Role of interleukin 1 in promoting human monocyte-mediated tumor cytotoxicity

Human peripheral blood monocytes from normal donors obtained by separation on a Percoll gradient showed considerable cytotoxicity against tumor cells when preincubated in vitro for 24 hr with human monocyte-derived interleukin 1 (IL 1). In contrast, monocytes after pretreatment in medium alone had low cytotoxic activity. All the IL 1 preparations, including IL 1 which was purified by high-performance liquid column chromatography (HPLC), as well as crude culture supernatant from human monocytes promoted monocyte-mediated cytotoxicity in the same dose-dependent manner as the thymocyte growth-promoting activity. There was no endotoxin or interferon (IFN) activity in the highly purified IL 1, suggesting that IL 1 itself was the active moiety. The effect of IL 1 on monocyte-mediated cytotoxicity was partially inhibited by indomethacin, whereas pretreatment of monocytes with prostaglandin (PG) E1 or E2 rather than IL 1 also resulted in substantial monocyte cytotoxicity. Thus, the effect of IL 1 on monocyte-mediated cytotoxicity is presumably mediated by PGE. Since fresh monocytes that were not preincubated exhibited levels of spontaneous cytotoxic activity similar to that of monocytes preincubated with IL 1, it seemed likely that the effect of IL 1 was to maintain the spontaneous level of activity rather than to induce cytotoxic activity. To elucidate this possibility, monocytes were first preincubated in medium alone for a longer period, and after losing their spontaneous activity they were further incubated with or without IL 1. Such "aged" monocytes did not develop cytotoxic activity in response to IL 1 but did in response to other agents known to induce macrophage cytotoxicity, such as endotoxin or lymphokine-containing supernatants. Therefore, the major effect of IL 1 actually seemed to prolong the cytotoxic state of monocytes. These results also suggest that IL 1 released by macrophages or monocytes may play a role in host defense against neoplastic cells by acting on monocytes as an autostimulating factor.     

Cytotoxicity of activated monocytes on endothelial cells

Unstimulated human monocytes did not express appreciable levels of cytotoxicity on normal human umbilical vein endothelial cells (EC) in a 24-48 hr TdR release assay. On activation with IFN-gamma and LPS, monocytes had appreciable cytotoxicity on EC. Monocyte cytotoxicity on EC was not dependent on the presence of contaminating lymphoid cells. Recombinant TNF, IL-1, and IL-6 as well as monocyte supernatants did not exert a cytotoxic effect on EC. Moreover, anti-TNF, anti-IL-1, and anti-IL-6 antibodies, as well as scavengers of reactive oxygen intermediates, did not affect the cytotoxicity of activated monocytes on EC. Antibodies against the beta-chain (CD18) of leukocyte integrins inhibited the adhesion and cytotoxicity of activated monocytes on EC. Pretreatment of EC with IL-1 augmented the adhesion of monocytes on EC. Normal monocytes were not cytotoxic on IL-1-pretreated EC and IL-1 treatment did not increase the susceptibility of EC to activated monocytes. Thus adhesion is necessary but not sufficient for monocyte killing of EC. Anti-alpha L (LFA-1) antibodies markedly reduced monocyte cytotoxicity on EC, although anti-alpha X (p150) antibodies had only a modest effect. Anti-alpha M (Mac-1/CR3) antibodies were intermediate inhibitors of EC killing by activated monocytes. Thus, alpha L, beta 2 (LFA-1), and, to a lesser extent, alpha M, beta 2 (Mac-1/CR3) and alpha X, beta 2 (p 150, 95) integrins are the main adhesive structures involved in the cytotoxic interaction of activated monocytes with EC. Monocyte-mediated damage of EC could play a role as a mechanism of tissue injury under conditions of local or systemic activation of mononuclear phagocytes.     

Biological effect of interferon-gamma during the course of experimental infection of rat by Schistosoma mansoni

We demonstrate here that a second mechanism of platelet activation dependent on lymphokine could also take place in the expression of platelet cytotoxicity against Schistosoma mansoni in vitro. Indeed, IgE, as previously described, but also IFN-gamma, present in the sera of infected rats, together induce platelets from normal rats into cytotoxic effectors for the parasitic larvae. This second mechanism appears also effective in vivo since the passive transfer of normal platelets treated by recombinant IFN-gamma (rIFN-gamma) and the administration of rIFN-gamma to rats conferred a protective immunity to S. mansoni.     

Functional and phenotypic changes associated with the in vitro development of human monocytes into activated macrophages

Freshly isolated human peripheral blood monocytes had minimal cytotoxic effect in vitro on the schistosomula of Schistosoma mansoni. However, stimulation of the cells with either interferon gamma (IFN) or specific anti-parasite antiserum caused an increase in cytotoxicity. Additionally, the normal development of monocytes into macrophages over 7 days was associated with a sharp increase in cytotoxicity. The non-cytotoxic monocytes were compared with activated macrophages to assess whether cytotoxicity was associated with changes in immunophenotype. As monocytes developed into macrophages there were marked increases in transferrin receptors (HB21), macrophage cellular integrin (3.9), and Fc receptors (KB61). A further three markers showed increased expression in 7-day-old macrophages stimulated by IFN, namely a high affinity Fc gamma receptor (10.1), MHC Class II (1B5) and tumour necrosis factor (TNF).     

TGF-Beta inhibits the in vitro induction of lymphokine-activated killing activity

Summary Employing serum-free media, human peripheral blood mononuclear cells, and purified recombinant interleukin-2 (IL-2), conditions were observed in which the development of IL-2-driven cytotoxic activity was suppressed. The cytotoxic activity of such IL-2-generated lymphokine activated killing (LAK) was tested against natural killer-resistant cultured tumor cells (Daudi, Raji, and a glioma). LAK generation was inhibited by addition of some normal sera, normal platelets, or some tumor cells. Because recent reports have indicated that transforming growth factor-beta (TGF-beta)-like factors are often secreted by tumors and the acidic alpha granules of platelets and can be present in sera, we tested the effect of purified human TGF-beta on the activation of LAK. Our results indicated that TGF-beta is very suppressive for LAK induction, and can completely prevent both the IL-2-driven proliferation and cytotoxicity at concentrations as low as 5 ng/ml. Titrations of IL-2 and of TGF-beta indicated that the suppression is dose-dependent and can be avoided by employing higher levels of IL-2. It was also found that the suppressive effect of TGF-beta can be overcome by washing suppressed cell populations and further culture in low levels of IL-2. Collectively, these data indicate that TGF-beta can be a potent inhibitor of LAK generation under standard activation conditions, but that this effect is regulated by the relative level of IL-2 and may be overcome and/or reversed in vitro.     

Modulation of cytokine production by monocytes and developing-dendritic cells under the influence of leukemia and lymphoma cell products

Cytokines and other soluble factors released by tumor cells play an important role in modulating immune cells to favor tumor development. Monocyte differentiation into macrophages or dendritic cells (DCs) with specific phenotypes is deeply affected by tumor signals and understanding this context is paramount to prevent and propose new therapeutic possibilities. Hence, we developed a study to better describe the modulatory effects of leukemia and lymphoma cell products on human monocytes and monocyte-derived DCs secretion of cytokines such as interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-12. Except with the promyelocytic leukemia cell supernatants (HL-60), the other two tumor supernatants (chronic myeloid leukemia, K562 and Burkitt lymphoma, DAUDI) increased both TNF-α and IL-1β production by monocytes and monocytes undergoing differentiation. This effect was neither explained by alterations of cell number in culture nor by the high amount of vascular endothelial growth factor (VEGF) present in the tumor supernatants. Moreover, all supernatants used were able to induce drastic reduction of IL-12 secretion by cells induced to activation, suggesting a negative interference with Th1 antitumoral responses that should be a huge advantage for tumor progression.     

The effect of RU 41.740 (Biostim) on the production of interleukin-1 by monocytes and enriched large granular lymphocytes in normals and patients with hepatocellular carcinoma

Summary The effect of RU 41.740 on natural killer (NK) cell cytotoxicity from normals and patients with advanced hepatocellular carcinoma (HCC) was studied. RU 41.740-treated normal enriched large granular lymphocytes (LGLs) showed increased cytotoxic activity against ⁵¹Cr-labelled K562 cells. Although the drug increased the cytotoxic activity of LGLs from HCC patients, these results were not statistically significant. As LGLs have been shown to release interleukin-1 (IL-1) after suitable stimulation the effects of RU 41.740 on production of this cytokine from both monocytes and LGLs was studied. The drug stimulated IL-1 release from normal cells but monocyte and LGL IL-1 production from HCC patients was not influenced by RU 41.740 by RU 41.740. It is concluded, therefore, that although RU 41.740 can stimulate natural cytotoxicity and IL-1 production by normal LGLs, it is unable to correct the defect which exists in these functions in patients with large mass tumours.     

Monocyte- and natural killer cell-mediated spontaneous cytotoxicity against human noncultured solid tumor cells

Unstimulated human peripheral blood mononuclear cells from healthy donors exhibited spontaneous cytotoxicity against noncultured solid tumor targets in a 12- to 24-hr 51Cr release or 111In release assay. Both purified monocytes (greater than 99% monocytes) and natural killer (NK)-enriched lymphocytes exhibited comparable levels of spontaneous cytotoxicity against fresh melanoma tumor targets. This cytotoxicity was observed under endotoxin-free conditions. NK-depleted lymphocytes did not lyse the melanoma targets. Culture supernatants of monocytes incubated with the melanoma tumor cells did not exhibit cytotoxic activity against these targets. Purified monocytes lacked NK activity against the K562 targets in a 4-hr 51Cr release assay. Treatment of the monocytes with anti-Leu 1 1b and anti-Leu7 monoclonal antibodies plus complement did not reduce monocyte-mediated lysis of the melanoma targets, demonstrating that contaminating NK cells, if any, were not responsible for the lysis of noncultured melanoma targets by monocytes. In contrast, Leu 1 1b+ NK cells were responsible for the lysis of the melanoma targets by NK-enriched lymphocytes. The addition of recombinant interferon-gamma (rIFN-gamma), but not lipopolysaccharide, into the 51Cr release assay or pretreatment of monocytes with rIFN-gamma significantly increased their cytotoxicity against noncultured solid tumor cells. Monocytes cultured for 3 days with medium alone lost their cytotoxic activity. The addition of rIFN-gamma from the beginning of these cultures prevented the loss of the cytotoxic activity of monocytes. In summary, both unstimulated monocytes and NK-enriched lymphocytes exhibit comparable levels of spontaneous cytotoxicity against fresh solid tumor targets.     

Functional and phenotypic changes associated with the in vitro development of human monocytes into activated macrophages

Abstract Freshly isolated human peripheral blood monocytes had minimal cytotoxic effect in vitro on the schistosomula of Schistosoma mansoni . However, stimulation of the cells with either interferon gamma (IFN) or specific anti-parasite antiserum caused an increase in cytotoxicity. Additionally, the normal development of monocytes into macrophages over 7 days was associated with a sharp increase in cytoxocity. The non-cytotoxic monocytes were compared with activated macrophages to assess whether cytotoxicity was associated with changes in immunophenotype. As monocytes developed into macrophages there were marked increases in transferrin receptors (HB21), macrophage cellular integrin (3.9), and Fc receptors (KB61). A further three markers showed increased expression in 7-day-old macrophages stimulated by IFN, namely a high affinity Fx γ receptor (10.1), MHC Class II (1B5) and tumour necrosis factor (TNF).     

Role of cytokines in the monocyte-mediated augmentation of human natural killer cell activity

Previous work has shown that normal human monocytes can augment natural killer (NK) cell activity both when mixed with enriched null cells in the assay and when precultured with enriched null cells and removed prior to testing. The data presented here show that a 4-hr preculture period is superior to slightly longer periods (10-12 hr) for demonstrating the augmentation. The role of cytokines in the monocyte effect was then investigated using a variety of antibody and recombinant reagents. Both monoclonal and rabbit polyclonal antibodies to IL-1 and IL-2 inhibited the monocyte effect, whereas antibodies against IFN-alpha and IFN-gamma from both sources had no effect. Of these cytokines, only IL-1 could be demonstrated (using a sensitive IL-1-dependent-IL-2 synthesis assay) in the supernatants of 4-hr cultures of monocytes plus null cells or null cells only. The ability to detect IL-1 was specifically inhibited by rabbit antibody to human IL-1 at 1:20 and 1:200 dilutions, but only the greater concentration inhibited the monocyte effect on NK activity. In contrast, the detection of soluble IL-1 was not inhibited by including monoclonal anti-IL-1 (1:20 dilution) in the 4-hr culture, although the same reagent abrogated the monocyte effect under these conditions. Recombinant IL-1 (up to 100 units/ml) did not augment NK activity either when added to the assay or when precultured for 4 hr with enriched null cells, whereas either recombinant IL-2 or monocytes were effective under these conditions. These results provide the first evidence for a cellular, and potentially physiologic, basis for the regulation of NK activity by IL-1 and IL-2, which had been previously known to act at pharmacologic levels in vitro.     

Endothelin-1 Stimulates Monocytes in vitro to Release Chemotactic Activity Identified as Interleukin-8 and Monocyte Chemotactic Protein-1

In the present study we examined whether endothelin-1 stimulation of human monocytes causes release of chemotactic factors. It was found that monocytes released neutrophil- and monocyte-chemotactic activity in a dose- and time-dependent manner in response to ET-1. ET-1 did not show any chemotactic activity by itself. NCA was detected in monocyte supernatants in response to ET-1 (0.01-100 nM) after 1, 4, 8 and 24 h stimulation. MCA was detected only after 24 h stimulation with ET-1 (0.1-100 nM). Preincubation of the monocyte cultures with the lipoxygenase inhibitors nordihydroguaiaretic acid (10(-4) M) or diethylcarbamazine (10(-9) M) completely abolished the appearance of NCA and MCA. NCA was neutralized by > 75% using a polyclonal antibody against human interleuktn-8. The ET-1 induced release of IL-8 was confirmed by IL-8 ELISA. A monoclonal antibody against human monocyte chemotactic protein-1 neutralized MCA by > 80%. It is concluded that ET-1 stimulation of monocytes in vitro causes release of neutrophil- and monocyte-chemotactic activity identified as IL-8 and MCP-I respectively. An intact lipoxygenase pathway is crucial for this effect of ET-1 to occur.     

Recombinant tumor necrosis factors mediate platelet cytotoxicity to Schistosoma mansoni larvae

The involvement of platelets in the immunity directed toward the parasite Schistosoma mansoni has been demonstrated in vitro and in vivo. The killing properties of platelets were shown previously to involve specific IgE after their binding to a membrane receptor. More recently, we have demonstrated that lymphokines released by S. mansoni Ag- or lectin-stimulated CD4+/CD8-T cells were able to induce the platelet cytotoxicity. The isoelectric focusing of the T lymphocyte supernatants showed that two factors were able to stimulate the platelet killing functions. One of them was clearly identified as being IFN-gamma. The present work demonstrates that the second lymphokine was TNF. Indeed the rTNF-beta and, to a lesser extent, rTNF-alpha, induce normal platelets into killer cells for the young larvae of schistosome. Moreover, an additive effect of the TNF-alpha and IFN-gamma has been observed.     

Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes.

Infections by coxsackievirus B3 (CVB3) have previously been shown to cause acute and chronic myocarditis characterized by a heavy mononuclear leukocyte infiltration and myocyte necrosis. Because clinical and experimental evidence suggested that cardiac damage may result from immunologic rather than viral mechanisms, we examined in this study the in vitro interaction of CVB3 with human monocytes. CVB3 was capable of infecting freshly harvested monocytes as revealed by immunofluorescence and release of infectious virus particles. Virus infection did not reduce monocyte viability but, on the contrary, enhanced spreading and adherence. In a dose-dependent manner, CVB3 stimulated the release of cytokines from monocytes. Whereas a potent production of TNF-alpha, IL-1 beta, and IL-6 was dependent on exposure to infectious CVB3, IFN release was also induced by UV-inactivated virus. On a molecular level, CVB3 stimulated cytokine gene expression as shown by a marked TNF-alpha, IL-1 beta, and IL-6 mRNA accumulation. Supernatants of CVB3-infected monocytes displayed cytotoxic activity against Girardi heart cells which could be abrogated by an anti-TNF-alpha antiserum. These data suggest that CVB3-induced cytokine release from monocytes may participate in virus-induced organ damage such as myocarditis, which may either occur by a direct cytotoxicity of cytokines or by activation of cytotoxic lymphocytes.     

Cytolysis of tumor necrosis factor (TNF)-resistant tumor targets. Differential cytotoxicity of monocytes activated by the interferons, IL-2, and TNF

Herein we demonstrate that IFN-alpha, IFN-gamma, and IL-2 can induce human peripheral blood monocyte-mediated lysis of tumor cells that are resistant to both the direct effects of TNF and to monocytes activated by TNF. Monocytes activated by TNF kill only TNF-sensitive tumor targets, whereas those activated by IFN and IL-2 can lyse both TNF-sensitive and TNF-resistant tumor targets. Monocyte cytotoxicity against TNF-sensitive lines induced by the IFN, IL-2, or TNF can be completely abrogated by the addition of anti-TNF antibodies. In contrast, anti-TNF antibodies have no effect on IFN- or IL-2-induced monocyte cytotoxicity against TNF resistant targets, confirming non-TNF-mediated lysis induced by lymphokine-activated monocytes. Neither induction of TNF receptors by IFN-gamma nor inhibition of RNA synthesis by actinomycin D increased the susceptibility of TNF-resistant tumor targets to TNF-mediated monocyte cytotoxicity. Thus, non-TNF-mediated modes of monocyte cytotoxicity are induced by IFN and IL-2, but not by TNF, indicating that different cytotoxic mechanisms are responsible for the lysis of TNF-sensitive and TNF-resistant tumor cells. In addition, these findings also suggest that TNF-sensitive lines are susceptible only to TNF-mediated killing and apparently insensitive to non-TNF-mediated monocyte cytotoxicity.     

Aggregated immunoglobulin and Fc fragment of IgG induce IL-6 release from human monocytes

The Fc fragment of immunoglobulin (Ig) has been shown to play an important role in the regulation of humoral immunity, cellular immunity, lymphocyte and monocyte activation, and immune mediator secretion. We wished to determine if Ig or Fc fragments would induce IL-6 production from monocytes. Incubation of monocytes purified from human peripheral blood mononuclear cells with aggregated Ig or Fc fragments of Ig induced interleukin-6 (IL-6) activity in the supernatants. Monomeric Ig taken from an intravenous preparation of Ig, from which all aggregated Ig are removed, would not induce IL-6 production from monocytes whereas as a heat-treated aliquot, presumably containing aggregates, did induce IL-6. The supernatants were assayed according to their ability to induce growth in a murine hybridoma cell line B9, or enhance Ig secretion of B cells stimulated with Staphylococcus aureus Cowan 1 (SAC). The IL-6 activity in the supernatants could be neutralized by a polyclonal rabbit anti-human IL-6 antiserum in both assays of IL-6 activity. Exposure of T-enriched or B-enriched lymphocyte subpopulations to Fc fragments did not induce the release of any IL-6 after 12 hr of incubation, but small amounts of IL-6 were produced by B-enriched cells after 60 hr of exposure to Fc fragments. Hence Fc fragments and aggregated Ig induce peripheral blood monocytes to rapidly secrete large quantities of interleukin-6.     

Interferon gamma encapsulated into liposomes enhances the activity of monocytes and natural killer cells and has antiproliferative effects on tumor cells in vitro

The effects of human interferon gamma (IFN gamma) encapsulated into liposomes were investigated in vitro. Monocytes were induced to release a cytotoxic factor with either IFN gamma encapsulated into liposomes, free IFN gamma or lipopolysaccharide (LPS). If IFN gamma was applied in the liposomal form, less IFN activity was required to stimulate monocytes. Most of the cytotoxic factor was secreted during the first 4 h of stimulation. The cytotoxic factor in supernatants from PMNLs was completely neutralized by a monospecific polyclonal antiserum to tumor necrosis factor (TNF). Combining subthreshold doses of IFN gamma liposomes or IFN gamma with lipopolysaccharide synergistically enhanced the release of TNF. In fluorescence analysis, altered expression of the class II HLA-DR antigen on LeuM3 positive monocytes was induced with IFN gamma liposomes as well as with IFN gamma. Not only monocytes but also natural killer (NK) cells were stimulated to higher cytotoxicity by IFN gamma liposomes in a dose-dependent manner. In comparison with IFN gamma, the same amount of activity was necessary for adequate stimulation of NK-cells against the K562 target cells. Furthermore, the antiproliferative effects of IFN gamma liposomes and free IFN gamma on several human tumor cell lines was compared. Among several cell lines tested, U937 and A549 turned out to be sensitive to IFN gamma, and both cell lines reacted with 50% growth inhibition at a lower amount of gamma presented by liposomes than in the free form. These data show production of IFN gamma liposomes which possess immunomodulatory and antiproliferative activity in vitro. In several of the test systems studied, liposome-encapsulated IFN gamma was more effective than free IFN gamma.     

Evaluation of the antibody-dependent cytotoxic capabilities of individual human monocytes. Role of Fc gamma RI and Fc gamma RII and the effects of cytokines at the single cell level.

In this report we present evidence that not all human peripheral blood monocytes mediate antibody-dependent cellular cytotoxicity (ADCC), and that this function may be determined on an individual cell by both the type and level of expression of FcR, and by the state of cellular activation and/or differentiation. Although the diverse range of effector and regulatory functions performed by human monocytes suggests the possibility of distinct subsets, it is not clear whether observed functional heterogeneity reflects the presence of true monocyte subpopulations, or whether this diversity represents a continuum of maturational states present in the peripheral circulation. In an attempt to address this question, we investigated the ability of human monocytes to carry out ADCC at the single cell level, with emphasis on the role of the three FcR for IgG (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) in mediating cytotoxicity. Using a modified plaque assay, 58.3% +/- 4.9 of freshly isolated monocytes mediated ADCC, as evidenced by the formation of lytic plaques in monolayers of ox erythrocyte (oxE) target cells. Significant increases in the number of plaque-forming cells were observed after positive selection by flow microfluorimetry for those monocytes expressing high levels of Fc gamma RI and Rc gamma RII, but not Fc gamma RIII. Bispecific antibodies composed of Fab fragments of anti-oxE antibody covalently coupled to Fab fragments of anti-Fc gamma R antibodies were used to independently evaluate the ability of Fc gamma RI, Fc gamma RII, and Fc gamma RIII to mediate single cell cytotoxicity. Significant increases in the number of plaque-forming cells were observed in the presence of anti-Fc gamma RI x anti-oxE and anti-Fc gamma RII x anti-oxE bispecific antibodies, confirming the efficiency of Fc gamma RI and Fc gamma RII as cytotoxic trigger molecules on human monocytes. Incubation of monocytes with purified rIFN-gamma and granulocyte macrophage-CSF, but not IL-2, IL-3, IL-4, IL-6, or TNF-alpha, also resulted in significant increases in the number of monocytes mediating cytotoxicity, suggesting that cytotoxic ability at the single cell level may be influenced by factors which effect monocyte activation and differentiation, respectively. Overall, these studies demonstrate that freshly isolated human monocytes are heterogeneous in their ability to mediate ADCC, and suggest that this functional diversity arises not from discrete subpopulations of cells, but from a continuum of maturational/activational states present within the peripheral circulation.     

Immunocytochemical analysis of interleukin-1 beta production by human monocytes]

IL-1 was localized within the cytoplasm of human blood monocytes by indirect immunofluorescence using polyclonal rabbit antibodies. After LPS stimulation first IL-1-positive cells appeared at 2 hours and maximal intracellular IL-1 concentration was observed at 10-24 hours when nearly 90% monocytes were labeled with subsequent decline at 48 hours. The highest intracellular IL-1 content preceded its maximal level in cell supernatants.     

Spontaneous cytotoxicity and tumor necrosis factor production by peripheral blood monocytes from AIDS patients

Peripheral blood monocytes (PBM) from AIDS patients have exhibited defects in some but not all of the immune functions yet tested. This study has examined the capacity of AIDS PBM to lyse tumor target cells as well as their ability to secrete TNF. Untreated PBM from AIDS patients were significantly cytotoxic to U937 target cells and responded to IFN-gamma pretreatment with augmented cytotoxicity. Both the spontaneous and IFN-gamma-stimulated cytotoxic activity was significantly (p less than 0.01) higher than that observed with normal PBM. The cytotoxic activity depended on the E:T ratio used and was higher in AIDS PBM at all ratios tested (10:1 to 40:1). Because TNF has been implicated in macrophage cell-mediated cytotoxicity, we examined whether the elevated cytotoxic activity of AIDS PBM was associated with an increase in TNF production. Supernatants from PBM cultured overnight with or without IFN-gamma were tested in a bioassay measuring cytotoxicity against U937 target cells as well as in an RIA specific for TNF. Supernatants derived from either unstimulated or IFN-gamma-treated AIDS PBM exhibited significantly higher levels of cytotoxicity than supernatants from normal macrophages. Both normal and AIDS PBM produced higher levels of cytotoxic factors in response to IFN-gamma. As determined by the RIA, AIDS PBM spontaneously released high levels of TNF whereas little TNF was produced by normal PBM. Treatment with IFN-gamma augmented the level of TNF production in both AIDS and normal PBM. These results demonstrate that PBM from AIDS patients have undergone in vivo activation as manifested by both cytotoxicity against tumor target cells and production of TNF. Target cell lysis by both AIDS PBM and their supernatants was inhibited by monoclonal anti-rTNF, suggesting that the increase in PBM cell-mediated cytotoxicity was caused by an increase in TNF production. The significance of these findings in the pathogenesis of the disease is discussed.     

Studies of the mechanisms for the induction of in vivo tumor immunity. IV. Enhancement of the in vitro generation of secondary cell-mediated cytotoxic response by normal peritoneal macrophages and their culture supernatants.

The effect of macrophages on the induction of the cell-mediated cytotoxicity against a leukemia in a syngeneic system was investigated. The addition of exogenous peritoneal cells from normal C57BL/6 MIce enhanced the in vitro secondary cell-mediated cytotoxic response of both spleen and lymph node cells as responding cells against syngeneic FBL-3 leukemia. Peritoneal phagocytic macrophages seemed to be responsible for the enhancement. No inhibitory effect was demonstrated by the addition of peritoneal macrophages at a concentration as high as 20%, whereas the primary cytotoxic allograft response was significantly suppressed. In the present studies, there was no absolute restriction of macrophage-T cell interaction by an H-2 barrier. Supernatants of peritoneal macrophage cultures also enhanced this cell-mediated cytotoxic response. There was no difference between the effects of syngeneic or allogeneic peritoneal macrophage culture supernatants.