Regulation of macrophage-mediated tumor cell killing by prostaglandins: comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated in vitro by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), the latter detected as its stable metabolite, 6-keto PGF1 alpha. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10(-7)M for PGI2 and 3 x 10(-8)M for PGE2. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE2 and PGI2; however, only PGE2 had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell killing

The effects of mouse interferon-alpha (MuIFN-alpha), -beta (MuIFN-beta), and -gamma (MuIFN-gamma) on macrophage activation for tumor cell killing were determined by using proteose peptone-elicited peritoneal macrophages from C3H/HeN and C3H/HeJ mice under conditions that either included or were free of detectable endotoxin. Alone, under the conditions used, none of the interferons was able to activate macrophages directly for tumor cell killing. However, with a second signal provided to responsive macrophages by contaminating endotoxin, added bacterial lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM), all three types of interferon induced cytolytic activity, with MuIFN-gamma approximately 500 to 1000-fold more active than either MuIFN-alpha or -beta. Thus, all three interferons were able to prime macrophages for killing but required a second signal before cytolytic activity could be expressed. When MuIFN-gamma was mixed with either MuIFN-alpha or -beta and placed on macrophages, little or no killing developed. Mixtures of MuIFN-gamma with either MuIFN-alpha or -beta did increase the sensitivity of macrophages to triggering by LPS, however, compared with macrophages treated with MuIFN-gamma alone. The results are collectively important because they i) confirm that significant quantitative differences exist between the various interferons with regard to their capacity to prime macrophages for tumor cell killing; ii) indicate that to be an efficient activator each type of interferon must be combined with a second stimulus, such as LPS or HKLM; iii) show that neither MuIFN-alpha nor -beta can provide an efficient second triggering signal for macrophages that are primed by MuIFN-gamma; and iv) document that mixtures of MuIFN-gamma with either MuIFN-alpha or -beta are most efficient at inducing priming, compared with any one of the interferons used alone.     

Lipopolysaccharide-mediated macrophage activation: the role of calcium in the generation of tumoricidal activity

As we have reported, calcium ionophore A23187 activates macrophages for tumor cell killing, and the activated macrophages produced a soluble cytotoxic factor (M phi-CF) that is similar, if not identical, to tumor necrosis factor. Based on these observations, we have investigated whether calcium is involved in the activation mediated by another potent macrophage activator, namely lipopolysaccharide (LPS). We first showed that A23187 caused uptake of extracellular calcium-45 by macrophage monolayers, whereas LPS did not. Because in this system rapid changes would not have been detected, several other approaches also have been used. We have examined the effect of depleting extracellular calcium by using medium containing no added calcium, supplemented with 1 mM EGTA. In no case did depletion result in decreased M phi-CF production by LPS-treated macrophages. Measurements using the fluorescent intracellular calcium indicator Quin 2 have also been performed. The calcium ionophore ionomycin caused a rapid change in the intracellular Quin 2 signal. LPS, even at a concentration in vast excess of that required to activate the macrophages, caused no change in the signal during a 2-hr period. If the macrophages were loaded with high doses of Quin 2 or another intracellular chelator, TMB-8, M phi-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M phi-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium.     

Identification and characterization of monoclonal antibodies specific for macrophages at intermediate stages in the 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. A number of mediators have been identified which have activating capability, including in particular IFN-gamma and bacterial LPS. Although the synergistic functional response of normal macrophages to sequential incubation with these activation signals has been well-established, characterization of the intermediate stages in the activation pathway has been difficult. We have developed a model system for examination of various aspects of macrophage activation, through 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 IFN-gamma and LPS in the development of tumor cytolytic activity. In addition, these cells can be stably primed by the administration of gamma-radiation. In the studies reported here, we have used RAW 264.7 cells treated with IFN-gamma alone or with IFN-gamma plus LPS to stimulate the production of rat mAb probes recognizing cell surface changes occurring during the activation process. In this way we have identified three Ag associated with intermediate stages of the activation process. One Ag, TM-1, is expressed on RAW 264.7 cells primed by IFN-gamma or gamma-radiation. This surface Ag thus identifies cells at the primed cell intermediate stage of the tumoricidal activation pathway regardless of the mechanism of activation. A second Ag, TM-2, is expressed on IFN-treated RAW 264.7 cells but not on RAW 264.7 cells primed with gamma-radiation alone. Expression of this Ag can be induced by treatment of irradiated cells with IFN-gamma, but is not induced by IFN-gamma treatment of a noncytolytic cell line, WEHI-3. This Ag thus appears to be an IFN-inducible cell surface protein associated specifically with macrophage activation for tumoricidal activity. Finally, Ag TM-3 is detectable on RAW 264.7 cells primed by either IFN-gamma or gamma-radiation, after subsequent triggering of the primed cells with LPS. The addition of the mAb recognizing this antigen to the function assay of tumor cell killing can inhibit they lytic activity of both triggered cells. Thus, this Ag may play a role in the antitumor effector functions of activated macrophages. Overall, the results suggest that these mAb can serve as useful tools for identification of molecules associated with the process of macrophage activation for tumor cell killing.     

Lipopolysaccharide and polyribonucleotide activation of macrophages: implications for a natural triggering signal in tumor cell killing

There is evidence that activation of macrophages for tumor cell killing can involve either two signals (interferon/lipopolysaccharide, for example) or one signal (lipopolysaccharide or double-stranded RNA, for example). We investigated the apparent one-signal activation of bone marrow-derived macrophages for P815 mastocytoma killing by treatment with lipopolysaccharide (LPS) or by the synthetic double-stranded polyribonucleotide polyinosinic acid-polycytidylic acid (poly I:C). We found that "direct" activation of macrophages by either LPS or poly I:C was still a two-signal process. Based on antibody neutralizations, the first signal was probably mediated by LPS or poly I:C induced alpha/beta interferon in the macrophage cultures, and the second signal was that of a direct effect of the LPS or poly I:C on the cell. The fact that poly I:C can provide the triggering signal for macrophage activation suggests a possible role for double-stranded RNA structures in macrophage triggering. Such double-stranded RNA requirements could be met by single-stranded RNAs that possess significant double-strandedness in their structures.     

Stimulation of macrophage tumoricidal activity by the growth and differentiation factor CSF-1

The effect of the macrophage growth and differentiation factor CSF-1 on the tumoricidal capacity of murine peritoneal exudate macrophages was investigated. Pretreatment of peptone-elicited macrophages 1 day with 300-1200 U/ml CSF-1 induced moderate killing and greatly stimulated lymphokine (LK)-induced killing of [3H]thymidine-labeled TU5 sarcoma cells to levels above that seen with fresh macrophages. Further addition of CSF-1 at Day 1 at the time of the tumor lysis assay promoted moderate increases in spontaneous and LK-induced activity. CSF-1 did not stimulate freshly harvested exudate macrophages to lyse TU5 targets in the presence or absence of lymphokine (LK) activators. Lipopolysaccharide (LPS) at 0.1-1000 ng/ml did not stimulate cytotoxicity, and the low endotoxin content and the use of polymyxin B and C3H/HeJ mice excluded a role for LPS in these experiments. Incubation of the macrophages with IFN and the myeloid growth factors IL-3 and GM-CSF did not stimulate tumoricidal activity. CSF-1 has been proposed as a therapeutic agent to restore myeloid cell numbers in induced (cancer chemotherapy, bone marrow transplantation, etc.) and natural aplastic anemias. These studies show that CSF-1 also may be useful in combination with LK activators to promote resistance to cancer in mature mononuclear cells. CSF-1 may have similar effects in LK-activated macrophages to enhance resistance to infectious diseases.     

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E₂ (PGE₂) and prostaglandin I₂ (PGI₂), the latter detected as its stable metabolite, 6-keto PGF_1α. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10⁻⁷M for PGI₂ and 3 × 10⁻⁸M for PGE₂. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE₂ and PGI₂; however, only PGE₂ had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Macrophage activation for intracellular killing as induced by calcium ionophore. Correlation with biologic and biochemical events

Changes in the concentration of cytosolic Ca2+ are known to affect various macrophage functions; in particular, exposure in vitro to the Ca2+ ionophore A23187 primes macrophages for tumor cell killing. In the present report, it is shown that treatment with this ionophore similarly mimics IFN-gamma as a priming signal for induction of microbicidal activity. Incubation of mouse bone marrow-derived macrophages with 10(-7) to 10(-6) M A23187 (in the presence of Ca2+) led to intracellular killing of the protozoan parasite Leishmania enriettii within 24 h, provided LPS (1 ng/ml) was also present; no microbicidal activity was observed using either compound alone. A 4-h exposure to the ionophore in the presence of Ca2+ (priming phase) was sufficient to induce leishmanicidal activity upon reincubation with LPS, here acting as a necessary second signal. Addition of EGTA during the priming phase blocked intracellular killing upon subsequent LPS treatment; microbicidal activity could be restored by excess Ca2+, but not Mg2+, suggesting that changes in the concentration of cytosolic Ca2+ are sufficient to mediate the molecular events that lead to acquisition of microbicidal potential. Ionophore-induced leishmanicidal activity was paralleled by a stimulation of the hexosemonophosphate shunt pathway and production of nitrites, which are biochemical correlates of the activated state. In addition, sequential exposure to A23187 and LPS markedly stimulated macrophages to release TNF and PGE2, two agents thought to act as modulators of macrophage activation.     

Suppression of both macrophage-mediated tumor cell lysis and cytolytic factor production by a factor (CIF) derived from normal embryonic fibroblasts

Summary We had previously established a murine bone marrow-derived cell line, designated JBM1.1, which displayed properties of normal macrophages, including the ability to perform macrophage-mediated cytolysis. It was also found that these cells could be induced by lipopolysaccharide (LPS) to produce reproducibly high levels of a cytolytic factor (CF) resembling tumor necrosis factor (TNF). This cell line was therefore selected for further studies on macrophage-mediated tumor cell lysis and CF production. Moreover, the CF production during incubation with LPS was higher in the absence of serum than in its presence, with a maximum at days 2–3 following the addition of LPS. A factor inhibitory to CF production (CIF) was detected in our laboratory in the supernatant of embryonic fibroblast cultures. We established the experimental conditions required for the optimal production and suppressive effect of CIF. High levels of CIF activity were obtained under conditions that promote fibroblast proliferation. Addition of embryonic fibroblast culture supernatant to the macrophages shortly before LPS suppressed both LPS-induced CF production and tumoricidal activity. CIF did not affect macrophage protein synthesis in the presence or absence of LPS. However, LPS-induced interleukin 1 release was partially (55%) suppressed by embryonic fibroblast culture supernatant. Our results show that CIF does not exert a general inactivating effect on the macrophages, although it may possibly affect other functions in addition to CF production and tumor cell lysis. The strong inhibition of both the latter properties further indicates that TNF-like CF is an important mediator in macrophage-mediated tumor cell lysis.     

IFN-gamma differentially modulates the susceptibility of L1210 and P815 tumor targets for macrophage-mediated cytotoxicity. Role of macrophage-target interaction coupled to nitric oxide generation, but independent of tumor necrosis factor production

IFN-gamma primes murine macrophages to render them responsive for triggering by subactivating concentrations of bacterial LPS to mediate nonspecific tumor cytotoxicity. However, IFN-gamma also has direct anti-proliferative effects on transformed cells that serve as sensitive tumor targets for cytotoxic macrophages. We investigated the effects of preexposure of L1210 mouse leukemia and P815 mouse mastocytoma targets to rIFN-gamma on changes in their susceptibility to cytotoxicity by LPS-activated mouse peritoneal macrophages (PM). Co-incubation of inflammatory PM and either L1210 or P815 targets with IFN-gamma and LPS produced a classical synergistic cytotoxicity for both targets over that of IFN-gamma or LPS alone. Similar synergistic augmentation of cytotoxicity occurred when effector PM were preprimed for 24 h with IFN-gamma before testing for cytotoxicity of untreated targets. However, pretreatment of L1210 and P815 targets for 24 h with IFN-gamma (50 U) before assay produced divergent results in that L1210 was more susceptible, whereas P815 was less susceptible to cytotoxicity by LPS-activated macrophages. Similar results were obtained when both macrophages and targets were pretreated separately with IFN-gamma for 24 h before their combined assay for tumor cytotoxicity. Pretreatment of L1210 targets for 1, 4, or 24 h with IFN-gamma produced similar effects on their increased susceptibility to macrophage cytotoxicity. In contrast, P815 pretreated for 1 and 4 h with IFN-gamma showed an early increased susceptibility to macrophage cytotoxicity followed by a decrease after 24 h pretreatment. The pretreatment of L1210 or P815 targets with IFN-gamma before their exposure to LPS-activated macrophages had no effect on the production of TNF. However, there was a corresponding increase in nitric oxide generation by LPS-activated macrophages after their exposure to IFN-gamma pretreated L1210 targets and a decrease in the presence of IFN-gamma-pretreated P815 targets that correlated with their changes in susceptibility to macrophage killing. Nitric oxide generation by macrophages alone in response to LPS was found to be greater than when effector macrophages were exposed to the tumor targets and this was either increased by L1210 or decreased by P815 that had been pretreated with IFN-gamma. Our results indicate that IFN-gamma may act directly and differentially on tumor targets to alter their susceptibility for macrophage cytotoxicity, which was coupled to changes in the generation of cytotoxic nitric oxide, rather than TNF production by the macrophage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of tumor necrosis factor in cytotoxicity mediated by human monocytes

Human monocytes cultured in a specially prepared medium free of lipopolysaccharide (LPS) constitutively produced a small, though significant, amount of tumor necrosis factor (TNF). Upon addition of LPS, the amount produced remained constant until the LPS concentration reached 1-10 ng/ml, whereupon the production of TNF dramatically increased, eventually becoming 100-fold greater than when the LPS concentration was below 1 ng/ml. Priming the monocytes with recombinant interferon-gamma (rIFN-gamma) before LPS exposure resulted in a 2- to 10-fold increase in TNF production, the highest relative increase being obtained at lower LPS concentrations and in the absence of LPS. Monocyte-produced TNF appears to be the effector molecule in monocyte-mediated killing of some target cell types, since antiserum against recombinant TNF inhibited killing of both actinomycin D-treated and untreated WEHI 164 cells by human monocytes. However, it also appears that TNF may not in all cases be an effector molecule in monocyte-mediated killing, since cytolysis of K562 cells mediated by IFN-gamma/LPS-activated monocytes was not inhibited by antiserum against recombinant TNF. Antiserum which was raised against a monocyte-derived cytotoxic factor and which neutralized recombinant TNF did, however, inhibit monocyte-mediated cytolysis of K562 cells, suggesting that an extracellular factor, perhaps related to TNF, was also involved in monocyte-mediated killing of K562 cells. A TNF-like activity was associated with the monocyte surface membrane, since paraformaldehyde-fixed monocytes expressed cytotoxic activity which was neutralized by antiserum against recombinant TNF. Fixed monocytes activated with rIFN-gamma in addition to LPS before fixation were generally more cytotoxic than those exposed to LPS alone, and those exposed to LPS were much more cytotoxic than those not exposed to LPS. Thus it is possible that high local TNF concentrations may be generated near the target cell upon direct contact between effector and target cells, and that also monocyte-associated TNF may in this way be involved in monocyte-mediated cytotoxicity.     

Lymphokine maintains macrophage activation for tumor cell killing by interfering with the negative regulatory effect of prostaglandin E2

Mouse resident peritoneal macrophages activated with bacterial lipopolysaccharide (LPS) rapidly lost their ability to kill tumor cells in vitro. Such loss of killing has previously been attributed to the effects of prostaglandin E (PGE) produced by the LPS-stimulated macrophages. Macrophages exposed in the current study to both LPS and partially purified lymphokine did not lose cytolytic activity, in spite of the fact that these cells produced undiminished amounts of PGE, compared to controls. Cytolytic activity was shown to be retained under these conditions because lymphokine decreased the sensitivity of activated macrophages to the negative regulatory effects of PGE. The mechanism responsible for the lymphokine effect is not known; however, generalized inhibition of macrophage responsiveness to the hormone does not appear to be involved because lymphokine did not reduce the cyclic AMP response of macrophages, measured on a whole cell basis, after they were exposed to PGE.     

Effect of porcine respiratory coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs

Porcine respiratory coronavirus (PRCV) potentiates respiratory disease and proinflammatory cytokine production in the lungs upon intratracheal inoculation with lipopolysaccharide (LPS) at 1 day of infection. This study aimed to quantify LPS-binding protein (LBP), CD14 and haptoglobin in the lungs throughout a PRCV infection. LBP and CD14 recognize LPS and enhance its endotoxic activity, whereas haptoglobin dampens it. Gnotobiotic pigs were inoculated intratracheally with PRCV (n = 34) or saline (n = 5) and euthanized 1-15days post inoculation (DPI). Virus was detected in the lungs from 1 to 9DPI. Cell-associated CD14 in lung tissue increased up to 15 times throughout the infection, due to an increase in highly CD14+ monocyte-macrophages from 1 to 12DPI and CD14+ type 2 pneumocytes from 7 to 9DPI. LBP and soluble CD14 levels in bronchoalveolar lavage fluids were elevated from 1-12DPI, with up to 35- and 4-fold increases, respectively. Haptoglobin levels increased significantly (x4.5) at 7DPI. In addition, we found that PRCV could sensitize the lungs to LPS throughout the infection, but the response to LPS appeared less enhanced at the end of infection (7DPI). The marked increases in LBP, CD14 and haptoglobin were not correlated with the extent of the LPS response.     

The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38

Mitogen-activated protein (MAP) kinases are critical mediators of innate immune responses. In response to lipopolysaccharide (LPS), MAP kinases are rapidly activated and play an important role in the production of proinflammatory cytokines. Although a number of MAP kinase phosphatases (MKPs) have been identified, their roles in the control of cytokine production have not been well defined. In the present report, we investigated the role of MKP-1 in alveolar macrophages stimulated with LPS. We found that LPS triggered transient activation of three MAP kinase subfamilies, ERK, JNK, and p38, in both immortalized and primary murine alveolar macrophages. MKP-1 was rapidly induced by LPS, and its induction correlated with the dephosphorylation of these MAP kinases. Blocking MKP-1 with triptolide prolonged the activities of both JNK and p38 in immortalized alveolar macrophages. Stimulation of primary alveolar macrophages isolated from MKP-1-deficient mice with LPS resulted in a prolonged p38 phosphorylation compared with wild type alveolar macrophages. Accordingly, these MKP-1-deficient alveolar macrophages also mounted a more robust and rapid tumor necrosis factor alpha production than their wild type counterparts. Adenovirus-mediated MKP-1 overexpression significantly attenuated tumor necrosis factor alpha production in immortalized alveolar macrophages. Finally, MKP-1 was induced by a group of corticosteroids frequently prescribed for the treatment of inflammatory lung diseases, and the anti-inflammatory potencies of these drugs closely correlated with their abilities to induce MKP-1. Our studies indicated that MKP-1 plays an important role in dampening the inflammatory responses of alveolar macrophages. We speculate that MKP-1 may represent a novel target for therapeutic intervention of inflammatory lung diseases.     

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.     

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.     

LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1.

The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).     

Involvement of CD14 in the inhibitory effects of dimethyl-alpha-cyclodextrin on lipopolysaccharide signaling in macrophages

The potential use of alpha-cyclodextrin and its hydrophilic alpha-cyclodextrin derivatives (alpha-CyDs) as antagonists against lipopolysaccharide (LPS), which stimulates the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production as well as nuclear factor-kappaB (NF-kappaB) activation in macrophages was examined. Of three alpha-CyDs used in the present study, 2,6-di-O-methyl-alpha-CyD (DM-alpha-CyD) had greater inhibitory activity than did the other CyDs against NO and TNF-alpha production through an impairment of gene expression in macrophage cell lines and primary macrophages stimulated with LPS and lipid A in a concentration-dependent manner. Concomitantly, DM-alpha-CyD inhibited NF-kappaB translocation into nucleus. These inhibitory effects of DM-alpha-CyD could be attributed to the release of CD14 from lipid rafts caused by an efflux of phospholipids, but not cholesterol. These results suggest that DM-alpha-CyD may have promise as a potent and unique antagonist for excess activation of macrophages stimulated with LPS.     

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.     

NAC Attenuates LPS-Induced Toxicity in Aspirin-Sensitized Mouse Macrophages via Suppression of Oxidative Stress and Mitochondrial Dysfunction

Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC), an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.