-homogenate was unable to activate them. It was ascertained from the above-mentioned results that macrophages are activated initially by direct action of CA in a nonspecific way and subsequently by a soluble factor elaborated by CA-sensitized lymphocytes in an immunological way.     

Cytophilic binding of IgE to the macrophage. II. Immunologic release of lysosomal enzyme from macrophages by IgE and anti-IgE in the rat: a new mechanism of macrophage activation.

Rat peritoneal macrophages release lysosome granule-associated β-glucuronidase, but not cytoplasmic leucine aminopeptidase, after successive incubation with purified IgE protein and ?-specific anti-IgE antibody or anti-IgE F(ab′)₂ fragments. The selective release of β-glucuronidase was shown to proceed by a first step of binding of the purified IgE to the cell surface, followed by IgE-anti-IgE reaction on the macrophage, whereas the possibility of cell activation by IgE-anti-IgE complexes in the bulk phase was ruled out. Heating rat IgE destroyed its ability to mediate lysosomal enzyme release. The characteristics of macrophage activation, insofar as the binding of IgE is concerned, were in agreement with those reported for the fixation of IgE to the mononuclear phagocyte, optimal binding of IgE being achieved with 20 min incubation. Preincubation of rat macrophages with rat IgG, either aggregated or not aggregated, did not inhibit the selective release of β-glucuronidase by the successive addition of IgE and anti-IgE antibody. Simultaneous incubation of macrophage monolayers with rat IgE and aggregated rat IgG did not reduce the subsequent activation by addition of anti-IgE. These studies indicated that rat macrophages can bind rat IgE through a specific receptor, with no interference of the classical Fc (γ) receptor, and are triggered to release lysosomal enzymes upon conformational changes of the IgE molecule by anti-IgE antibody. Antibody-dependent macrophage cytotoxicity in rat schistosomiasis is mediated by IgE antibody to the parasite, which may therefore function by activating the macrophage to an efficient effector cell.     

Activation of macrophages for ADCC in vitro: effects of IL-4, TNF, interferons-alpha/beta, interferon-gamma, and GM-CSF.

Macrophages in varying states of activation differ in their ability to perform antibody-dependent cellular cytotoxicity (ADCC) and antibody-independent macrophage-mediated tumor cytotoxicity (MTC). To define further the activation requirements for macrophages to perform various cytolytic functions, we stimulated peptone-elicited peritoneal macrophages, which are only poorly cytolytic, with one of a panel of cytokines and then quantified three distinct cytolytic capacities. The peptone-elicited macrophages, after stimulation with IFN-alpha/beta, IL-4, or TNF, had increased ability to perform both the rapid and slow variants of ADCC but not to perform MTC. Stimulation with high doses of IFN-gamma, however, increased the macrophages' ability to perform all three cytolytic functions. GM-CSF had no effects on any cytolytic capacity. The effects of IL-4, TNF, IFN-gamma, and IFN-alpha/beta on the macrophages' capacity for both forms of ADCC were dose-dependent. IFN-gamma and IFN-alpha/beta increased the macrophages' capacity for both variants of ADCC within 4 hr of treatment, whereas IL-4 and TNF did so only after prolonged treatment. These results suggest that three forms of macrophage cytolytic capacity can be enhanced by cytokine treatment but that the requirements for enhancing each of the three forms of macrophage cytolytic capacity differ.     

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.     

Effect of recombinant IFN-gamma (rIFN-gamma) on the mechanism of human macrophage IgG FcRI-mediated cytotoxicity. rIFN-gamma decreases inhibition by cytophilic human IgG and changes the cytolytic mechanism.

Different classes of receptors for the Fc moiety of IgG (Fc gamma R) have been defined on human monocytes and macrophages: Fc gamma RI, Fc gamma RII, and Fc gamma RIII. All three classes are capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Fc gamma RI, which binds monomeric human IgG (hIgG) with high affinity, was shown an effective cytotoxic trigger molecule on different types of cells. In vitro, the inhibition of Fc gamma RI-mediated ADCC by hIgG is well documented. The low affinity receptor classes, Fc gamma RII and Fc gamma RIII, are not blocked by monomeric hIgG. Because monomeric hIgG is present at high concentrations in plasma and interstitial fluids it has been postulated inhibitory in vivo. We investigated the effect of rIFN-gamma on macrophage Fc gamma RI-mediated ADCC in the presence of low doses hIgG. With human E sensitized with hIgG as target cells, Fc gamma RI was studied selectively. We found that rIFN-gamma enhances both expression and cell surface density of Fc gamma RI on cultured peripheral blood monocytes. Furthermore, this cytokine partially reversed the inhibitory effect of monomeric hIgG on ADCC. More interestingly, we found that the cytolytic mechanism of monocyte-derived macrophages changed completely after prolonged culture with rIFN-gamma. Monocytes cultured for 9 days in control medium mediate predominantly phagocytosis. After long term rIFN-gamma stimulation (9 days), monocyte-derived macrophages almost completely lost the capacity to perform phagocytosis. Interestingly, they became highly efficient in mediating extracellular lysis of human E sensitized with hIgG. Short term rIFN-gamma stimulated monocyte-derived macrophages (for the last 40 h of culture) were found to mediate both phagocytosis and extracellular lysis. Our findings suggest that in vivo rIFN-gamma-stimulated macrophages may be most efficient in Fc gamma RI-mediated cytolysis as a consequence of a changed cytolytic mechanism in combination with enhanced Fc gamma RI density.     

Alloreactive cloned T cell lines. VI. Multiple lymphokine activities secreted by helper and cytolytic cloned T lymphocytes

Culture supernatants generated by alloantigenic or lectin stimulation of a cloned helper T lymphocyte, designated L2, contain interleukin 2 (IL 2), granulocyte/macrophage colony-stimulating factor (CSF), B cell stimulating factor (BCSF), macrophage (Ia+)-recruiting factor (MIRF), (Ia+)-inducing activity, gamma-interferon, Fc receptor-enhancing activity, macrophage migration inhibitory factor (MIF), macrophage activation factor (MAF), interleukin 3 (IL 3), and a factor responsible for prolonging the synthesis and secretion of the fourth and second components of complement by guinea pig peritoneal macrophages. Erythropoietin was not detected. A spontaneously arising variant of L2, designated L2V, produces much lower quantities of macrophage-stimulating activities, IL 2, and interferon. However, when compared to L2, L2V produces much higher levels of BCSF, equivalent amounts of IL 3, and slightly smaller amounts of CSF. Unlike L2V, a cytolytic clone, designated L3, secretes lymphokines that primarily affect macrophage function. The time course of lymphokine production by L2 cells indicates that for the six lymphokine activities studied there are three different times at which maximal or near maximal levels are reached, as follows: 1) IL 2, 12 to 24 hr; 2) IL 3 and CSF, 24 to 48 hr; and 3) (Ia+)-inducing activity, MAF, and interferon, 48 hr or later. Only IL 2 activity disappears during the 8-day culture cycle. The time course data and the differential production of activities by the three types of lymphocyte clones suggest that at least four terminal effector lymphokine molecules account for the ten biologic activities tested.     

Differences in the tumor necrosis factor-alpha-mediated lysis by fixed natural cytotoxic cells and fixed cytotoxic macrophages

TNF-alpha has been shown to be associated with macrophage cell membranes in such a way as to retain cytolytic activity despite fixation of the macrophage effector cells with paraformaldehyde. In this paper we report that, similar to cytotoxic macrophages, natural cytotoxic (NC) cells also use cell-associated TNF to lyse sensitive target cells. However, in contrast to fixed cytotoxic macrophages, NC cells do not retain cytolytic activity after fixation with paraformaldehyde. Additionally, the cytolytic activity of paraformaldehyde-fixed NC cells is not increased by incubation with LPS or by incubation with rTNF before fixation. Western blot analysis indicates that, unlike macrophages, NC cells use a smaller (17 kDa) constitutively active form of TNF. These results indicate that, although both macrophages and NC cells use effector cell-associated TNF to mediate lysis of sensitive targets, the way in which TNF is associated with these two types of effector cells must be different.