Tumor necrosis factor and IL-1 associated with plasma membranes of activated human monocytes lyse monokine-sensitive but not monokine-resistant tumor cells whereas viable activated monocytes lyse both

The purpose of our study was to determine some of the mechanisms involved in macrophage-mediated lysis of tumorigenic cells. A375 human melanoma cells (A375-R) resistant to lysis mediated by TNF and IL-1 were selected from the TNF- and IL-1-sensitive A375 parental melanoma cells subsequent to continuous (2 mo) exposure to rTNF. Peripheral blood monocytes isolated by centrifugal elutriation from healthy donors were incubated with rIFN-gamma and muramyl dipeptide, with a lipoprotein derived from Escherichia coli (CG-31362) or with LPS for 24 h. These activated monocytes lysed both the A375 (monokine-sensitive) and A375-R (monokine-resistant) melanoma cells. Activated tumoricidal macrophages fixed in 2% paraformaldehyde lysed only the TNF- and IL-1-sensitive A375 cells. These fixed monocytes contained both IL-1 and TNF activities as determined by D10 cell proliferation and L929 cytolysis assays, respectively. Nearly identical results were obtained with preparations of plasma membranes from activated human monocytes. Anti-IL-1 and/or anti-TNF sera neutralized the cytolysis of tumor cells mediated by free monokines, by fixed monocytes, or by plasma membrane preparations. In contrast, anti-TNF and/or anti-IL-1 sera did not inhibit tumor cell lysis by viable activated monocytes. We conclude that IL-1 and TNF molecules associated with the plasma membranes of activated monocytes mediate lysis of susceptible target cells. However, because activated monocytes lysed IL-1-and TNF-resistant target cells, molecules other than these monokines must also be involved in the antitumor activity of monocytes.     

Plasma membrane-associated tumor necrosis factor. A non-integral membrane protein possibly bound to its own receptor

Purified plasma membranes from LPS-activated human blood monocytes produced significant lysis and growth inhibition of the TNF-sensitive L929 murine fibroblast cell line. Unactivated human monocyte plasma membranes did not display either activity. Anti-TNF serum specifically inhibited the anti-tumor activity of activated monocyte membranes whereas anti-IL-1 serum or non-specific rabbit serum decreased neither the lysis nor growth inhibition of L929 cells. Membrane-associated TNF did not behave as an integral protein as it could be eluted from the plasma membranes by either high salt or low pH treatment. Plasma membranes cleared of membrane-associated TNF by high salt treatment were able to bind TNF, and this binding was specifically inhibited by preincubation of rTNF with specific anti-TNF serum. Western blot analysis of plasma membranes showed a membrane-associated TNF with a m. w. of approximately 17 kDa present only in the activated monocytes. When the plasma membranes were preincubated with the cross-linker agent dissuccinimidyl suberate, Western blot analysis revealed the presence of a TNF-binding protein with a Mr of approximately 102 kDa. These studies indicate that unlike IL-1, membrane-associated TNF is not an integral membrane protein and that TNF may be associated with the monocyte membrane by occupying the TNF R.     

Production of TNF-alpha and TNF-beta by staphylococcal enterotoxin A activated human T cells

Staphylococcal enterotoxin at concentrations of less than 1 pg/ml induces significant TNF activity in human peripheral blood T cells and monocytes. Maximal TNF activity is routinely detected after 48 to 72 h of culture. IL-2 and IL-4 were both growth promoting for human T cells but only IL-2 could efficiently induce TNF production. The production of TNF-alpha and TNF-beta differed greatly in kinetics. An early intracytoplasmatic production of TNF-alpha after 6 h was detected in both monocytes and T cells whereas a late production of TNF-beta (lymphotoxin) after 48 h, occurred in the T cell population. Induction of TNF-alpha and TNF-beta production by Staphylococcal enterotoxin requires the presence of both monocytes and T cells. The CD4+45R- but not CD4+45R+ and CD8+ cells supported TNF-alpha production in monocytes. The main lytic component from Staphylococcal enterotoxin-activated mononuclear cells is TNF-beta. CD4+ and CD8+ T cells produced about equal amounts of biologically active TNF into the culture supernatants but a fourfold higher frequency of TNF-beta producing cells was demonstrated among CD4+ vs CD8+ cells. The CD4+45R- T cell subset was an efficient producer of TNF-beta and IFN-gamma whereas the CD4+45R+ T cell subset produced significant amounts of TNF-beta but only marginal amounts of IFN-gamma.     

Accessory function of human fibroblasts in mitogen-stimulated interferon-gamma production by T lymphocytes. Inhibition by interleukin 1 and tumor necrosis factor

Highly purified human T cells from peripheral blood fail to produce interferon (IFN)-gamma in the absence of accessory cells. The ability of T cells to produce IFN-gamma upon stimulation with phytohemagglutinin (PHA) or concanavalin A could be restored by the addition of cultured allogeneic human foreskin fibroblasts. Addition of antibodies specific for HLA-DR, DQ, and DP antigens failed to block this accessory function of the fibroblasts. In contrast, antibodies to HLA-DR and DQ antigens inhibited the accessory cell activity of autologous monocytes. Allogeneic fibroblasts failed to exert accessory activity when exogenous interleukin 2 (IL-2) was used as the stimulus for IFN-gamma production. In contrast, autologous monocytes were active as accessory cells for IL-2-stimulated T cells. Addition of recombinant human interleukin 1 alpha (IL-1 alpha) or IL-1 beta to PHA-stimulated T cells co-cultured with fibroblasts stimulated IFN-gamma production. In contrast, preincubation of fibroblasts with IL-1 alpha or IL-1 beta caused a dose-dependent suppression of the ability of fibroblasts to augment PHA- and concanavalin A-induced IFN-gamma production by T cells. Preincubation of fibroblasts with recombinant human tumor necrosis factor (TNF) also reduced their accessory activity. Incubation of fibroblasts with IFN-gamma produced some reduction in their accessory activity and the inhibitory effect of TNF was further enhanced in the presence of IFN-gamma. A 4- to 10-hr incubation of fibroblasts with IL-1 or TNF was sufficient to produce a maximal suppression of accessory activity. Fixation of fibroblasts with formaldehyde decreased their accessory activity, but fixation did not abolish the suppression of accessory function induced by earlier incubation with IL-1. Supernatants of IL-1-treated fibroblast cultures had less suppressive activity than the IL-1-treated fibroblasts per se, and no suppressive activity at all was detected in the supernatants of TNF-treated fibroblasts. Enhanced prostaglandin synthesis may play a role in the IL-1- and TNF-induced suppression of accessory cell function, but other factors are likely to be involved. Our results show that fibroblasts can have a marked effect on T cell function and that IL-1 and TNF can exert immunoregulatory activities indirectly by altering the interactions of fibroblasts with T cells.     

Expression of two distinct cytolytic mechanisms among murine CD4 subsets

A TNF (TNF-alpha and TNF-beta)-sensitive target, L929, and two TNF-resistant targets, P815 and LK were used to compare the cytolytic activity among subsets of CD4+ (Th) clones. Cytolytic activity was induced with either Con A, CD3-mAb, or Ag-pulsed LK cells. Six Th1 clones are strongly cytolytic against all three targets. In contrast, Th2 clones are either noncytolytic or weakly cytolytic. Although there is an apparent correlation between TNF production, killing of L929 cells, and the killing of TNF-resistant targets, an anti-TNF serum (capable of neutralizing both TNF-alpha and TNF-beta) selectively inhibits CD4 clones to lyse L929 cells, whereas the lysis of P815 or LK cells was unaffected. The continuous presence of noncytotoxic levels of Actinomycin D (AcD) and cycloheximide, but not mitomycin C, cyclosporin A (CsA), or cholera toxin (ChT) inhibits the lysis of Ag-pulsed, Ia-bearing LK cells; indicating a requirement for de novo synthesis of RNA and protein for cytolytic activity. Although pretreatment with AcD, CsA, or ChT strongly inhibits production of IL-2, TNF and IFN-gamma, only clones pretreated with AcD lose cytolytic activity against Ag-pulsed, Ia-bearing LK cells. These observations support a model of TNF-independent killing of TNF-resistant targets. The TNF-independent cytolytic activity does not correlate with serine esterase activity released into media upon activation of CD4 clones. Moreover, the effects of metabolic inhibitors on serine esterase release do not correlate with their effects on cytolytic activity. Collectively, the data demonstrate that activated CD4 cells express two distinct cytolytic activities; a TNF (and IFN-gamma)-mediated cytotoxicity and a TNF (and IFN-gamma)-independent cytolytic activity. Both pathways require de novo synthesis of RNA and protein and appear to be independent of granule enzyme release. Only the TNF-independent cytolytic activity is resistant to CsA and ChT inhibition.     

Analysis of rat natural killer cytotoxic factor (NKCF) produced by rat NK cell lines and the production of a murine monoclonal antibody that neutralizes NKCF

Natural killer cytotoxic factor (NKCF) is produced as a result of the interaction of murine, rat, or human natural killer (NK) cells with NK-susceptible targets. This factor has been linked to the target cell lysis mediated by the NK effector cell. In the present results, culture supernatants from rat large granular lymphocyte (LGL) tumors exhibited NKCF activity which lysed the susceptible targets, MBL-2 and YAC-1. NKCF production from these rat tumor lines was spontaneous and was not significantly increased by co-incubation of the LGL tumors with target cells, target cell membranes, or by preincubation of the LGL tumor cells with interferon or interleukin 2. In addition to NKCF activity, the supernatants lysed L929, indicating the presence of tumor necrosis factor (TNF) in these preparations. The presence of this latter cytokine was verified using specific antibodies to recombinant murine TNF which neutralized the L929 activity while not affecting the NKCF activity against MBL-2 or YAC-1. Mouse monoclonal antibodies (mAb) A0287, A0462, and A0316) which significantly inhibit the NKCF cytolytic activity of these LGL-derived supernatants were also produced. These antibodies were shown to cross-react with human NKCF in a manner similar to that seen in the rat. Interestingly these same mAb demonstrated no inhibition of L929 cytotoxicity from either LGL-derived supernatants or by recombinant murine or human TNF. To examine further the specificity of these antibodies, they were chemically linked to Sepharose 4B and found to remove a significant proportion of the NKCF cytolytic activity from LGL supernatants, while not affecting the TNF reactivities in these preparations. In addition, these antibodies demonstrated significant inhibition of cell-mediated cytotoxicity by rat LGL against YAC-1 target cells. Biochemical analysis of labeled NKCF-containing supernatants indicated the major protein recognized by these anti-NKCF mAb to be approximately 12,000 m.w. The use of these mAb against NKCF should be very useful in further purification and biochemical characterization of NKCF and in studying its role in a variety of cell-mediated cytotoxicity assays.     

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.     

Production of interleukin-1 and tumor necrosis factor by cisplatin-treated murine peritoneal macrophages

Supernatants collected from cisplatin-treated macrophages demonstrated enhanced cytotoxicity against actinomycin-D-treated L929 cells and also enhanced the thymocyte proliferation in response to concanavalin A, showing that cisplatin-treated macrophages release interleukin-1 (IL-1) and tumor necrosis factor (TNF) into the culture supernatant. The supernatant collected from untreated macrophages showed little TNF and IL-1 activity. The release of TNF and IL-1 was observed to be dependent on the dose and duration of cisplatin treatment. Medium alone containing cisplatin did not enhance thymocyte proliferation and had little cytotoxic effect on actinomycin-D-treated L929 cells. Cisplatin-treated macrophage culture supernatants were chromatographed over a Superose 12 column on an FPLC system. TNF activity eluted in two major peaks with apparent molecular weights of 50-55 and 15-20 kilodaltons, respectively. The kinetics of IL-1 release was also studied. Maximum production and release of IL-1 were observed up to 24 h after cisplatin treatment and then gradually declined. Freeze-thaw lysates of cisplatin-treated macrophages also showed enhanced IL-1 activity. Paraformaldehyde (PFA)-fixed cisplatin-treated macrophages showed significantly enhanced cytotoxic activity against L929 cells as compared to PFA-fixed untreated macrophages. PFA-fixed cisplatin-treated macrophages also enhanced thymocyte proliferation. These results suggest that cisplatin treatment of murine macrophages also results in increased expression of membrane-associated IL-1 and TNF activity.     

Induction of monocyte antitumor response by human cancer cells transduced with TNF-GFP fusion gene: possible implications for immunotherapy of cancer

This study was undertaken to determine how human pancreatic cancer (HPC-4) cells transduced with the TNF-GFP fusion gene (TLG) alter the antitumor response of human monocytes in vitro and whether they could act as an antitumor vaccine. In our model, HPC-4 cells were transduced with retroviral vector harboring TLG gene and designated as HPC-4(TLG). The TLG protein expression was confirmed by Western blot and flow cytometry analysis. Monocytes were co-cultured with transduced and control HPC-4 cells. The secretion of TNF, IL-10 and IL-12 was measured by ELISA. The cytotoxicity of monocytes against HPC-4 cells was determined by MTT test. The results show that the HPC-4(TLG) cells expressed membrane-bound, intracellular and secretory TLG protein. When cultured with HPC-4(TLG) cells, monocytes released a higher amount of TNF, but IL-10 and IL-12 secretion was inhibited. The pre-exposure of monocytes to HPC-4(TLG), but not to HPC-4, cells did not decrease TNF nor increase IL-10 production, thus not leading to monocyte deactivation. Also, the antitumor cytotoxicity of monocytes stimulated with HPC-4(TLG) was not downregulated, which occurred when non-transduced HPC-4 cells were used. In conclusion, compared to parental HPC-4 cells, TLG gene transduced HPC-4 cells induced stronger antitumor response of monocytes in vitro and prevented deactivation of monocytes.     

乳酸杆菌发酵液RNA的抗肿瘤及对巨噬细胞功能的调节作用

目的研究乳酸杆菌DM9811发酵滤液中存在的100200 bp长的RNA片段（Ls-RNA）的免疫调节与抗肿瘤作用。方法采用中性红吞噬试验检测巨噬细胞吞噬功能,用L929细胞检测TNF,用免疫保护试验检测体内抗肿瘤作用。结果Ls-RNA可增强脾巨噬细胞的吞噬活性,对小鼠肝癌Hca-F的生长有抑制作用,延长小鼠的存活时间,但对TG诱导的腹腔巨噬细胞产生TNF无明显调节作用。结论Ls-RNA有一定免疫调节和抗肿瘤作用。     

Enhancement of human monocyte tumoricidal activity by recombinant M-CSF

Activated monocytes are an important component of immunologic defense against neoplastic disease. A variety of agents capable of inducing tumoricidal activity have been described, including bacterial LPS, IFN-gamma, IL-1, IL-2, TNF, and GM-CSF. We now show that pretreatment of monocytes with recombinant human macrophage-specific colony stimulating factor (M-CSF) augments the tumoricidal activity of human peripheral blood monocytes induced by other activating agents. Monocytes were preincubated for three days with M-CSF at 10(3) U/ml, washed, and treated for an additional two days with secondary activators. Tumoricidal activity was measured in a 6-h 51Cr-release assay using NK-resistant WEHI 164 cells that had been treated with actinomycin D. Pretreatment of monocytes with M-CSF significantly increased tumoricidal activity induced by LPS, IFN gamma, LPS plus IFN gamma, and LPS plus PMA. Pretreatment with IL-1, IL-2, IL-3, IL-4, or GM-CSF was not as effective as M-CSF in increasing tumoricidal activity. Enhanced tumoricidal activity was directly correlated to the increased TNF production resulting from M-CSF pretreatment. TNF antiserum completely blocked tumoricidal activity, demonstrating that TNF was responsible for the M-CSF-mediated increase in tumor cell lysis. M-CSF pretreatment also enhanced non-TNF mediated tumoricidal activity by monocytes, as seen by increased killing of the TNF-resistant target P815. This study demonstrated that in addition to the role of M-CSF in the proliferation and differentiation of monocyte/macrophage precursors, M-CSF also augments an effector function of mature blood monocytes.     

Maximal T cell IL-13 production requires monocyte or monokine participation

The T cell-secreted lymphokine interleukin 13 (IL-13) exerts pleiotropic effects on monocytes (Mphi) and B cells. Since accessory cells, like Mphi and B cells, also act in antigen-presenting and lymphokine augmentation of T cells, Mphi and B cells may be able to effect T cell IL-13 production. Purified T cells produced slightly less IL-13 than the lower T cell numbers contained in peripheral blood mononuclear cell population, further suggesting accessory cell augmentation. Addition of 10% B cells [either unstimulated or pokeweed mitogen (PWM)-stimulated] to autologous T cells only moderately augmented T cell IL-13 levels. PWM-stimulated B cell culture supernates had even less augmenting effect on T cell IL-13 levels and unstimulated B cell culture supernates did not augment T cell IL-13 production. In contrast to the moderately augmenting effect of B cells or their stimulated culture supernates, addition of 10% Mphi, either unstimulated or muramyl dipeptide (MDP)+IFN-gamma stimulated, to autologous T cells produced a highly significant increase in T cell IL-13 production. Mphi culture supernates were equally effective in augmenting T cell IL-13 levels, suggesting both that cell-to-cell contact is not critical for Mphi augmentation of T cell IL-13 levels, and that Mphi secreted factors are pivotal. CD64(+) Mphi (or their culture supernates), which are known as poor antigen-presenting cells, also effectively augmented T cell IL-13 production, further supporting the involvement of Mphi secreted factors. Finally, experiments with exogenous addition of recombinant monokines, as well as neutralization experiments with different cytokine antibodies, suggested IL-1beta as a primary cytokine involved in the augmentation of T cell IL-13 levels by accessory cells. However, these experiments also indicated other unidentified Mphi factors as playing a significant role in producing maximal T cell IL-13 production.     

Cellular models of macrophage tumoricidal effector mechanisms in vitro. Characterization of cytolytic responses to tumor necrosis factor and nitric oxide pathways in vitro

The recently described L-arginine-dependent nitric oxide (NO) pathway has been proposed to interact synergistically with the TNF pathway in murine macrophage-mediated tumor cytotoxicity in vitro. We have employed an experimental construct in which these two pathways were independently expressed by two different effector cell populations. The TNF-dependent pathway was committed by murine 3T3 cells transfected with the cDNA encoding human pro-TNF. The NO pathway was executed by the murine EMT-6 mammary adenocarcinoma cell line treated with murine rIFN-gamma and LPS. Controls for the TNF pathway committed by the transfectant included lysis of the TNF-sensitive murine L929 cell in coculture, secretion of TNF, and absence of nitrite synthesis. For the NO pathway controls included lysis of the murine P815 mastocytoma cocultured with activated EMT-6 cells that had been pretreated with murine rIFN-gamma and LPS, production of nitrite by this activated effector cell, and an absence of TNF secretion. The target cell panel included L929, EMT-6, P815, and murine B16 melanoma and TU-5 sarcoma cell lines. All targets on this panel were susceptible to lysis by LPS-triggered murine bacillus Calmette-Guérin-activated macrophages. The 3T3 transfectant caused significant lysis of cocultured L929 and TU-5 targets. The EMT-6 effector cell only caused significant lysis of the P815 target. When both effector cells were cocultured with these target cells, lysis of the P815 target was observed to be additive or superadditive; however, for all the other targets, cytotoxicity was comparable with or subadditive compared with that seen with the 3T3 transfectant effector cell alone. Thus, these two pathways do not appear to account for the broad, potent tumoricidal activity observed for activated macrophages in vitro.     

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.     

Tumor target-derived soluble factor synergizes with IFN-gamma and IL-2 to activate macrophages for tumor necrosis factor and nitric oxide production to mediate cytotoxicity of the same target.

Inflammatory mouse peritoneal macrophages were activated by IFN-gamma in synergy with IL-2 or Lipid A to mediate TNF production for autocrine generation of cytotoxic nitric oxide (NO) to kill P815 or L1210 tumor targets. It was determined that for IL-2, but not Lipid A, to effectively trigger activation of IFN-gamma-primed macrophages, the tumor targets must be also present for interaction with effector macrophages to mediate the production of TNF and NO. IFN-gamma- and IL-2-activated macrophages from syngeneic DBA/2 and allogeneic C3H mice had identical MHC-unrestricted requirements for interaction with DBA/2 mouse-derived P815 and L1210 targets to mediate production of TNF and NO for tumor cytotoxicity. To further define the mechanistic requirements for macrophage-tumor target interaction, IFN-gamma- and IL-2-activated macrophages were separated from P815 targets in culture by a semipermeable membrane. Under these conditions, both TNF and NO were produced by the macrophage, which indicated that the requirement for tumor target-macrophage interaction may be due to a soluble factor produced by the target rather than to direct physical contact. This was confirmed by experiments in which 24-h cell-free culture fluids, derived from either P815 or L1210 tumor targets, substituted for the intact tumor cells in the stimulation of TNF mRNA synthesis and secretion with NO generation of TNF mRNA synthesis and secretion with NO generation by IFN-gamma- and IL-2-activated C3H or DBA/2 macrophages. The activity in 24-h culture fluids derived from P815 and L1210 tumor targets was tentatively designated as tumor-derived recognition factor(s) (TDRF) since it was produced constitutively by the tumor targets and synergized with IFN-gamma and IL-2 to induce macrophage production of TNF and NO for death of the same targets. A variety of nontransformed human and mouse fibroblasts, mouse spleen lymphocytes, and two adherent mouse fibrosarcomas did not produce detectable TDRF activity, whereas two mouse T lymphomas, EL4 and EL4.IL-2, produced TDRF activity similar to L1210 mouse leukemia and P815 mastocytoma. The C3H/MCA, a TDRF-nonproducing mouse fibrosarcoma, was susceptible to cytotoxicity mediated by macrophages activated by IFN-gamma and Lipid A, but not by IL-2 triggering. Exogenous TDRF derived from L1210 targets reconstituted the cytotoxic activity for C3H/MCA MCA targets mediated by IFN-gamma- and IL-2-activated macrophages accompanied by the production of TNF and cytotoxic NO.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipopolysaccharide and interleukin 1 inhibit interferon-gamma-induced Fc receptor expression on human monocytes

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.     

Heterogeneous nature of the acute phase response. Differential regulation of human serum amyloid A, C-reactive protein, and other acute phase proteins by cytokines in Hep 3B cells

Because a number of different cytokines have been reported to regulate the synthesis of human, murine, and rat acute phase proteins (APP), we studied the effect of cytokines on production of several major human APP in a single system, the human hepatoma cell line Hep 3B. Conditioned medium (CM) prepared from human blood monocytes activated with LPS in the presence of dexamethasone led to substantial induction of serum amyloid A (SAA) and C-reactive protein (CRP) synthesis whereas the defined cytokines IL-1 beta, TNF alpha, and medium from a human keratinocyte cell line (COLO-16), containing hepatocyte-stimulating factor activity, failed to induce these two major APP. Induction of SAA and CRP was accompanied by an increase in concentration of their specific mRNA. Size fractionation of CM from activated monocytes by fast protein liquid chromatography indicated that SAA- and CRP-inducing activity eluted as a single peak with a Mr of approximately 18 kDa. alpha 1-Antitrypsin, which also failed to respond to IL-1 beta or TNF alpha, was induced by both CM and medium from COLO-16 cells. The induction of AT by CM was accompanied by an increase in specific mRNA. Induction of ceruloplasmin and alpha 1-antichymotrypsin and decrease in the synthesis of albumin was achieved by both CM and IL-1 beta. Ceruloplasmin and albumin responded in a comparable fashion to both TNF alpha and medium from COLO-16 cells; the response of ACT to these cytokines was not evaluated. These results indicate that human SAA and CRP are induced in Hep 3B cells by products of activated monocytes but not by IL-1 beta, TNF-alpha, or some hepatocyte-stimulating factor preparations and that a group of heterogeneous mechanisms are involved in the induction of the various human APP.     

Role of glucose in interleukin-1β production by lipopolysaccharide-activated human monocytes

When monocytes are activated with endotoxin (lipopolysaccharide [LPS]), they make and release several mediators, including interleukin-1β (IL-1β). This study was undertaken to investigate the role of glucose in IL-1β production by these cells. IL-1β was produced in a dose-dependent manner to glucose concentration in the culture medium. The uptake of (³H)2-deoxyglucose in monocytes was stimulated by LPS 1,554% after 10 minutes, 6,095% after 2 hours, then gradually declined after 4 hours of incubation. The inhibition of the uptake of (³H)2-deoxyglucose by either 10 μM cytochaiasin B or phloretin, added at the time of monocyte activation, was accompanied by significant reduction in ATP/ADP ratio and the inhibition of the production of IL-1β by activated monocytes. The synthesis of total protein did not change in monocytes activated in the absence of glucose in the culture medium, nor in the presence of either 10 μM cytochalasin B or phloretin. The export of IL-1β from LPS-activated monocytes was not inhibited by either 10 μM cytochalasin B or phloretin, nor in the absence of glucose in the culture medium. These data suggest that (1) glucose is required for LPS-induced IL-1β production by monocytes; (2) glucose is the major source of ATP for IL-1β production; (3) glucose transporter (GLUT 1) does not control the export of IL-1β. © 1993 Wiley-Liss, Inc.