Cells Of Macrophage Lineage Research Articles

Osteoclast stimulatory transmembrane protein and dendritic cell–specific transmembrane protein cooperatively modulate cell–cell fusion to form osteoclasts and foreign body giant cells

Cell–cell fusion is a dynamic phenomenon promoting cytoskeletal reorganization and phenotypic changes. To characterize factors essential for fusion of macrophage lineage cells, we identified the multitransmembrane protein, osteoclast stimulatory transmembrane protein (OC-STAMP), and analyzed its function. OC-STAMP–deficient mice exhibited a complete lack of cell–cell fusion of osteoclasts and foreign body giant cells (FBGCs), both of which are macrophage-lineage multinuclear cells, although expression of dendritic cell specific transmembrane protein (DC-STAMP), which is also essential for osteoclast/FBGC fusion, was normal. Crossing OC-STAMP–overexpressing transgenic mice with OC-STAMP–deficient mice restored inhibited osteoclast and FBGC cell–cell fusion seen in OC-STAMP–deficient mice. Thus, fusogenic mechanisms in macrophage-lineage cells are regulated via OC-STAMP and DC-STAMP.

Immunity to Human Immunodeficiency Virus (HIV) Infection

Immunity to Human Immunodeficiency Virus (HIV) Infection

Immunzellen in primären und metastasierten Gastrointestinalen Stromatumoren

AIM: This study investigated the immune cells within in the tumor stroma of primary and metastatic gastrointestinal stromal tumors (GISTs) by immunohistochemistry. GISTs appear usually as histological homogeneous tumors with a dense cell structure. However, GISTs have been shown to have a tumor stroma, in which different immune cells of the innate and acquired immune system are embedded. The frequency and function of these cells in GISTs remain widely unknown. In this work the various immune cells are described morphologically and their frequency in primary GISTs as well as peritoneal and liver metastases is being compared. Furthermore, the criteria proliferation activity, morphology, tumor size, organ localization and malignancy are examined and compared with the number of immune cells in the primary and metastatic GISTs. In a small number of metastases real time RT-PCR was performed to gain an overview of the functionality of immune cells using RT-PCR.MATERIAL & METHODS: A total of 188 untreated primary GISTs and 52 untreated GIST metastases were immunohistochemically processed to determine the percentage of tissue-associated fibrohistiocytic cells (Kim-1P) and cells of macrophage lineage (CD68). In addition, the number of several lymphocytes (T-Lymphocytes: CD3, B-Lymphocytes: CD20, NK-cells: CD56) was studied in these samples. For this purpose punches of the paraffin blocks of resected specimen or biopsies were lined up site by site in 22 tissue microarryas (TMAs) using a manual tissue arrayer. The photographs of 3-6 punches have been analysed computer-assisted with subsequent statistical evaluation of the results. This analysis resulted in very precise numbers of the percentage of immune cells in the respective tissue preparations. Furthermore, reverse transcription and real-time RT-PCR were used to detect the expression of the proinflammatory cytokines interleukin 1β, interleukin 6 and tumor necrosis factor α in a small sample size of snap-frozen tissue samples of metastases.RESULTS: GISTs have a tumor stroma, in which many immune cells are embedded. In the primary GISTs Kim-1P+ cells (28,8% ±7,1) dominated, but also lymphocytic cells (CD3+(2,2% ±1,8), CD20+(0,6% ±0,7), CD56+(1,1% ±0,9)) and macrophages (CD68+(3,6% ±2,1)) were present. Interestingly, the number of immune cells differ in the metastases of GISTs. On the one hand metastases show more lymphocytic cells than the primary GISTs (CD3+: 7,3% ±2,3 (p<0,01); CD20+: 1,8% ±0,3 (p<0,05)), on the other hand, the peritoneal metastases differ from the liver metastases. Peritoneal metastases have a significant higher number of Kim-1P+ cells (31,8% ±7,5) than liver metastases (18,2% ±3,8; p<0,01), while the latter in turn contain more CD3+ T-lymphocytes (11,7% ±1,8) than the peritoneal metastases (4,4% ±2,6; p<0,01). At the same time, the two sites differ in proliferation activity. Liver metastases have a lower proliferation activity (12,9% ±8,2) than peritoneal metastases of GISTs (18,3% ±7,3; p<0,05). In the RT-PCR of fresh-frozen tissue from metastases of a small cohort, one patient with a high percentage of Kim 1P+ cells (49,5% ±17) did not only show a comparatively high expression of Il-6 (CT value 26,8) and Il-1β (CT value 26,1) but also the lowest expression of TNF (CT 34,1) as well as a rapid clinical progress.CONCLUSION: The different numbers of the various immune cells in primary GISTs compared with metastatic GISTs as well as within the two metastatic sites, suggest a location specific microenvironment, which may play a part in the tumor growth of primary and secondary GISTs. Further studies will be needed to understand the type of communication between the immune cells of the GIST stroma and the tumor itself. In the future, the individuality of this tumor will come to the fore, elucidating that tumors with a different composition of their stromal cells, for example immune cells, might also need a different and individual treatment. Thus, not only the description of the immune cells is necessary, but also the understanding of GISTs as a family of tumors, which should not be seen as uniform, homogeneous tumors.

Comparative analysis of macrophage associated vectors for use in genetic vaccine

BackgroundAntigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used.MethodsThree promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries.ResultsAll the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines.ConclusionsBased on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.

Anti-Inflammatory Properties of High Density Lipoprotein (HDL) on Intestinal Epithelial Cells

G A A b st ra ct s and label-free detection. Results: Treatment of MM6 cells with TNF, but none of the other cytokines (IFN-γ, IL-1β, IL-6, TGF-β) tested, led to significant upregulation of GPR68 mRNA. Induction of GPR68 mRNA expression by TNF was dose-dependent. Macrophagic differentiation of MM6 cells with PMA also led to a significant increase in GPR68 mRNA expression. Dose-dependence of TNF and PMA induction of GPR68 mRNA was confirmed in primary human monocytes. In agreement with these findings, GPR activity in MM6 cells was increased upon treatment with TNF or PMA, only at acidic pH. In hypoxia, both 0.2% and 2% oxygen conditions synergized with TNF, PMA, and LPS in the induction of GPR68 mRNA expression. Furthermore, TNF-mediated induction of GPR68 mRNA expression was reversed by simultaneous treatment of cells with NF-κB inhibitors AICAR, BAY-11-7082, CAY10512, and SC-514. No induction of mRNA expression of the related GPRs, GPR4 and GPR65, was observed upon treatment with TNF, PMA, or LPS. Conclusion: GPR68 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF, PMA, and LPS. NF-κB inhibition reverses the induction of GPR68 mRNA expression by TNF. Interestingly, hypoxia, also known to activate the NF-κB pathway, synergizes with TNF in the induction of GPR68 expression. TNF-mediated stimulation of GPR68 expression and subsequent pH-sensing activity may play a role in the molecular pathogenesis of IBD.

Mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish

AbstractMacrophages and neutrophils play important roles during the innate immune response, phagocytosing invading microbes and delivering antimicrobial compounds to the site of injury. Functional analyses of the cellular innate immune response in zebrafish infection/inflammation models have been aided by transgenic lines with fluorophore-marked neutrophils. However, it has not been possible to study macrophage behaviors and neutrophil/macrophage interactions in vivo directly because there has been no macrophage-only reporter line. To remove this roadblock, a macrophage-specific marker was identified (mpeg1) and its promoter used in mpeg1-driven transgenes. mpeg1-driven transgenes are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. Using these lines, the different dynamic behaviors of neutrophils and macrophages after wounding were compared side-by-side in compound transgenics. Macrophage/neutrophil interactions, such as phagocytosis of senescent neutrophils, were readily observed in real time. These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology.

Antitumor Immunity Produced by the Liver Kupffer Cells, NK Cells, NKT Cells, and CD8+CD122+T Cells

Mouse and human livers contain innate immune leukocytes, NK cells, NKT cells, and macrophage-lineage Kupffer cells. Various bacterial components, including Toll-like receptor (TLR) ligands and an NKT cell ligand (α-galactocylceramide), activate liver Kupffer cells, which produce IL-1, IL-6, IL-12, and TNF. IL-12 activates hepatic NK cells and NKT cells to produce IFN-γ, which further activates hepatic T cells, in turn activating phagocytosis and cytokine production by Kupffer cells in a positive feedback loop. These immunological events are essentially evoked to protect the host from bacterial and viral infections; however, these events also contribute to antitumor and antimetastatic immunity in the liver by activated liver NK cells and NKT cells. Bystander CD8+CD122+ T cells, and tumor-specific memory CD8+T cells, are also induced in the liver by α-galactocylceramide. Furthermore, adoptive transfer experiments have revealed that activated liver lymphocytes may migrate to other organs to inhibit tumor growth, such as the lungs and kidneys. The immunological mechanism underlying the development of hepatocellular carcinoma in cirrhotic livers in hepatitis C patients and liver innate immunity as a double-edged sword (hepatocyte injury/regeneration, septic shock, autoimmune disease, etc.) are also discussed.

Tyrosine Kinase Receptor Flt/VEGFR Family: Its Characterization Related to Angiogenesis and Cancer

Ligands and their tyrosine kinase (TK) receptors regulate a variety of biological systems in animals. Vascular endothelial growth factor (VEGF) and its receptor (Flt/VEGFR family) system play a crucial role not only in physiological but also in most parts of pathological angiogenesis including cancer. Flt-1/VEGFR-1 and KDR/VEGFR-2 bind VEGF-A but have different functions on angiogenesis at early embryogenesis: Flt-1 has a negative role by trapping ligands, whereas KDR (Flk1 in mice) exerts a strong positive signal, resulting in a balance in blood vessel formation. At adult stages, however, both VEGFRs contribute to pathological angiogenesis either directly or through stimulation of migration/activation of macrophage lineage cells and stimulate tumor growth, metastasis, and inflammation. VEGFRs activate downstream signaling of the phospholipase Cγ-protein kinase C-MAP kinase pathway but not Ras pathway for cell proliferation. The VEGF-C/D and Flt-4/VEGFR-3 system regulates lymphangiogenesis. Thus, VEGFs as well as these receptor TKs are attractive targets for suppressing pathological angiogenesis.

Vascular Endothelial Growth Factor Receptor-1 Signaling Promotes Mobilization of Macrophage Lineage Cells from Bone Marrow and Stimulates Solid Tumor Growth

Vascular endothelial growth factor and its receptors, including Flt-1 and Flk-1, are involved in angiogenesis under physiologic and pathologic conditions. Recently, Flt-1-expressing cells were reported to contribute to the intracranial growth of glioma cells. However, the role of Flt-1 signaling in solid tumor growth in s.c. tissue has not been elucidated. To investigate how Flt-1 signaling is involved in the proliferation of solid tumors, we implanted tumor cells into wild-type (Wt) and Flt-1 tyrosine kinase (TK)-deficient (Flt-1 TK(-/-)) mice. Growth of HSML and B16 but not Lewis lung carcinoma cell in s.c. tissue was significantly decreased in Flt-1 TK(-/-) mice. Angiogenesis in HSML and B16 tumors was remarkably reduced in Flt-1 TK(-/-) mice. Moreover, the infiltration of macrophage lineage cells into HSML and B16 tumors was clearly suppressed in Flt-1 TK(-/-) mice. Pericyte marker(+) cells were also reduced in Flt-1 TK(-/-) mice. However, in the border area of tumor, angiogenesis and the infiltration of macrophage lineage cell were basically similar between Wt and Flt-1 TK(-/-) mice. In bone marrow (BM) transplantation experiments, tumor angiogenesis, infiltration of macrophage lineage cells, and tumor growth were significantly suppressed in Wt/Flt-1 TK(-/-) mice implanted with Flt-1 TK(-/-) BM cells compared with those implanted with Wt BM cells. We conclude that Flt-1 signaling is involved in the function of BM-derived cell, such as the migration of macrophages into cancerous tissues, and significantly contributes to angiogenesis and tumor progression.

Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice

Liver Kupffer cells have been suggested to be heterogeneous macrophage lineage cells. We explored this possibility by classifying the mouse Kupffer cells into subpopulations and characterizing them by their phenotype and function. Liver mononuclear cells (MNCs) from C57BL/6 mice were isolated and their phenotypes and functions were analyzed. The effects of clodronate liposomes and gadolinium chloride (GdCl(3)) on Kupffer cells were also investigated. Approximately 25% of liver MNCs were F4/80(+) Kupffer cells. Of these, 46% were CD11b(-)CD68(+), 22% were CD11b(+)CD68(-), and 6% were CD11b(+)CD68(+). CD68(+) cells showed potent phagocytic activity and reactive oxygen species (ROS) production capacity after lipopolysaccharide (LPS) stimulation, whereas CD11b(+) cells did not. CD11b(+) cells showed a strong capacity for the production of cytokines (TNF and IL-12), which was much less prominent in CD68(+) cells. At 24h after LPS or Escherichia coli injection into mice, the proportions of CD11b(+)CD68(-) and CD11b(+)CD68(+) cells increased but that of CD11b(-)CD68(+) cells decreased. The increase in CD11b(+)CD68(+) cells appeared to be derived from the CD11b(+)CD68(-) subset. Although the CD11b(+) cells augmented phagocytic activity after LPS injection, they did not increase ROS production, suggesting their weak lytic activity. Injection of clodronate or GdCl(3) into mice depleted the CD68(+) cells but increased CD11b(+) cells proportionally because CD68(+) cells may phagocytose these toxic reagents and undergo apoptosis. GdCl(3)-treated mice also consistently increased serum TNF after LPS challenge. Two F4/80(+) Kupffer cell subsets may exist, a CD68(+) subset with phagocytic activity and a CD11b(+) subset with cytokine-producing capacity.

Methylmercury Induces Acute Oxidative Stress, Altering Nrf2 Protein Level in Primary Microglial Cells

The neurotoxicity of methylmercury (MeHg) is well documented in both humans and animals. MeHg causes acute and chronic damage to multiple organs, most profoundly the central nervous system (CNS). Microglial cells are derived from macrophage cell lineage, making up approximately 12% of cells in the CNS, yet their role in MeHg-induced neurotoxicity is not well defined. The purpose of the present study was to characterize microglial vulnerability to MeHg and their potential adaptive response to acute MeHg exposure. We examined the effects of MeHg on microglial viability, reactive oxygen species (ROS) generation, glutathione (GSH) level, redox homeostasis, and Nrf2 protein expression. Our data showed that MeHg (1-5 microM) treatment caused a rapid (within 1 min) concentration- and time-dependent increase in ROS generation, accompanied by a statistically significant decrease in the ratio of GSH and its oxidized form glutathione disulfide (GSSG) (GSH:GSSG ratio). MeHg increased the cytosolic Nrf2 protein level within 1 min of exposure, followed by its nuclear translocation after 10 min of treatment. Consistent with the nuclear translocation of Nrf2, quantitative real-time PCR revealed a concentration-dependent increase in the messenger RNA level of Ho-1, Nqo1, and xCT 30 min post MeHg exposure, whereas Nrf2 knockdown greatly reduced the upregulation of these genes. Furthermore, we observed increased microglial death upon Nrf2 knockdown by the small hairpin RNA approach. Taken together, our study has demonstrated that microglial cells are exquisitely sensitive to MeHg and respond rapidly to MeHg by upregulating the Nrf2-mediated antioxidant response.

Abstract 1301: Involvement of VEGFR-1 signal in solid tumor growth and tumor angiogenesis via recruitment of macrophage-lineage cells

Abstract VEGF/VEGFR signaling is essential for pathological angiogenesis. It has been reported that Flt-1 (VEGFR-1) is expressed not only in vascular endothelial cells but also in monocyte/macrophages. Previously, we and others reported that bone marrow derived cells which expressed Flt-1 were involved in the formation of pre-metastatic niche in mouse tumor model. However, the role of Flt-1 signal in tumorigenesis is not fully understood. To clarify the involvement of Flt-1 in solid tumor growth, we used wild type (Wt) mice and Flt-1 tyrosine kinase-deficient (Flt-1 TK-/-) mice which developed basically normal blood vessels and survived. In allograft cancer model, tumor growth was significantly slower in Flt-1 TK-/- mice compared with that in Wt mice. Survival rate of Flt-1 TK-/- mice were also higher than Wt mice under tumor-bearing condition. The degree of tumor angiogenesis and infiltration of macrophage-lineage cells in tumor tissues were remarkably suppressed in Flt-1 TK-/- mice compared with those in Wt mice. Surprisingly, endothelial cells and macrophage-lineage cells at the surrounding tissue of tumors were similar in both mice. The number of pericyte marker-positive cells covered with vascular endothelial cells was reduced in tumor tissue in Flt-1 TK-/- mice compared with that in Wt mice. To distinguish which Flt-1 signal on the vascular endothelial cell or on the macrophage is important, we performed bone marrow transplantation. Tumor volume in Wt mice implanted with bone marrow derived from Flt-1 TK-/- mice was significantly smaller than that in Wt mice implanted with bone marrow derived from Wt mice. In tumor tissue inoculated in Wt mice implanted with Flt-1 TK-/- bone marrow, angiogenesis and infiltration of macrophage-lineage cells were significantly decreased compared with Wt mice implanted with Wt bone marrow. Based on these results, we suggest that, in some tumors, Flt-1 signaling is important for bone marrow-derived macrophage-migration into tumors which promotes tumor angiogenesis and tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1301.

Macrophage responses to interleukin-17 are regulated by location and inflammation (37.35)

Abstract The purpose of this study was to examine how macrophage-lineage cells participate in T helper 17 (Th17)-mediated inflammatory responses. We were interested in how this response may be affected by compartmentalization, and the presence of an inflammatory microenvirionment. We isolated primary murine F4/80+ macrophages (Mϕ) from a broad variety of compartments including spleen, peripheral blood, peritoneum, gut, lung, liver, and CNS. We assayed the expression of the receptors IL17RA and IL17RC on these cells by flow cytometric methods, and observed the highest levels of IL17RA and IL17RC expression on Mϕ associated with mucosal surfaces, particularly gut and lung. In order to examine the influence of inflammatory stimuli on the expression of IL17RA and IL17RC in vitro, bone marrow-derived macrophages, which express low levels of IL17RA and IL17RC were stimulated with combinations of TLR ligands, and proinflammatory cytokines. We observed coordinate expression of both IL17 receptor subunits controlled by TNFα and peptidoglycan signaling. Following encounter with CFA adjuvant in vivo, the expression of both IL17RA and IL17RC was most dramatically increased on Mϕ in the liver. We further wished to determine biological consequences of IL17 signaling, and have begun to identify genes regulated by IL17 in macrophage-lineage cells. Together, these data indicate that macrophages participate in Th17-mediated immunity in a manner regulated by localization and inflammation.

Demonstration of T cell and macrophage progenitors in carp ( Cyprinus carpio) kidney hematopoietic tissues. Development of clonal assay system for carp hematopoietic cells

Single hematopoietic cells from carp ( Cyprinus carpio) kidney were seeded to each well of 96-well plates and cultured in the presence of a supporting cell layer and conditioned media (CM). The CM were obtained from bulk-cultured carp hematopoietic cells, in which T and macrophage-lineage cells rapidly proliferated as previously reported. After 2–3 weeks, colony formation was found in 0–4 wells of each plate. Three different morphological types of colonies were observed: “type I colonies”, “type II colonies” and “mixed-type colonies”. Type I colony cells were interpreted as composed by macrophage-lineage cells, since they expressed a specific macrophage marker, M-CSFR/csf1r gene, and most of them phagocytosed latex particles. Type II colony cells were interpreted as composed by T lineage cells, since they expressed several T cell marker genes including gata3, lck and TCRβ, but did not engulf latex particles. Mixed-type colonies were interpreted as composed by both macrophages and T lineage cells. They expressed not only the M-CSFR gene but also a T cell marker gene, gata3, but not other T cell markers, such as lck and TCRβ. These results indicated that the mixed-type colonies were developed from immature common progenitors of macrophage and T cell. In contrast, type I and type II colonies were developed from more mature and mono-potent progenitors of macrophage and T cell, respectively.

PCR detectable Y chromosome-specific DNA but no intact Y chromosome-bearing cells in polymyositis biopsies of two women with male offspring

Pregnancy-related and idiopathic adult polymyositis are inflammatory myopathies of unknown aetiology in which CD8 positive T cells are found in close association with the up-regulation of human leukocyte antigen (HLA) class I on affected muscle cells. A similar polymyositis can also occur in patients with chronic graft versus host disease, wherein graft lymphocytes may be involved in the myositis. We investigated whether polymyositis that was temporally related to pregnancy, contained Y chromosome-bearing cells or signals using polymerase chain reaction (PCR) in biopsies of lesional muscle from two women who had given birth to sons. Furthermore, if Y chromosome material was present, we investigated whether it was contained in the intact inflammatory cells (CD8 positive lymphocytes for example), fetal macrophages, or differentiated fetal stem cells engrafted in the lesional skeletal muscle, and thus whether fetal cells played a role in the pathogenesis of the myositis. PCR analysis was used for the Y chromosome in lesional tissue and fluorescence in situ hybridisation (FISH) for intact cells carrying the Y chromosome. Small amounts of Y chromosome material were detected on second round PCR in fresh frozen tissue. No Y chromosome-bearing intact cells of lymphocytic, macrophage or muscle lineage were detected. Our results suggest that microchimeric fetal cells are not found in the lesional tissue of pregnancy-related polymyositis.

Mindin is upregulated during colitis and may activate NF-κB in a TLR-9 mediated manner

To investigate the regulation of mindin expression and the signaling pathway involved during inflammation. C57BL/6 mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 6 d to induce acute colitis, and then the colon was harvested for histological analysis or for RNA isolation. mRNA expression of mindin and nuclear factor (NF)-kappaB p65 was analyzed by quantitative real time polymerase chain reaction (RT-PCR) and mindin expression construct was confirmed by Western blotting. Mouse macrophage and intestinal epithelial lineage cells were stimulated with different cytokines and toll-like receptor (TLR) ligands, before pNF-kappaB-luciferase activity was assessed using the Dual-Luciferase reporter assay system. mRNA expression of mindin was upregulated 4.7 + or - 1.1 fold compared with the baseline during DSS-induced intestinal inflammation in the mice. Stimulation with CpG-ODN (a known TLR-9 ligand) induced 4.2 + or - 0.3 fold upregulation of mindin expression in RAW 264.7 cells. Full-length of mindin was cloned from cDNA of mouse mesenteric lymph node, then the pCMV-Mindin-Flag expression vector was established and the protein expression level was confirmed. Transfection of the mindin construct and stimulation with CpG-ODN significantly increased the NF-kappaB-luciferase activity by 2.5 + or - 0.3 and 4.5 + or - 0.5 fold in RAW264.7 and CMT93 cells, respectively (P < 0.01). Mindin expression is upregulated during intestinal inflammation and may induce NF-kappaB promoter activation in a TLR-9 mediated manner.

Mice with a Selective Impairment of IFN-γ Signaling in Macrophage Lineage Cells Demonstrate the Critical Role of IFN-γ–Activated Macrophages for the Control of Protozoan Parasitic Infections In Vivo

IFN-gamma has long been recognized as a cytokine with potent and varied effects in the immune response. Although its effects on specific cell types have been well studied in vitro, its in vivo effects are less clearly understood because of its diverse actions on many different cell types. Although control of multiple protozoan parasites is thought to depend critically on the direct action of IFN-gamma on macrophages, this premise has never been directly proven in vivo. To more directly examine the effects of IFN-gamma on cells of the macrophage lineage in vivo, we generated mice called the "macrophages insensitive to IFN-gamma" (MIIG) mice, which express a dominant negative mutant IFN-gamma receptor in CD68+ cells: monocytes, macrophages, dendritic cells, and mast cells. Macrophage lineage cells and mast cells from these mice are unable to respond to IFN-gamma, whereas other cells are able to produce and respond to this cytokine normally. When challenged in vitro, macrophages from MIIG mice were unable produce NO or kill Trypanosoma cruzi or Leishmania major after priming with IFN-gamma. Furthermore, MIIG mice demonstrated impaired parasite control and heightened mortality after T. cruzi, L. major, and Toxoplasma gondii infection, despite an appropriate IFN-gamma response. In contrast, MIIG mice displayed normal control of lymphocytic choriomeningitis virus, despite persistent insensitivity of macrophages to IFN-gamma. Thus, the MIIG mouse formally demonstrates for the first time in vivo, the specific importance of direct, IFN-gamma mediated activation of macrophages for controlling infection with multiple protozoan parasites.

Coordinated Regulation of SIV Replication and Immune Responses in the CNS

Central nervous system (CNS) invasion during acute-stage HIV-infection has been demonstrated in a small number of individuals, but there is no evidence of neurological impairment at this stage and virus infection in brain appears to be controlled until late-stage disease. Using our reproducible SIV macaque model to examine the earliest stages of infection in the CNS, we identified immune responses that differentially regulate inflammation and virus replication in the brain compared to the peripheral blood and lymphoid tissues. SIV replication in brain macrophages and in brain of SIV-infected macaques was detected at 4 days post-inoculation (p.i.). This was accompanied by upregulation of innate immune responses, including IFNβ, IFNβ-induced gene MxA mRNA, and TNFα. Additionally, IL-10, the chemokine CCL2, and activation markers in macrophages, endothelial cells, and astrocytes were all increased in the brain at four days p.i. We observed synchronous control of virus replication, cytokine mRNA levels and inflammatory markers (MHC Class II, CD68 and GFAP) by 14 days p.i.; however, control failure was followed by development of CNS lesions in the brain. SIV infection was accompanied by induction of the dominant-negative isoform of C/EBPβ, which regulates SIV, CCL2, and IL6 transcription, as well as inflammatory responses in macrophages and astrocytes. This synchronous response in the CNS is in part due to the effect of the C/EBPβ on virus replication and cytokine expression in macrophage-lineage cells in contrast to CD4+ lymphocytes in peripheral blood and lymphoid tissues. Thus, we have identified a crucial period in the brain when virus replication and inflammation are controlled. As in HIV-infected individuals, though, this control is not sustained in the brain. Our results suggest that intervention with antiretroviral drugs or anti-inflammatory therapeutics with CNS penetration would sustain early control. These studies further suggest that interventions should target HIV-infected individuals with increased CCL2 levels or HIV RNA in the CNS.

Unique signal transduction of the VEGF family members VEGF-A and VEGF-E

Both VEGF (vascular endothelial growth factor)-A and Orf-virus-encoded VEGF-E bind and activate VEGFR (VEGF receptor)-2; however, only VEGF-A binds VEGFR-1. To understand the biological differences between VEGF-A and VEGF-E in vivo, we established transgenic mouse models. K14 (keratin-14)-promoter-driven VEGF-E transgenic mice showed a significant increase in mature blood vessels. However, K14-VEGF-A transgenic mice exhibited severe inflammation and oedema with increased angiogenesis, as well as lymphangiogenesis and lymph vessel dilatation. K14-VEGF-A transgenic mice deficient in VEGFR-1 signalling (K14-VEGF-A-tg/VEGFR-1 TK(-/-) mice) showed decreases in oedema and inflammation with less recruitment of macrophage-lineage cells, suggesting an involvement of VEGFR-1 in these adverse effects. VEGFE might be more useful than VEGFA for pro-angiogenic therapy.

Histogenesis‐specific expression of fibroblast activation protein and dipeptidylpeptidase‐IV in human bone and soft tissue tumours

Aims:Fibroblast activation protein (FAP)/seprase and dipeptidylpeptidase-IV (DPP-IV)/CD26 are serine integral membrane proteases. They are involved in tissue remodelling, cancer invasion and metastases, mechanisms that are controversial. The aim was to identify cell types that express FAP and DPP-IV in human bone and soft tissue tumours, and to determine whether there are any correlations between the expression of FAP and DPP-IV and the malignant potential of tumours.Methods and results:This study analysed in situ expression in 25 malignant and 13 benign human bone and soft tissue tumours. Reverse transcriptase-polymerase chain reaction analyses confirmed mRNA expression of FAP and DPP-IV in all individuals. Immunohistochemistry using pre-fixed frozen sections revealed that FAP was positive in low-grade myofibroblastic sarcoma, the fibroblastic component of osteosarcomas, and malignant fibrous histiocytomas, but negative in Ewing’s sarcomas and rhabdomyosarcomas. DPP-IV showed similar immunohistochemical results. Among benign tumours, non-ossifying fibromas, desmoid tumours and chondroblastomas expressed both FAP and DPP-IV. Giant cells expressed DPP-IV in giant cell tumours.Conclusions:Our data suggest that FAP and DPP-IV are consistently expressed in bone and soft tissue tumour cells that are histogenetically related to activated fibroblasts and/or myofibroblasts, irrespective of their malignancy. DPP-IV is also expressed in monocyte–macrophage lineage cells.