Inhibition of macrophage activation by calcium channel blockers and calmodulin antagonists

The biochemical mechanisms by which macrophages become activated to the tumoricidal state are poorly understood. To investigate the role of calcium in this process, the effect of calcium channel blockers and calmodulin antagonists on the acquisition of tumoricidal properties by macrophages activated by a number of different agents was examined. Activation of thioglycollate-stimulated C57BL/6 mouse peritoneal macrophages by macrophage activation factor (MAF) plus LPS, IFN-gamma plus LPS or the calcium ionophore, A23187, was inhibited in a dose-dependent fashion by the calcium channel blockers nifedipine and verapamil. These agents blocked the influx of 45Ca into macrophages activated by MAF plus LPS. Macrophage activation was also inhibited by chlorpromazine, W-7, and calmidazolium at concentrations known to perturb calmodulin function. The data suggest that activation of macrophages to the tumoricidal state is a calcium-dependent process involving the participation of calcium-regulated biochemical reactions whose activities can be modulated by pharmacological agents that frustrate transmembrane calcium fluxes and/or inhibit calmodulin function.     

Abrogation of species specificity for activation of tumoricidal properties in macrophages by recombinant mouse or human interferon-gamma encapsulated in liposomes

Highly purified human blood monocytes, isolated by continuous Percoll density gradients under endotoxin-free conditions, and mouse peritoneal exudate macrophages (PEM) were activated in vitro by the combination of muramyl dipeptide (MDP) and recombinant interferon-gamma (r-IFN-gamma) to become tumoricidal against their respective tumorigenic target cells. The activation of human monocytes or mouse PEM by free unencapsulated r-IFN-gamma and MDP was species specific: human r-IFN-gamma activated human blood monocytes to lyse allogeneic melanoma cells, but did not activate mouse PEM. Mouse r-IFN-gamma activated mouse PEM to lyse syngeneic melanoma cells, but did not activate cytotoxic properties in human monocytes. The encapsulation of either mouse or human r-IFN-gamma with MDP within the same liposome preparation produced synergistic activation of cytotoxic properties in both PEM and monocytes without apparent species specificity. The activation of tumoricidal properties in macrophages by r-IFN-gamma and MDP occurred as a consequence of intracellular interaction. We base this conclusion on the data showing that whereas free r-IFN-gamma and MDP did not activate macrophages pretreated with pronase, liposome-encapsulated r-IFN-gamma and MDP did. Moreover, the i.v. injection of liposomes containing human or mouse r-IFN-gamma and MDP produced in vivo activation of mouse alveolar macrophages. These data suggest that in contrast to activation with free r-IFN-gamma, which requires binding to macrophage surface receptors, the intracellular interaction of r-IFN-gamma, which produces tumoricidal activity in macrophages, is not species specific.     

IL-4 inhibits the costimulatory activity of IL-2 or picolinic acid but not of lipopolysaccharide on IFN-gamma-treated macrophages

We reported previously that IL-2 induces tumoricidal activity in IFN-gamma-treated murine macrophages. The present study was performed to investigate the regulation of IL-2-dependent tumoricidal activity in murine macrophage cell lines. The v-raf/v-myc-immortalized murine macrophage cell lines ANA-1, GG2EE, and HEN-CV did not express constitutive levels of cytotoxic activity against P815 mastocytoma cells. Moreover, these macrophage cell lines did not become tumoricidal after exposure to IL-4, IFN-gamma, IL-2 or LPS. However, these macrophages developed cytotoxic capabilities after incubation with either IFN-gamma plus IL-2 or IFN-gamma plus LPS. IL-4 inhibited IFN-gamma plus IL-2- but not IFN-gamma plus LPS-induced tumoricidal activity. This effect of IL-4 was not restricted to v-raf/v-myc-immortalized macrophage cell lines because similar results were obtained by using a macrophage cell line that was established from a spontaneous histiocytic sarcoma. The suppressive activity of IL-4 on the ANA-1 macrophage cell line was dose-dependent (approximately 12-200 U/ml) and was neutralized by the addition of anti-IL-4 mAb. IL-4 decreased the IFN-gamma-induced expression of mRNA for the p55 (alpha) subunit of the IL-2R in ANA-1 macrophages. Therefore, at least one mechanism by which IL-4 may have inhibited IFN-gamma plus IL-2-induced tumoricidal activity was by reducing macrophage IL-2R alpha mRNA expression. We have previously reported that picolinic acid, a tryptophan metabolite, is a costimulator of macrophage tumoricidal activity. We now report that IL-4 also inhibited IFN-gamma plus picolinic acid-induced cytotoxicity in ANA-1 macrophages. We propose that IL-2 and picolinic acid may have a common mechanism of action that is susceptible to IL-4 suppression.     

Developmental pattern of poly (ADP-ribose) synthetase and NAD glycohydrolase in the brain of the fetal and neonatal rat

Poly (ADP-ribose) synthetase and NAD glycohydrolase were examined in nuclear fractions from rat brain at sequential times during late fetal and the first two weeks of neonatal life. In whole brain, both enzymes were demonstrable at all stages of development, but followed separate patterns. Activity of the synthetase which was greatest in fetal life, fell steadily with fetal maturation from 3.90±0.06 nmol/mg DNA at 16 days, to reach a nadir of 1.36±0.09 nmol/mg DNA on the 4th postnatal day. Subsequently it underwent a non sustained neonatal rise reaching a peak of 2.46±0.07 nmol/mg DNA on the 8th day. By contrast, NAD glycohydrolase activity increased steadily throughout late fetal and during the first two weeks of neonatal life, from 12.77±0.40 nmol/mg DNA on day 16 of gestation to 25.80±.95 nmol/mg DNA on neonatal day 12. In neonatal cerebellum the activity of poly (ADP-ribose) synthetase was greater at 8 than at 4 days, could be stimulated with graded concentrations of sonicated DNA up to 100 g, but was inhibited by higher concentrations of DNA and by all concentrations of exogenous histone. In an in vitro culture system of fetal rat brain cells, the activity of poly (ADP-ribose) synthetase increased steadily over six days. Cycloheximide 10^–3 M completely inhibited the activity of this enzyme. NAD glycohydrolase activity increased progressively in vitro, and after 6 days in cycloheximide (10^–3 M), the cultures contained significantly greater levels of enzyme activity. It is suggested that changing activities of poly (ADP-ribose) synthetase and NAD glycohydrolase could both provide potential markers for brain cell differentiation in this system.     

Role of protein kinase C and intracellular calcium mobilization in the induction of macrophage tumoricidal activity by interferon-gamma

These studies were designed to test the hypothesis that changes in intracellular Ca2+ levels and activation of the calcium ion- and phospholipid-dependent protein kinase C were required for the induction of macrophage tumoricidal activity by interferon-gamma (IFN-gamma). Phenothiazines and R24571, known antagonists of calcium-binding proteins and therefore nonspecific inhibitors of protein kinase C, blocked in a dose-dependent manner the induction of macrophage cytocidal activity by either natural or recombinant IFN-gamma. Macrophages depleted of intracellular Ca2+ by chelation with Quin 2, were also unresponsive to IFN-gamma. These treatments effected neither the binding of IFN-gamma to its cell surface receptor nor the normal intracellular processing of IFN-gamma. Activators of protein kinase C (such as phorbol esters) and Ca2+ ionophores when added alone did not effect the activation state of the macrophage population. However, macrophages exposed to both drugs in combination were elevated into the primed activation state such that in the presence of a second signal (lipopolysaccharide or heat killed Listeria monocytogenes), the cells were triggered to express full levels of tumoricidal activity. The capacity of phorbol esters to induce cellular activation correlated with their ability to bind and to activate protein kinase C. No synergistic effect was observed between IFN-gamma and protein kinase C activators and/or Ca2+ ionophores, indicating that the drugs could only prime and could not trigger macrophages for tumor cell killing. These results thus support the concept that protein kinase C activation and mobilization of intracellular Ca2+ are essential steps in the pathway of IFN-gamma-dependent induction of non-specific tumoricidal activity in macrophages.     

Role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma. II. Down-regulation of macrophage-mediated cytotoxicity by tumor-derived granulocyte-macrophage colony-stimulating factor

Peritoneal elicited macrophages (PEM) from mammary tumor-bearing mice have a decreased capacity to become cytotoxic against syngeneic, allogeneic, and xenogeneic target cells upon in vitro stimulation with LPS, as compared with PEM of normal mice. A regulatory mechanism other than PG release is suggested because the addition of both indomethacin and LPS to macrophage cultures from tumor-bearing mice caused no changes in their cytotoxic capability. Because tumor products have been implicated in the down-regulation of immune responses, we investigated whether pretreatment with supernatants from the tumor cell line DA-3, derived from the in vivo mammary adenocarcinoma D1-DMBA-3, affects the cytolytic capacity of macrophages. This treatment inhibits, in a dose-dependent fashion, the ability of stimulated normal PEM to kill target cells. Partial purification of DA-3 cell line supernatant showed that most of the inhibitory activity was exerted by factors with a molecular mass greater than 10 kDa and less than 30 kDa. However, slight inhibition could also be observed with fractions containing molecules less than 10 kDa. The data suggest that more than one factor released by the mammary tumor cells may be involved in the down-regulation of macrophage-mediated cytotoxicity. Because the DA-3 cells constitutively produce granulocyte-macrophage CSF (GM-CSF), which has a molecular mass of 27 kDa, we pretreated PEM from normal mice in vitro with rGM-CSF for 24 h. This resulted in a dose-dependent decrease in their capacity to kill tumor target cells upon LPS stimulation. Furthermore, PEM from normal mice injected with rGM-CSF for 25 days displayed a profound decrease in their cytolytic ability against DA-3 targets upon in vitro stimulation with increasing amounts of LPS. The pretreatment of PEM from normal mice with a combination of DA-3 cell supernatants and specific anti-GM-CSF partially neutralized the inhibitory effect of the DA-3 supernatant on macrophage tumoricidal capability. These results indicate that tumor-derived GM-CSF is an important factor involved in the decreased macrophage cytotoxicity during mammary adenocarcinoma progression.     

Inhibition of macrophage tumoricidal activity by glucocorticoids

In this study, the effect of corticosteroids on the activation of macrophages to a fully tumoricidal state was examined. Thioglycolate-elicited peritoneal exudate macrophages from C3H/HeJ mice were rendered cytolytic for P815 mastocytoma cells in a two-signal tumoricidal assay that used recombinant interferon-gamma (rIFN-gamma; 1 to 10 U/ml) as a "priming" signal and butanol-extracted lipopolysaccharide (But-LPS; 0.1 to 5 micrograms/ml) as a "trigger" signal. Treatment of macrophages with either rIFN-gamma alone or But-LPS alone failed to result in significant cytolytic ability. Tumoricidal activity was markedly inhibited in a dose-dependent fashion when glucocorticoids were added simultaneously to the cultures with rIFN-gamma and But-LPS at concentrations ranging from 1 X 10(-10) to 1 X 10(-5) M. Nonglucocorticoid sex hormones failed to inhibit tumoricidal activity in this system under identical culture conditions. Inhibition was most effective if the glucocorticoids were added simultaneously with the priming and triggering signals (rIFN-gamma and But-LPS); however, if the glucocorticoids were added 24 hr after the signals were provided to the cultures, suboptimal inhibition was observed. Experiments that dissociated the priming phase of activation from the triggering phase showed that glucocorticoids inhibited both the rIFN-gamma-induced priming stage as well as the But-LPS-induced triggering stage of activation. These observations provide evidence that glucocorticoids, but not other steroid hormones, inhibit the activation of macrophages to a fully tumoricidal state by interfering with either the priming or triggering signals in this two-signal model of macrophage activation.     

Acidic polysaccharide isolated from Phellinus linteus induces nitric oxide-mediated tumoricidal activity of macrophages through protein tyrosine kinase and protein kinase C

Mushroom polysaccharides are increasingly being utilized to treat a wide variety of diseases. Aqueous extracts from the Phellinus linteus have been reported to have anti-tumor and immunomodulatory properties. In particular, acidic polysaccharide (PL) isolated from P. linteus induced a secretory and cellular macrophage response. However, the exact mechanism by which PL regulates the macrophage functions remains unclear. PL-treated murine peritoneal macrophages in vitro and in vivo dramatically induced the production of NO. PL enhanced the lytic death of B16 cells through the production of NO. The present study examined signal molecules that may participate in PL-elicited responses by macrophages. The data demonstrated that a protein kinase C (PKC) inhibitor, staurosporine, and a protein tyrosine kinase (PTK) inhibitor, genistein, inhibited the tumoricidal activity of macrophages induced by PL. In addition, these inhibitors blocked the production of NO and the expression of surface molecules in PL-stimulated macrophages. Furthermore, CD11b/CD18 possibly mediates PL-induced cell activation. These results suggest that PL stimulates NO production for tumoricidal activity and induces cell-mediated immunity by increasing surface molecules, and the process may be a mechanism by which PL produces its therapeutic effects.     

Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.     

Murine recombinant IL-4 is a bifunctional regulator of macrophage growth induced by colony-stimulating factors.

Murine peritoneal exudate macrophage (PEM) coexpress receptors for both granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) and can be induced by both factors, either alone or in combination, to undergo extensive proliferation in vitro. In this study the effect of murine rIL-4 (MurIL-4) on the proliferation of PEM was examined. MurIL-4 alone did not support macrophage proliferation but prolonged their survival in vitro. When MurIL-4 was combined with human (Hu)rM-CSF, it enhanced the proliferative response of PEM to rHuM-CSF in a dose-dependent manner, reaching a maximum at approximately 10 ng/ml. Contrarily, MurIL-4 suppressed the proliferative response of PEM to MurGM-CSF. Receptor binding assays using radiolabeled ligands showed that MurIL-4 selectively enhanced the expression of M-CSF receptors; suggesting that at least part of the synergistic effect of MurIL-4 is mediated at the receptor level. Of relevance to this effect is the finding that MurIL-4 greatly promoted the responsiveness of PEM to low concentrations of HurM-CSF. Unlike M-CSF receptors, however, MurIL-4 treatment failed to modulate the levels of GM-CSF receptors in PEM. The proliferative responses of PEM to both MurGM-CSF and HurM-CSF could be inhibited by MurIFN-gamma with similar sensitivity. This inhibitory effect of MurIFN-gamma was partially neutralized by MurIL-4 in cultures containing HurM-CSF but not those containing MurGM-CSF. This study demonstrates that IL-4 is involved directly in the regulation of macrophage production by modulating their responsiveness to various cytokines.     

A dual role for poly-ADP-ribosylation in spatial memory acquisition after traumatic brain injury in mice involving NAD+ depletion and ribosylation of 14-3-3gamma

Poly(ADP-ribose) polymerase-1 (PARP-1) is a homeostatic enzyme that paradoxically contributes to disturbances in spatial memory acquisition after traumatic brain injury (TBI) in transgenic mice, thought to be related to depletion of its substrate nicotinamide adenine dinucleotide (NAD+). In this study, systemic administration of the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP) after TBI preserved brain NAD+ levels and dose-dependently reduced poly-ADP-ribosylation 24 h after injury. While moderate-dose INH2BP improved spatial memory acquisition after TBI; strikingly, both injured- and sham-mice receiving high-dose INH2BP were unable to learn in the Morris-water maze. Poly-ADP-ribosylated peptides identified using a proteomics approach yielded several proteins potentially associated with memory, including structural proteins (tubulin alpha and beta, gamma-actin, and alpha-internexin neuronal intermediate filament protein) and 14-3-3gamma. Nuclear poly-ADP-ribosylation of 14-3-3gamma was completely inhibited by the dose of INH2BP that produced profound memory disturbances. Thus, partial inhibition of poly-ADP-ribosylation preserves NAD+ and improves functional outcome after TBI, whereas more complete inhibition impairs spatial memory acquisition independent of injury, and is associated with ribosylation of 14-3-3gamma.     

Superoxide dismutase as a target of clioquinol-induced neurotoxicity

Subacute myelo-optico-neuropathy (SMON) is a progressive neurological disorder affecting the spinal cord, peripheral nerves and optic nerves. Although it has been assumed that SMON was caused by intoxication of clioquinol, the mechanism underlying clioquinol-induced neurotoxicity is not fully understood. This study aimed to clarify the relevance of oxidative stress to clioquinol-induced neurotoxicity and the cause of the enhanced oxidative stress. Clioquinol induced cell death in human-derived neuroblastoma cell line, SH-SY5Y, in a dose-dependent manner. This process was accompanied by activation of caspase-3 and enhanced production of reactive oxygen species (ROS). We examined whether clioquinol inhibited the activity of superoxide dismutase-1 (SOD1), based on its metal chelating properties. Clioquinol inhibited activities of purified SOD1 in a dose-dependent manner. Cytosolic SOD activities were also inhibited in SH-SY5Y cells treated with clioquinol. Finally, addition of exogenous SOD1 to the culture significantly reduced enhanced ROS production and cell death induced by clioquinol in SH-SY5Y cells. These findings suggested that enhanced oxidative stress caused by inhibition of SOD1 undelay clioquinol-induced neurotoxicity and was relevant to the pathogenesis of SMON.     

Hsp70 attenuates hypoxia/reoxygenation-induced activation of poly(ADP-ribose) synthetase in the nucleus of adult rat cardiomyocytes

Effects of heat shock protein 70 (Hsp70) translocated to nuclear fraction on hypoxia/reoxygenation injury was examined by using adult cardiomyocytes isolated from rats. Cardiomyocytes were exposed to heat shock at 42°C for 15 min (HS group), and then incubated at 37°C for 6–24 h. Hsp70 production increased and the protein translocated from cytosol to nucleus. The maximum level of Hsp70 in the nuclear fraction was observed 12 h after HS. When cardiomyocytes without exposure to HS (nHS group) were subjected to 120 min hypoxia/15 min reoxygenation (Hypo/Reoxy), post-hypoxic cell viability was approximately 25% of the pre-hypoxic value. A rise in poly(ADP-ribose) synthetase (PARS) activity in the nuclear fraction was observed in nHS group, associated with an increase in polyADP-ribosylated protein. In contrast, post-hypoxic cell viability of HS group was approximately 60% of the pre-hypoxic value. Hypo/reoxy-induced rise in PARS activity and increase in polyADP-ribosylated protein were attenuated in HS group. To confirm the relationship between an increase in cell viability after Hypo/Reoxy and attenuation of PARS activation, cardiomyocytes without exposure to HS were subjected to Hypo/Reoxy in the presence of 1 mM 3-aminobenzamide, an inhibitor of PARS. Treatment of cells with 3-aminobenzamide attenuated Hypo/Reoxy-induced decrease in cell viability. These results suggest that Hsp70 translocated into nucleus after HS may attenuate PARS activation during Hypo/Reoxy, leading to the cytoprotection of cardiomyocytes against Hypo/Reoxy injury.     

Macrophage activation by interferon alpha + beta is associated with a loss of proliferative capacity: role of interferon alpha + beta in the regulation of macrophage proliferation and function

Murine peritoneal exudate macrophages (PEM) can be induced by colony-stimulating factor (CSF-1) to undergo extensive proliferation and colony formation in vitro. In the presence of interferon alpha + beta (IFN alpha + beta) the proliferative capacity of PEM was greatly suppressed in a dose-dependent manner. The antiproliferative activity of IFN alpha + beta appears to be noncytocidal and reversible at low concentrations. At higher concentrations, exposure to IFN alpha + beta was sufficient to cause growth inhibition in PEM. Tissue-derived PEM were at least 25-fold more sensitive than bone marrow GM-CFC to the antiproliferative activity of IFN alpha + beta. The fact that bone marrow-derived adherent cells also exhibited a higher degree of sensitivity than the less differentiated nonadherent counterparts suggests that the primary targets of IFN alpha + beta are cells derived from a later stage of development. Concomitantly with the loss of proliferative activity, both the tumoricidal and Fc receptor-mediated phagocytic activities in IFN alpha + beta treated PEM were greatly enhanced. These effects could be completely neutralized by the addition of anti-IFN alpha + beta immunoglobulin, indicating that they are mediated by the same molecule. This remarkable dichotomy in the actions of IFN alpha + beta (stimulates functional activities but suppresses proliferative capacity) suggests that IFN alpha + beta may play a role in the regulation of macrophage production and function.     

Temporally different poly(adenosine diphosphate-ribosylation) signals are required for DNA replication and cell division in early embryos of sea urchins

To analyze the temporal relationship of poly(adenosine diphosphate [ADP]-ribosylation) signal with DNA replication and cell divisions, the effect of 3 aminobenzamide (3ABA), an inhibitor of the poly(ADP-ribose)synthetase, was determined in vivo during the first cleavage division of sea urchins. The incorporation of ³H-thymidine into DNA was monitored and cleavage division was examined by light microscopy. The poly(ADP-ribose) neosynthesized on CS histone variants was measured by labeling with ³H-adenosine during the two initial embryonic cell cycles and the inhibitory effect of 3ABA on this poly(ADP-ribosylation) was determined. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) de novo during the initial cell cycles of embryonic development. The synthesis of poly(ADP-ribose) is decreased but not abolished by 20 mM of 3ABA. The incubation of zygotes in 3ABA at the entrance into S1 phase decreased ³H-thymidine incorporation into DNA in phase S2, while S1 was unaltered. Alternatively, when the same treatment was applied to zygotes at the exit of S1 phase, a block of the first cleavage division and a retardation of S2 phase were observed. The inhibitory effect of 3ABA on both DNA replication and cell division was totally reversible by a release of the zygotes from this inhibition. Taking together these observations it may be concluded that the poly(ADP-ribosylation) signals related to embryonic DNA replication are not contemporaneous with S phase progression but are a requirement before its initiation. These results also indicate that a poly(ADP-ribosylation) signal is required for cell division; such signal is temporally different from that related to S phase initiation and occurs at the exit of S phase. © 1993 Wiley-Liss, Inc.     

Production of C3 as a marker of lymphokine-mediated macrophage activation

C3 production was assayed using an enzyme-linked immunosorbent assay (ELISA) in cell-free supernatants harvested from thioglycollate-elicited macrophages exposed to a variety of macrophage stimulating and activating agents. Macrophage monolayers treated with the stimulating agents starch, glycogen, and zymosan secreted three- to four-fold less C3 (mean 12 ng/10(5) cells/12 hr) than macrophages exposed to lymphokines containing macrophage-activating factor (MAF) (mean C3 production 44 ng/10(5) cells/12 hr). The increased production of C3 in macrophages exposed to MAF parallels the ability of these macrophages to acquire tumoricidal capacity as monitored in an in vitro 72 hr tumor cell cytotoxicity assay using B16 melanoma cells. Macrophages previously rendered tumoricidal by exposure to MAF and which are refractory to further challenge by MAF following decay of their tumoricidal properties, do not produce C3 on rechallenge with MAF. Exposure of refractory macrophages to liposome-encapsulated MAF overcomes the refractory state and induces re-expression of the tumoricidal phenotype and C3 production. We conclude that quantitative detection of macrophage-generated C3 antigen provides a useful biochemical marker for monitoring the acquisition of tumoricidal properties in macrophages exposed to MAF and offers a sensitive assay for screening novel agents that activate macrophages via mechanisms similar to MAF.     

Calpain activation is not required for AIF translocation in PARP-1-dependent cell death (parthanatos)

Apoptosis-inducing factor (AIF) is critical for poly(ADP-ribose) polymerase-1 (PARP-1)-dependent cell death (parthanatos). The molecular mechanism of mitochondrial AIF release to the nucleus remains obscure, although a possible role of calpain I has been suggested. Here we show that calpain is not required for mitochondrial AIF release in parthanatos. Although calpain I cleaved recombinant AIF in a cell-free system in intact cells under conditions where endogenous calpain was activated by either NMDA or N -methyl- N '-nitro- N -nitrosoguanidine (MNNG) administration, AIF was not cleaved, and it was released from mitochondria to the nucleus in its 62-kDa uncleaved form. Moreover, NMDA administration under conditions that failed to activate calpain still robustly induced AIF nuclear translocation. Inhibition of calpain with calpastatin or genetic knockout of the regulatory subunit of calpain failed to prevent NMDA- or MNNG-induced AIF nuclear translocation and subsequent cell death, respectively, which was markedly prevented by the PARP-1 inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-iso-quinolinone. Our study clearly shows that calpain activation is not required for AIF release during parthanatos, suggesting that other mechanisms rather than calpain are involved in mitochondrial AIF release in parthanatos.     

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.     

Inhibition of retroviral mRNA expression in the murine macrophage cell line GG2EE by biologic response modifiers

We immortalized the GG2EE macrophage (M phi) cell line by infection of freshly isolated bone marrow cells with the recombinant J2 retrovirus carrying v-raf and v-myc oncogenes. We investigated the expression of J2 virus mRNA in relationship with the proliferative ability and tumoricidal activity of GG2EE cells exposed to biologic response modifiers (BRM). Calcium ionophore (Ca2+I), picolinic acid (PA), or IFN-gamma were employed to activate GG2EE cells. Each BRM was due to inhibit the proliferation of GG2EE cells in a dose-dependent manner, whereas only Ca2+I or the combined treatment with PA plus IFN-gamma induced tumoricidal GG2EE cells. J2 virus mRNA expression was not affected by PA or IFN-gamma, but it was dramatically decreased by Ca2+I or PA plus IFN-gamma. These results indicated that the expression of J2 mRNA can be inhibited in GG2EE cells by appropriate BRM such as Ca2+I or IFN-gamma plus PA. In contrast, the expression of 2'5'-oligoadenylate synthetase mRNA was augmented to similar levels by treatment of the GG2EE cells with IFN-gamma alone or in combination with PA. The down-regulation of J2 mRNA expression was not associated with the antiproliferative activity of the BRM but rather with their ability to induce tumoricidal activity. These results suggest that the process of activation of tumoricidal macrophages also triggers a mechanism(s) of resistance to viral mRNA expression. Moreover, the finding that IFN-gamma or PA inhibit cell proliferation but not J2 mRNA expression indicates that the intracellular targets of these BRM are intact, independent from and unaffected by J2 virus expression.