Biological and Biochemical Characterization of Macrophage Activating Factor (MAF) in Murine Lymphocytes: Physicochemical Similarity of MAF to Gamma Interferon (IFN-γ)

During the course of an investigation designed to separate macrophage activating factor (MAF) activity from interferon (IFN) antiviral activity in the lymphokine-rich fraction (LKF) produced by stimulation of murine splenic cells with concanavalin A (Con A), we found molecular evidence for the similarity of the two activities. MAF activity was expressed as the rate of inhibition of intracellular growth of Salmonella typhimurium in macrophages based on the linear correlation between relative MAF activity and LKF concentration. The antiviral substance in LKF was identified as IFN-γ based on the observation that its activity was inactivated at pH 2 and neutralized with anti-mouse IFN-γ serum but not with anti-mouse IFN-α/β serum. MAF and IFN antiviral activities displayed identical sensitivity to pH 2 and temperature. Further, neither activity was affected by β-mercapto-ethanol, but both were inactivated by guanidine hydrochloride and by sodium dodecyl sulfate, suggesting that the structures related to conformation of the protein of the two molecules may be similar. In affinity chromatography of the LKF on a Con A-Sepharose column, MAF and IFN activities were found in both the nonadsorbing (F I) and adsorbing (F II) fractions. However, the rates of F II of MAF and IFN activities increased proportionally when the sample was applied on a column of higher capacity, suggesting that the molecular structure of the mannose-containing glycosyl moiety of the two molecules may also be similar. Moreover, the intact or modified form of MAF and IFN activities of different LKF preparations showed a strong correlation, indicating that the production and denaturation of MAF activity were proportional to those of IFN antiviral activity. The results of this study provide strong evidence that MAF and IFN antiviral activities may reside in virtually the same molecular species.     

d-Mannose as a component of the macrophage surface receptor for macrophage-activating factor (MAF) in mice

Mouse peritoneal exudate macrophages were rendered cytostatic and cytolytic for various mouse tumor cells in vitro by exposure to partially purified lymphokines containing macrophage-activating factor (MAF) at 37 °C for 2 hr. The macrophage activation disappeared completely when either 0.1 Md-mannose or 0.1 M methyl-d-mannoside was present with MAF. On the other hand, neither d-galactose nor d-glucose inhibited the activation, and l-fucose, l-rhamnose, and N-acetyl-d-glucosamine inhibited it only partially. Incubation of either macrophages or MAF with 0.1 Md-mannose for 2 hr had no effect on activation of the macrophages by the MAF. Treatment of the macrophages by α-d-mannosidase rendered them no longer responsive to MAF. Macrophages treated by either neuraminidase or proteolytic enzymes, but not with β-d-galactosidase lost their ability to respond to MAF. Treatment of MAF with α-d-mannosidase did not affect MAF activity. In addition, MAF activity was not reduced by passage through a column of immobilized concanavalin A. In an absorption experiment, the presence of d-mannose was shown to prevent the adsorption of MAF to macrophages, while d-galactose did not. Treatment of macrophages with plant lectins having affinity for d-mannose, sialic acid or l-fucose prevented the adsorption of MAF, but the other lectins did not. Mouse MAF failed to adsorb to guinea pig peritoneal exudate macrophage, which were suggested as having a fucose-containing glycolipid as a lymphokine receptor. Taken together, these results strongly suggest that the receptor for MAF on mouse macrophages may be a glycoprotein containing d-mannose and sialic acid as essential components.     

Different Sensitivity of Complement to Salmonella typhimurium Accounts for the Difference in Natural Resistance to Murine Typhoid between A/J and C57BL/6 Mice

The difference in natural resistance to Salmonella typhimurium between S. typhimurium-resistant A/J mice and S. typhimurium-susceptible C57BL/6 mice was analyzed. In both strains, the growth of S. typhimurium was controlled in the spleen until 48 hr of infection, while serum C3b levels were increased in A/J mice immediately after infection but not in C57BL/6 mice. Incubation of A/J mouse serum with S. typhimurium or its lipopolysaccharide (LPS) generated sufficient amounts of C3b, but that of C57BL/6 mouse serum with them did not. A/J macrophages had higher intracellular killing activity in vitro than did C57BL/6 cells against S. typhimurium pre-opsonized with each corresponding fresh serum. However, the cells from both mice exhibited a similar level of killing activity against S. typhimurium pre-opsonized with fresh A/J serum or rabbit complement. The resistance of C57BL/6 mice was significantly increased by opsonizing S. typhimurium with fresh A/J serum or rabbit complement before inoculation. The serum level of interferon-γ (IFN-γ) in A/J mice was 2.7 times as high as in C57BL/6 mice at 48 hr post-infection. Recombinant murine IFN-γ enhanced the intracellular killing activity of macrophages from both mice when S. typhimurium was pre-opsonized with fresh A/J serum but not with fresh C57BL/6 serum. These findings suggest that A/J macrophages exhibit maximal killing activity against A/J serum-opsonized S. typhimurium in vivo when the cells are activated with IFN-γ. Therefore, the rapid and sufficient activation of complement by Salmonella LPS may render A/J mice more resistant against murine typhoid.     

Salmonella–Macrophage Interactions upon Manganese Supplementation

Various studies indicate the role of manganese (Mn) in the virulence of pathogens. Salmonella is an intracellular pathogen which is able to multiply within the macrophages. The present study was therefore, designed to assess the effect of Mn supplementation on Salmonella–macrophage interactions particularly in reference to Salmonella virulence and macrophage functions. A 50-fold decrease in the lethal dose 50 (LD₅₀) of Salmonella typhimurium was observed when mice were infected with Salmonella grown in the presence of Mn as compared to the LD₅₀ in the absence of Mn indicating an increase in the virulence of the organism. A significant increase was observed in the levels of superoxide dismutase (SOD) of S. typhimurium grown in presence of manganese. Upon Mn supplementation, macrophage functions were also found to be altered. Decreased phagocytic activity of macrophages interacted with Salmonella was observed in presence of Mn as compared to the activity in the absence of Mn. A significant increase was observed in the extent of lipid peroxidation along with significant decreases in the activities of SOD and catalase as well as nitrite levels of macrophages interacted with S. typhimurium upon supplementation with Mn. These observations indicate that Mn supplementation might have increased the expression of Mn transporters in Salmonella resulting in increased levels of its superoxide dismutase. The altered Salmonella function in turn might have been responsible for inhibiting phagocytosis and impairing the balance between the oxidant and antioxidant profile of macrophages, thus protecting itself by exhibiting exalted virulence.     

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor

The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.     

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.     

Glycoconjugates enhanced the intracellular killing of Bacillus spores, increasing macrophage viability and activation

Infections caused by Bacillus spores can be attenuated if the intracellular killing of the organism by macrophages can be enhanced. Glycoconjugate-bearing polymers, which selectively bind to Bacillus spores, were tested for modulation of intracellular killing when added prior to, during, and following macrophage exposure to B. cereus spores. In the absence of glycoconjugates, murine macrophages were ineffective at killing Bacillus spores. In presence of glycoconjugates, however, macrophages efficiently killed spores, whether the glycoconjugates were added to the cells prior to, during, and following spore addition. Glycoconjugates were shown to exert a protective influence on macrophages and increase their activation, as evidenced by viability and lactate dehydrogenase release assays. Increased levels of nitric oxide production by macrophages pretreated with glycoconjugates suggest that, under these conditions, glycoconjugates provide an activation signal to macrophages. These results indicate that glycoconjugates promote killing of Bacillus spores, while increasing macrophage activation level and viability. The selection of glycoconjugate ligands bearing immunomodulating properties could be exploited for vaccine and/or immunomodulator development and/or for the improvement of existing vaccines against B. cereus and B. anthracis.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses

Summary In the present study we investigated some of the physicochemical properties of macrophage-activating factor(s) (MAF) produced by the tumor-immune Lyt-1⁺2^– T cell subset. Supernatant from mixed culture of spleen and lymph node cells, obtained from C3H/HeN mice immunized with syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma, with the corresponding tumor cells exhibited the capability of activating peritoneal exudate macrophages to exert their cytostatic and cytolytic activities on tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 antibody plus complement (C) before culturing. Anti-Lyt-2.1 and/or anti-asialo GM1 plus C treatment, however, had only marginal effect on the generation of MAF by these cells, despite the complete disappearance of natural killer (NK) cell activity of spleen and lymph node cells after the treatment with anti-asialo GM1 plus C. Thus, the tumor-specific Lyt-1⁺2^– T cell subset could fulfill a crucial role in generating MAF without the support of NK cells. The MAF activity was heat, acid, and trypsin sensitive. On Sephacryl S-300 column, MAF activity was eluated in a broad single peak around a molecular weight (m.w.) of 70,000 daltons. Antiviral activity was detected in the concentrated pool of MAF-containing fractions from Sephacryl S-300. Gel permeation analysis using HPLC also showed a coincident peak of MAF and antiviral activities at a m.w. of approximately 70,000 daltons. In addition, MAF activity was almost completely neutralized by incubation with rabbit antiserum against recombinant murine -interferon (IFN). Taken together, these results indicate that MAF generated by tumor-immune Lyt-1⁺2^– T cell subset is closely related to IFN.     

Role of lymphokines in regulation of macrophage differentiation

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.     

The mechanism of cell surface changes of guinea pig macrophages activated with purified migration inhibitory factor/macrophage activation factor

Guinea pig MIF (MIF/MAF), which was purified by immunoadsorbent column chromatography using an antibody against MIF/MAF, was observed to induce characteristic cell surface changes in macrophages under scanning electron microscopy (SEM). MIF/MAF induced enlarged petal-like ruffles in both rounded and spreading macrophages. The changes were observed as early as 2 hr after stimulation with MIF/MAF and continued for 24 hr. These morphological changes appeared to be a good indicator of macrophage activation and migration inhibition in the early phase. The mechanism of the characteristic ruffle formation was studied using metabolic inhibitors and reagents known to affect microfilaments and microtubules. When macrophages were treated with MIF/MAF in the presence of mitomycin C, actinomycin D, or puromycin, formation of the petal-like ruffles was not affected. However, vinblastine and cytochalasin B inhibited the induction of these ruffles. These results indicate that microtubule and microfilament assembly, but not synthesis of DNA, RNA, and protein, are required for the formation of the petal-like ruffles. In addition, treatment with a Ca²⁺ ionophore induced the same petal-like ruffles in macrophages, while treatment with dibutyryl-cyclic AMP or-cyclic GMP did not. These findings suggest that Ca²⁺ plays an important role in macrophage activation by MIF/MAF, especially in the early phase.     

Macrophage activation factor from EL-4, a murine T-cell line: antigenic characterization by hamster monoclonal antibodies to murine interferon-gamma

A cloned variant of the EL-4 murine T-cell line treated with phorbol myristate acetate (PMA) releases a factor that activates macrophages for nonspecific tumor cytotoxicity. This macrophage activation factor (MAF) is both physicochemically (Mr 25,000; pH 2 stable) and biologically different from interferon-gamma (IFN-gamma). However, EL-4 MAF may represent a breakdown product or otherwise altered fragment of IFN-gamma. We examined this possibility with a unique pair of hamster monoclonal antibodies against different epitopes of murine IFN-gamma. Both antibodies inhibited IFN-gamma-induced fibroblast antiviral activity; H21 but not H1 antibody also inhibited lymphokine (LK)-induced macrophage-mediated tumor cytotoxicity. Neither antibody, however, had any effect on the EL-4 MAF throughout a broad dose response. Moreover, passage through a H21 immunoaffinity chromatography column or addition of staphylococcal protein A and antibody completely inhibited LK-induced macrophage tumoricidal activity but did not affect the activity in EL-4 MAF. Identical effects in both fluid and solid phase were observed with polyclonal rabbit antisera to murine IFN-gamma. Results with all of these antibodies strongly suggest that the EL-4 MAF and murine IFN-gamma are antigenically distinct.     

Separation of a Water-Soluble Adjuvant (MAF3) from Delipidated Cells of Mycobacterium tuberculosis Strain Aoyama B by Hydrogenolysis and Gel Filtration

A water-extract from hydrogenolyzed cells of Mycobacterium tuberculosis strain Aoyama B was separated into four portions (F-1 to F-4 fractions) by gel filtration with a Sephadex G-100 column. The third peak (called MAF3) eluted from the column was the most adjuvant-active fraction. The molecular weight determined by gel filtration was around 16 000 daltons. MAF3 consisted of heteropolymer(s) composed of approximately 76 to 79% neutral sugars (Ara, Gal, Man, and Glc) and 19% mucopeptide (MurN, GlcN, Glu, Ala, Dpm, Gly, Asp, Thr, Ser, Leu, Lys, Arg, His, Pro, Tyr, and Phe). The adjuvanticities of MAF3 and other fractions in water-in-oil emulsion were estimated by the enhancing effect on immune response to egg albumin (EA) in guinea pigs. MAF3 stimulated the production of humoral antibodies, particularly IgG2 antibody specific to the antigen, and induced delayed type hypersensitivity against EA in the skin and cornea of antigen-primed guinea pigs. These adjuvanticities of MAF3 were similar to the characteristics of mycobacterial cell wall in Freund's complete adjuvant.     

Macrophage activation: synergism between hybridoma MAF and poly(I). Poly(C) delivered by liposomes

High concentrations of a murine T cell hybridoma culture supernatant containing macrophage-activating factor (MAF) rendered resident mouse peritoneal macrophages cytotoxic for P815 mastocytoma cells. The capacity of the hybridoma-derived MAF (MAFH) to induce tumoricidal activity increased 10(3) to 10(4)-fold when the lymphokine was encapsulated into liposomes. Combinations of MAFH and poly(I) X poly(C) acted synergistically to render macrophages potently cytotoxic. Subthreshold (nonactivating) concentrations of free or liposome-encapsulated MAFH increased the potency of free poly(I) X poly(C) and liposome encapsulated poly(I) X poly(C). Either as free agent or encapsulated in liposomes, single-stranded poly(I) or poly(C) did not activate macrophages in the presence or absence of MAFH. Double-stranded poly(I) X poly(C) was thus required for macrophage activation and synergism with MAFH.     

Macrophage activation to kill Leishmania tropica: characterization of a T cell-derived factor that suppresses lymphokine-induced intracellular destruction of amastigotes

Factors obtained from phorbol myristate acetate (PMA)-stimulated EL-4 thymoma cells, a continuous T cell line, suppressed lymphokine-induced macrophage activation to kill intracellular Leishmania tropica amastigotes. Suppression of this macrophage effector activity was dependent upon concentration of EL-4 fluids admixed with lymphokines in infected macrophage cultures, and was not due to residual PMA or factors released from unstimulated EL-4 cells. Fluids from PMA-stimulated EL-4 cells did not affect the expression of microbicidal activity by macrophages activated in vivo as a consequence of infections with Mycobacterium bovis strain BCG, nor did they abrogate intracellular killing activities by C3H/HeJ macrophages primed by BCG infection and triggered by lymphokines in vitro. That the action of this EL-4 suppressor activity was at the priming stage of macrophage activation was confirmed by kinetic studies: EL-4 fluids added to lymphokine-treated cells in the first 4 hr of treatment completely suppressed intracellular killing of L. tropica; fluids added after 4 hr were not effective. The effects of these EL-4 factors appeared to be selective: of three effector activities of activated macrophages tested, induction of resistance to infection, tumor cytotoxicity, and intracellular destruction of L. tropica, only intracellular killing by lymphokine-treated macrophages was significantly suppressed. These T cell-derived soluble suppressor factor(s) may provide insight into mechanisms of immunosuppression during leishmanial disease and perhaps other intracellular parasitic infections.     

The Interaction of Naegleria fowleri Amoebae with Murine Macrophage Cell Lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D₁, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowler bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

Macrophage cell lines derived from major histocompatibility complex II-negative mice

Summary Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II-negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-γ, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphylococcal enterotoxins A or B. C2Dt cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.     

Macrophage binding of cells resistant and sensitive to contact-dependent cytotoxicity

We compared macrophage binding and killing of F5b cells to the binding and killing of P815 mastocytoma cells and to several other nontransformed and transformed cell lines. Formalin fixation of elicited or activated macrophages did not affect binding of F5b or 3T3 cells but did abrogate binding of P815 cells. However, formalin fixation abrogated resident macrophage binding of F5b and 3T3 cells. Therefore, depending on the type of macrophage or target cell, formalin fixation may affect binding. Only the binding of P815 cells was dependent upon activation; macrophage binding of target cells F5b and 3T3 was not. Even though macrophages bound F5b and 3T3 cells, macrophages only mediated contact-dependent cytotoxicity against F5b cells. Macrophages did not kill 3T3 cells. Experiments also compared macrophage binding and killing of the uv-light-induced tumor cell lines 1422, 2237, and 2237a46. Only the cell line 2237a46 was susceptible to contact-dependent killing. Both 1422 and 2237 cells were resistant. In contrast, cell lines 2237a46 and 1422 were bound by activated macrophages while 2237 cells were bound poorly.     

Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.     

Macrophage activation redirects yersinia-infected host cell death from apoptosis to caspase-1-dependent pyroptosis

Infection of macrophages by Yersinia species results in YopJ-dependent apoptosis, and naïve macrophages are highly susceptible to this form of cell death. Previous studies have demonstrated that macrophages activated with lipopolysaccharide (LPS) prior to infection are resistant to YopJ-dependent cell death; we found this simultaneously renders macrophages susceptible to killing by YopJ⁻ Yersinia pseudotuberculosis (Yptb). YopJ⁻ Yptb-induced macrophage death was dependent on caspase-1 activation, resulting in rapid permeability to small molecules, followed by membrane breakdown and DNA damage, and accompanied by cleavage and release of proinflammatory interleukin-18. Induction of caspase-1-dependent death, or pyroptosis, required the bacterial type III translocon but none of its known translocated proteins. Wild-type Yptb infection also triggered pyroptosis: YopJ-dependent activation of proapoptotic caspase-3 was significantly delayed in activated macrophages and resulted in caspase-1-dependent pyroptosis. The transition to susceptibility was not limited to LPS activation; it was also seen in macrophages activated with other Toll-like receptor (TLR) ligands and intact nonviable bacteria. Yptb infection triggered macrophage activation and activation of caspase-1 in vivo. Y. pestis infection of activated macrophages also stimulated caspase-1 activation. These results indicate that host signaling triggered by TLR and other activating ligands during the course of Yersinia infection redirects both the mechanism of host cell death and the downstream consequences of death by shifting from noninflammatory apoptosis to inflammatory pyroptosis.