The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. II. Differential Induction of Tumor Necrosis Factor-Alpha Production by Rickettsia tsutsugamushi and Rickettsia conorii

The present study was undertaken to investigate the ability of members of two different groups of Rickettsia to stimulate macrophages or immune lymphocytes to produce TNF. It was found that R. conorii, a spotted fever group rickettsia, readily induced murine peritoneal macrophages or the macrophage-like cell line P388D1 to produce relatively high levels of TNF. The interaction of macrophages with viable organisms or heat-killed organisms resulted in TNF production. In contrast, viable or killed R. tsutsugamushi did not stimulate the production of detectable TNF even though viable organisms grew to high numbers in both cell types. It was found that the appropriate immune spleen cells stimulated with heat-killed R. tsutsugamushi or R. conorii produced TNF, and TNF activity was found in the sera of immune mice after injection with rickettsial antigen. Infection of naive mice with viable R. tsutsugamushi resulted in high TNF levels in ascites, but TNF was not found in ascites obtained from infected athymic (nu/nu) mice. These data support the suggestion that spotted fever group rickettsiae, such as R. conorii, possess components perhaps on the surface that interact with macrophages to induce TNF production and this component is lacking in R. tsutsugamushi. Antigens of R. tsutsugamushi and R. conorii will stimulate immune cells to produce TNF activity. These data are compatible with the suggestion that the TH-1 subset of T cells is predominant in immunity to R. tsutsugamushi.     

Effect of tumor necrosis factor-α and interferon-γ on the growth of a human salivary gland cell line

Interferon-γ (IFN-γ) is a product of activated T-lymphocytes, and tumor necrosis factor-α (TNF-α) is a product of both lymphocytes and macrophages. These cell types are often present at sites of tissue damage secondary to chronic infection or autoimmune disease. The purpose of this study was to characterize the effects of TNF-α and IFN-γ on a human submandibular gland epithelial cell line (HSG). IFN-γ caused a concentration-dependent decrease in HSG cell growth (～70% in 6 days). Conversely, TNF-α alone had little effect on the growth of these cells. When these cytokines were added in combination (20 units/ml TNF-α and 1,000 units/ml of IFN-γ), there was a synergistic antiproliferative effect; no apparent cell growth was observed. The cytokine-induced antiproliferative effect was reversible. After the apparent cessation of cell growth for 3–6 days, removal of the cytokines permitted complete growth recovery. Further, cells that recovered and exhibited growth patterns that were similar to control cells remained susceptible to the antiproliferative effects of the cytokines. Flow cytometry revealed that the percentage of cells in G0/G1 with the combination of cytokines was significantly increased by 24 h. The antiproliferative effect of IFN-γ alone and that of IFN-γ and TNF-α in combination were blocked completely using an antibody to the IFN-γ receptor. A hypothesized mechanism of tissue damage in autoimmune inflammatory disorders is via up-regulation of cell surface markers such as intercellular adhesion molecule type I (ICAM-1) and histocompatibility antigen HLA-DR which can exacerbate the inflammatory process. Treatment of HSG cells with IFN-γ, with or without TNF-α, resulted in increased levels of ICAM-1 and the acquisition of HLA-DR expression. These aggregate data suggest that IFN-γ alone can regulate the expression of cell surface markers involved in the inflammatory process as well as cause a potent yet reversible inhibition of HSG cell growth that is modulated by the presence of TNF-α. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.     

Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

Characteristics of in vitro infection of human monocytes,by Rickettsia helvetica

Eighteen species of rickettsiae are reported to cause infections in humans. One of these is Rickettsia helvetica, which is endemic in European and Asian countries and transmitted by the tick Ixodes ricinus. Besides fever, it has been demonstrated to cause meningitis and is also associated with perimyocarditis. One of the initial targets for rickettsiae after inoculation by ticks is the macrophage/monocyte. How rickettsiae remain in the macrophages/monocytes before establishing their infection in vascular endothelial cells remains poorly understood. The main aim of the present study was to investigate the impact on and survival of R. helvetica in a human leukemic monocytic cell line, THP-1. Our results show that R. helvetica survives and propagates in the THP-1 cells. The infection in monocytes was followed for seven days by qPCR and for 30 days by TEM, where invasion of the nucleus was also observed as well as double membrane vacuoles containing rickettsiae, a finding suggesting that R. helvetica might induce autophagy at the early stage of infection. Infected monocytes induced TNF-α which may be important in host defence against rickettsial infections and promote cell survival and inhibiting cell death by apoptosis. The present findings illustrate the importance of monocytes to the pathogenesis of rickettsial disease.     

Inhibition of LPS-induced TNF-α production by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

The purpose of this study was to examine whether rCGRP has effects on TNF-α produced by mouse resident peritoneal macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5 × 10⁵ cells per well and allowed to adhere for 2 hr. Pretreatment with rCGRP (10 nM − 1 μM) for 24 hr, the macrophages were cultured with LPS 1 μg/ml for another 24 h. The medium was harvested for measuring TNF-α by ELISA kits. The results showed that rCGRP had no direct effects on TNF-α production, but it inhibited LPS-induced TNF-α production in a concentration-dependent manner. When rCGRP was at a concentration of 1 μM, the LPS-induced TNF-α production was inhibited by 39%. The effect of rCGRP was reversed by hCGRP8-37 (10 μM), an antagonist of CGRP₁ receptor. The LPS-induced TNF-α production from macrophages was also inhibited by forskolin 3 μM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 μM or Rp-cAMPS 100 μM, the inhibitors of cAMP-dependent protein kinase, the effect of rCGRP was abolished. These data suggest that the LPS-induced TNF-α production is inhibited by rCGRP via activation of cAMP responses in mouse resident peritoneal macrophages.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Caspase-3 is Involved in IFN-γ- and TNF-α-Mediated MIN6 Cells Apoptosis via NF-κB/Bcl-2 Pathway

TNF-α and IFN-γ are the major pro-inflammatory cytokines in the β-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic β-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-γ and TNF-α significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-κB activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-γ and TNF-α treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-γ- and TNF-α-induced cells apoptosis. Inhibition of NF-κB activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-γ- and TNF-α-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it’s activation is further confirmed to be related to the NF-κB-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-γ- and TNF-α-mediated MIN6 cells apoptosis.     

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-β-d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.     

Ceramide synthase 6 plays a critical role in the development of experimental autoimmune encephalomyelitis

Ceramides are mediators of apoptosis and inflammatory processes. In an animal model of multiple sclerosis (MS), the experimental autoimmune encephalomyelitis (EAE) model, we observed a significant elevation of C(16:0)-Cer in the lumbar spinal cord of EAE mice. This was caused by a transiently increased expression of ceramide synthase (CerS) 6 in monocytes/macrophages and astroglia. Notably, this corresponds to the clinical finding that C(16:0)-Cer levels were increased 1.9-fold in cerebrospinal fluid of MS patients. NO and TNF-α secreted by IFN-γ-activated macrophages play an essential role in the development of MS. In murine peritoneal and mouse-derived RAW 264.7 macrophages, IFN-γ-mediated expression of inducible NO synthase (iNOS)/TNF-α and NO/TNF-α release depends on upregulation of CerS6/C(16:0)-Cer. Downregulation of CerS6 by RNA interference or endogenous upregulation of C(16:0)-Cer mediated by palmitic acid in RAW 264.7 macrophages led to a significant reduction or increase in NO/TNF-α release, respectively. EAE/IFN-γ knockout mice showed a significant delay in disease onset accompanied by a significantly less pronounced increase in CerS6/C(16:0)-Cer, iNOS, and TNF-α compared with EAE/wild-type mice. Treatment of EAE mice with l-cycloserine prevented the increase in C(16:0)-Cer and iNOS/TNF-α expression and caused a remission of the disease. In conclusion, CerS6 plays a critical role in the onset of MS, most likely by regulating NO and TNF-α synthesis. CerS6 may represent a new target for the inhibition of inflammatory processes promoting MS development.     

CD1d-restricted IFN-γ-secreting NKT cells promote immune complex-induced acute lung injury by regulating macrophage-inflammatory protein-1α production and activation of macrophages and dendritic cells

Immune complex-induced acute lung injury (IC-ALI) has been implicated in various pulmonary disease states. However, the role of NKT cells in IC-ALI remains unknown. Therefore, we explored NKT cell functions in IC-ALI using chicken egg albumin and anti-chicken egg albumin IgG. The bronchoalveolar lavage fluid of CD1d(-/-) and Jα18(-/-) mice contained few Ly6G(+)CD11b(+) granulocytes, whereas levels in B6 mice were greater and were increased further by α-galactosyl ceramide. IFN-γ and MIP-1α production in the lungs was greater in B6 than CD1d(-/-) mice. Adoptive transfer of wild type (WT) but not IFN-γ-, MIP-1α-, or FcγR-deficient NKT cells into CD1d(-/-) mice caused recruitment of inflammatory cells to the lungs. Moreover, adoptive transfer of IFN-γR-deficient NKT cells enhanced MIP-1α production and cell recruitment in the lungs of CD1d(-/-) or CD1d(-/-)IFN-γ(-/-) mice, but to a lesser extent than WT NKT cells. This suggests that IFN-γ-producing NKT cells enhance MIP-1α production in both an autocrine and a paracrine manner. IFN-γ-deficient NKT cells induced less IL-1β and TNF-α production by alveolar macrophages and dendritic cells in CD1d(-/-) mice than did WT NKT cells. Taken together, these data suggest that CD1d-restricted IFN-γ-producing NKT cells promote IC-ALI by producing MIP-1α and enhancing proinflammatory cytokine production by alveolar macrophages and dendritic cells.     

Propagation of arthropod-borne Rickettsia spp. in two mosquito cell lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Host defense peptide LL-37 selectively reduces proinflammatory macrophage responses

The human cathelicidin peptide, LL-37, is a host defense peptide with a wide range of immunomodulatory activities and modest direct antimicrobial properties. LL-37 can exert both pro- and anti-inflammatory effects and can modulate the proinflammatory responses of human peripheral blood monocytes and epithelial cells. In this study, we evaluated the effect of LL-37 on mouse bone marrow-derived macrophages (BMDM) and tissue macrophages in vitro and in vivo. LL-37 dramatically reduced TNF-α and NO levels produced by LPS and IFN-γ-polarized M1-BMDM and slightly reduced reactive oxygen species production by these cells. LL-37 did not affect the ability of IL-4-polarized M2-BMDM to upregulate arginase activity, although it did inhibit LPS-induced TNF-α secretion in these cells. LL-37 did not compromise the ability of M1-polarized BMDM to phagocytose and kill bacteria and did not affect the uptake of apoptotic neutrophils by M2-polarized BMDM. However, LL-37-treated M1-BMDM were more efficient at suppressing tumor growth in vitro. LL-37 significantly reduced LPS-induced TNF-α secretion in ex vivo alveolar macrophages, whereas its effect on peritoneal macrophages was much less dramatic. Effective inhibition of LPS-induced TNF-α secretion by alveolar macrophages also occurred in vivo when LL-37 was administered by intratracheal injection. This demonstrates a selective ability of LL-37 to decrease M1-BMDM, M2-BMDM, and tissue macrophage production of the proinflammatory cytokine TNF-α in response to LPS while leaving other crucial anti-inflammatory M1 and M2 macrophage functions unaltered.     

Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

N-acetyl tryptophan glucopyranoside (NATG) as a countermeasure against gamma radiation-induced immunosuppression in murine macrophage J774A.1 cells

Radiation exposure to immune system induces imbalance in cytokines expression involved in Th1/Th2 homeostasis perturbations. In the present study, N-acetyl tryptophan glucoside (NATG), a bacterial secondary metabolite, was evaluated for its possible radioprotective potential to immune system using J774A.1 murine macrophages. In this study, expression of IFN-γ, TNF-α, IL-10, IL-2, IL-12, IL-13 and IL-17A cytokines was analyzed in irradiated and NATG pretreated cells using ELISA assay. Results of the study indicated that irradiated macrophages (NK-1R^+?cells) pretreated with NATG showed higher (p?相似文献   

Induction of Tumor Necrosis Factor Alpha (TNFα) and Enhancement of HIV-1 Replication in the J22HL60 Cell Line by Mycoplasma penetrans

Mycoplasma penetrans isolated from clinical specimens of AIDS patients showed potent activity in tumor necrosis factor alpha (TNFα) production in THP-1, U937 and J22HL60 cell lines, and in the enhancement of HIV-1 replication in a dormantly-infected J22HL60 cell line as compared with the activities of other mycoplasmas. Both activities were found in the methanol layer but not in the chloroform layer of the membrane extracted by the Bligh-Dyer method. TNFα production was observed in the peritoneal macrophages from both lipopolysaccharide-responsive and -unresponsive mouse strains, and was not inhibited by polymyxin B. The induction of TNFα production and enhancement of HIV-1 replication were strongly inhibited by Concanavalin A-Sepharose. The inhibitory effect of Concanavalin A-Sepharose was partially prevented by sugars in the order methyl-α-D -mannopyranoside and methyl-α-D -glucopyranoside but not methyl-α-D -galactopyranoside. Anti-human TNFα antibody, however, did not reduce the activity of the methanol layer to enhance HIV-1 replication, suggesting that the methanol layer could enhance HIV-1 replication directly. These results suggest that the carbohydrate derived from M. penetrans might be responsible for the progression of HIV-1 infection.     

Immunomodulatory Activities of Oat β-Glucan In Vitro and In Vivo

Previous studies have shown that β-glucans extracted from yeast or fungi potentiate immune responses. In the present study, the immunomodulatory activities of β-(1→3, 1→4)-glucan, derived from oats, were investigated. The ability of oat β-glucan (OβG) to stimulate IL-1 and TNF-α release from murine peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of macrophages with OβG resulted in the production of IL-1 in a dose and time-dependent manner, whereas only small amounts of TNF-α could be detected in the culture supernatants. OβG also induced the production of IL-2, IFN-γ and IL-4 secretion in a dose-dependent manner in cultured spleen cells. The intraperitoneal administration of OβG in mice resulted in the accumulation of leucocytes, predominantly macrophages, in the peritoneal cavity. Furthermore, OβG was tested for its ability to enhance non-specific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was enhanced by a single intraperitoneal administration of 500 μg of OβG 3 days prior to bacterial challenge. In conclusion, these studies demonstrated that OβG possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.     

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-β (IFN-β) or interferon-γ (IFN-γ). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-γ were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-β. When IFN-β-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-γ-treated cells. LPS and MTP also upregulated IFN-γ-mediated IDO activity when suboptimal amounts of IFN-γ were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1α (IL-1α), along with either maximum-stimulating amounts of IFN-β or suboptimal amounts of IFN-γ, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1α or IL-1β was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1α was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1α abolished the upregulatory effect of exogenous IL-1α, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1α, upregulation of IDO activity by these agents is independent of IL-1α production and may be mediated through distinct pathways.     

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.