M1- and M2-macrophage polarization in rat liver cirrhosis induced by thioacetamide (TAA), focusing on Iba1 and galectin-3

Introduction

Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1⁺ (Iba1⁺) and galectin-3⁺ (Gal-3⁺) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1⁺ and Gal-3⁺ macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1⁺ and Gal-3⁺ macrophages in chemical-induced liver cirrhosis.

Materials and methods

Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100 mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs.

Results

Activation of myofibroblasts was most pronounced in livers at week 15. CD68⁺ (M1), CD204⁺ (M2), Iba1⁺ and Gal-3⁺ macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1β, IL-6, and TNF-α) and M2-(TGF-β1, IL-4, and IL-10) related factors. Iba1⁺ and Gal-3⁺ macrophages showed both M1- and M2-immunophenotypes. CD163⁺ macrophages showed a persistent increase, consistent with TGF-β1 upregulation. MHC class II⁺ macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis.

Conclusion

Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1⁺ and Gal-3⁺ macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.     

Heterogeneity of macrophage populations and expression of galectin-3 in cutaneous wound healing in rats

The aim of this study was to investigate the properties of macrophages that infiltrated the sites of cutaneous wound healing in rats between 1 and 26 days post wounding (dpw). During the inflammation phase (1–3 dpw), ED1⁺ (CD68⁺) macrophages with enhanced lysosomal activity dominated. From 5 to 7 dpw there was formation of granulation tissue as indicated by the presence of myofibroblasts expressing α-smooth muscle actin. At this stage, ED2⁺ (CD163⁺) macrophages, capable of producing inflammatory factors, were dominant. The majority of ED1⁺ macrophages expressed galectin-3, a regulator of fibrosis. Corresponding to the increased numbers of ED1⁺ and ED2⁺ macrophages at 3–9 dpw, there was increased expression of genes encoding transforming growth factor-β1 (a major fibrogenic factor), monocyte chemoattractant protein-1 and colony stimulating factor-1. These macrophage-related factors might contribute to inflammation and formation of granulation tissue. OX6⁺ macrophages expressing class II molecules of the major histocompatibility complex became predominant in the healing stages (15–26 dpw), indicating important roles for antigen-presenting cells in tissue remodelling. The OX6⁺ macrophages were most likely derived from ED1⁺ macrophages. The results of this study show that infiltration of phenotypically- and functionally-distinct macrophage populations characterizes different stages of the wound healing process.     

Relationship of heat shock protein 25 with reactive macrophages in thioacetamide-induced rat liver injury

Heat shock protein 25 (Hsp25), which has anti-inflammatory activity, was examined for the relationship of its expression to macrophage appearance in thioacetoamide (TAA)-induced rat acute hepatic lesions. TAA-induced lesions, consisting of hepatocyte coagulation necrosis and reactive macrophages, developed in the centrilobular areas. Macrophages immuno-reacting to ED1 (CD68; exudative macrophages) were mainly seen within the lesions, whereas macrophages reacting to ED2 (CD163; resident macrophages and Kupffer cells), which have abundant cytoplasm, appeared mainly in the periphery of the lesions. Hsp25-immunopositivity was seen in hepatocytes around the lesions in relation to ED1- and ED2-positive macrophages in and around the centrilobular lesions, respectively. Because macrophages appearing in early stages of hepatic lesions produce various pro-inflammatory factors, mRNA expressions of tumor necrosis factor-α (TNF-α), monocyte chemoattractant factor-1 (MCP-1) and osteopontin (OPN) were examined in relation to Hsp25 mRNA expression. Hsp25 mRNA expression generally was correlated with TNF-α, MCP-1 and OPN expressions, suggesting their direct or indirect association with Hsp25 expression. Thus, Hsp25 might have a cytoprotection function against macrophages appearing in hepatic lesions, and factors produced by macrophages in the very early stages of hepatic lesions may influence Hsp25 expression. Hsp25 expression should be useful as an index of anti-inflammatory action for evaluation of hepatotoxicants in vivo.     

Galectin-3 deficiency enhances type 2 immune cell-mediated myocarditis in mice

Experimental autoimmune myocarditis (EAM) is a mouse model of immune-mediated myocarditis and cardiomyopathy. The role of Galectin-3 (Gal-3), a β-galactoside-binding lectin, in autoimmune myocarditis has not been studied. Therefore, the aim of this study was to delineate the role of Gal-3 in myosin peptide-induced autoimmune myocarditis in mice. EAM was induced in relatively resistant C57BL/6J mice (wild type, WT) and in mice with a targeted deletion of Gal-3 gene (Gal-3KO) by immunization with myosin peptide MyHCα_334–352. Gal-3KO mice developed more severe myocarditis and more pronounced heart hypertrophy than WT mice. Increased infiltration of CD45⁺ leucocytes, CD3⁺ T cells, F4/80⁺ macrophages, and eosinophils was observed in hearts of Gal-3KO mice compared to WT mice on day 21 after EAM induction. Moreover, hearts of Gal-3KO mice had more T helper type 2 (Th2) cells, alternatively activated M2 macrophages, higher amounts of IgG deposits, and higher serum levels of IL-4 and IL-33 than WT mice. Ablation of Gal-3 in Th1-dominant C57BL/6J mice that are relatively resistant to EAM resulted in more severe disease characterized by type 2 cardiac inflammation. The complex effects of Gal-3 on EAM progression might be important in the consideration of therapeutic options for the treatment of EAM.     

Identification of alternatively activated macrophages in new-onset paediatric and adult immunoglobulin A nephropathy: potential role in mesangial matrix expansion

Ikezumi Y, Suzuki T, Karasawa T, Hasegawa H, Yamada T, Imai N, Narita I, Kawachi H, Polkinghorne K R, Nikolic‐Paterson D J & Uchiyama M
(2011) Histopathology  58, 198–210
Identification of alternatively activated macrophages in new‐onset paediatric and adult immunoglobulin A nephropathy: potential role in mesangial matrix expansion Aims: New onset of the clinical symptoms of immunoglobulin A (IgA) nephropathy (IgAN) manifests with proliferative glomerular lesions in children, whereas adults exhibit mesangial matrix expansion and interstitial fibrosis. Alternatively, activated (M2) macrophages have been implicated in promoting tissue fibrosis in some settings. Therefore, the aim of this study was to investigate whether M2 macrophages are present in new‐onset IgAN and if they are related to pathological differences between paediatric and adult disease. Methods and results: Biopsy specimens from paediatric (<10 years, n = 14; >12 years, n = 15) and adult (n = 27) IgAN showed a significant infiltrate of CD68⁺ macrophages. M2 macrophages, identified by CD163 or CD204 expression, were detected in glomeruli and the interstitium, being more prominent in adults versus young children. CD163⁺ and CD204⁺ macrophages were present in areas of fibrosis containing myofibroblasts, and double staining showed that CD163⁺ cells produced the profibrotic molecule, connective tissue growth factor. In young children, total CD68⁺ macrophages, but not M2 macrophages, correlated with glomerular hypercellularity. In contrast, in adults and older children, mesangial matrix expansion correlated with M2 macrophages but not with the total CD68⁺ macrophage infiltrate. Conclusions: Alternatively activated M2 macrophages are present in new‐onset paediatric and adult IgAN, and this population may promote the development of fibrotic lesions.     

The origin and distribution of CD68, CD163, and αSMA+ cells in the early phase after meniscal resection in a parabiotic rat model

We previously reported that circulating peripheral blood-borne cells (PBCs) contribute to early-phase meniscal reparative change. Because macrophages and myofibroblasts are important contributors of tissue regeneration, we examined their origin and distribution in the reparative meniscus. Reparative menisci were evaluated at 1, 2, and 4 weeks post-meniscectomy by immunohistochemistry to locate monocytes and macrophages (stained positive for CD68 and CD163), and myofibroblasts (stained positive for αSMA). Of the total number of cells, 13% were CD68⁺ at 1 week post-meniscectomy, which decreased to 1% by 4 weeks post-meniscectomy; of these, almost half of CD68⁺ cells (49.4%: 98.8% as PBCs) were green fluorescent protein (GFP)-positive post-meniscectomy (1, 2, and 4 weeks), indicating that the majority of CD68⁺ cells were derived from PBCs. Of the total cells, 6% were CD163⁺ at 1 week post-meniscectomy, which decreased to 1% by week 4. Of the CD163⁺ cells, the majority were GFP-positive (42.5%: 85.0% as PBCs) after 1 week; however, this decreased significantly over time, which indicates that the majority of CD163⁺ cells are derived from PBCs during the early phase of meniscal reparative change, but are derived from resident cells at later time points. Of the total cells, 38% were αSMA⁺ at 1 week post-meniscectomy, which decreased to 3% by 4 weeks. The proportion of GFP-positive αSMA⁺ cells was 2.8% after 1 week, with no significant change over time, which indicates that the majority of αSMA⁺ cells originated from resident cells. Here, we describe the origin and distribution of macrophages and myofibroblasts during meniscal reparative change.     

Discovered on gastrointestinal stromal tumour 1 (DOG1): a useful immunohistochemical marker for diagnosing chondroblastoma

Akpalo H, Lange C & Zustin J
(2012) Histopathology  60, 1099–1106 Discovered on gastrointestinal stromal tumour 1 (DOG1): a useful immunohistochemical marker for diagnosing chondroblastoma Aims: Cellular areas of chondroblastoma are composed of polygonal chondroblasts with indented nuclei and scattered osteoclast‐type multinucleated cells. To learn more about the phenotype of chondroblasts, we investigated the expression of several established immunohistochemical markers in chondroblastomas. Methods and results: Nine chondroblastomas were analysed using immunohistochemical antibodies [CD34, α‐smooth muscle actin (α‐SMA), DOG1, CD117, AE1/AE3 and CD163]. Ten chondromyxoid fibromas, seven giant cell tumours of bone and four foetal proximal femurs were also analysed. The cellular areas of each chondroblastoma contained nests of DOG1⁺αSMA⁺ CD117^? CD34^? chondroblasts, a phenotype that was not detected in chondromyxoid fibroma cases or in giant cell tumours. Although AE1/AE3 was expressed in all chondroblastomas, the staining intensity and proportion of the positive cells varied widely. Intra‐lesional CD163⁺ macrophages were detected in all cases of chondroblastoma, chondromyxoid fibroma and giant cell tumours. Conclusions: Our results demonstrated nests of membranous DOG1⁺ chondroblasts located within cellular portions of chondroblastoma containing diffuse heterogeneous infiltrates of mostly DOG1^? chondroblasts, CD163⁺ macrophages and multinucleated osteoclastic giant cells. Thus, chondroblastoma can be added to the tumours that are usually positive for DOG1, alongside gastrointestinal stromal tumour (GIST), rare solid‐pseudopapillary neoplasms of the pancreas and exceptional mesenchymal tumours including uterine type retroperitoneal leiomyoma, peritoneal leiomyomatosis and synovial sarcoma.     

SIV Encephalitis Lesions Are Composed of CD163+ Macrophages Present in the Central Nervous System during Early SIV Infection and SIV-Positive Macrophages Recruited Terminally with AIDS

Macrophage recruitment to the central nervous system (CNS) during AIDS pathogenesis is poorly understood. We measured the accumulation of brain perivascular (CD163⁺) and inflammatory (MAC387⁺) macrophages in SIV-infected monkeys. Monocyte progenitors were 5-bromo-2′-deoxyuridine (BrdU) labeled in bone marrow, and CNS macrophages were labeled serially with fluorescent dextrans injected into the cisterna magna. MAC387⁺ macrophages accumulated in the meninges and choroid plexus in early inflammation and in the perivascular space and SIV encephalitis (SIVE) lesions late. CD163⁺ macrophages accumulated in the perivascular space and SIVE lesions with late inflammation. Most of the BrdU⁺ cells were MAC387⁺; however, CD163⁺BrdU⁺ macrophages were present in the meninges and choroid plexus with AIDS. Most (81.6% ± 1.8%) of macrophages in SIVE lesions were present in the CNS before SIVE lesion formation. There was a 2.9-fold increase in SIVp28⁺ macrophages entering the CNS late compared with those entering early (P < 0.05). The rate of CD163⁺ macrophage recruitment to the CNS inversely correlated with time to death (P < 0.03) and increased with SIVE. In SIVE animals, soluble CD163 correlated with CD163⁺ macrophage recruitment (P = 0.02). Most perivascular macrophages that comprise SIVE lesions and multinucleated giant cells are present in the CNS early, before SIVE lesions are formed. Most SIV-infected macrophages traffic to the CNS terminally with AIDS.HIV-associated neurological disorders are associated with central nervous system (CNS) pathologies and include motor, behavioral, and cognitive impairment.^1–3 Proposed explanations for the high prevalence of HIV-associated neurological disorders (approximately 50%), despite effective antiretroviral therapy (ART), include incomplete CNS drug penetrance, continued viral replication in the brain, persistent and chronic macrophage activation, CNS toxicity associated with ART, and the normal effects of aging.^2–7 In SIV-infected monkeys, HIV-infected humans pre-ART, and some HIV-infected humans after ART, infection of the CNS may be associated with encephalitic lesions composed of a focal accumulation of macrophages and microglia, and productive viral infection. Macrophages and microglia that drive CNS pathology are targets of productive viral infection.^3,8–16The timing of monocyte and macrophage entry and accumulation in the CNS as well as the entry of HIV and SIV quasispecies that are associated with pathogenesis are not well understood because of the technical limitations of such studies in humans. An increased rate of monocyte egress from the bone marrow, increased numbers and percentages of CD16⁺ monocytes in the blood, and the accumulation of macrophages in the CNS support the idea that a basal rate of monocyte and macrophage recruitment to the CNS is augmented with HIV infection.^16–22 In a macaque model of SIV infection, carboxyfluorescein succinimidyl ester–labeled autologous monocytes were shown to traffic to the cerebral perivascular space and choroid plexus during acute SIV infection [12 to 14 days postinfection (dpi)] at an accelerated rate compared with uninfected controls.²³ Analyses of macrophage recruitment to and turnover within the CNS from early infection up to the development of AIDS and SIV encephalitis (SIVE) have not been performed.CNS macrophages are heterogeneous and can be classified on the basis of tissue localization and/or immune phenotype.^9,24,25 Parenchymal microglia, the resident macrophages of the CNS, are yolk sac–derived, myeloid lineage cells that engraft the CNS during embryonic development and are then maintained as a stable population.^26,27 Perivascular, meningeal, and choroid plexus macrophages are of bone marrow origin and are thought to be replenished from circulating monocytes in rodents, nonhuman primates, and humans, likely at different rates.^17,28–32 In the normal CNS, microglia express the pan-macrophage marker CD68 and have low to undetectable hemoglobin-haptoglobin scavenger receptor CD163 (CD68⁺CD163⁻).²⁵ Perivascular macrophages express both CD163 and CD68 (CD163⁺CD68⁺).²⁵ A third phenotypic population of macrophages is labeled by the antibody MAC387, which recognizes migration inhibitory factor–related protein (MRP) 14 or the MRP8/MRP14 heterodimer, but does not have detectable CD68 or CD163 (MAC387⁺CD68⁻CD163⁻).¹⁴ MAC387⁺ macrophages are not present in the uninfected or noninflamed CNS but are recruited with inflammation.^14,33–36 Parenchymal microglia and CD163⁺ perivascular macrophages are considered primary targets of HIV and SIV infection in the CNS.^5,7 MAC387⁺ macrophages are rarely found to be productively infected.^{9,13,14,25,37} The timing of monocyte and macrophage entry into the CNS and the role of macrophage subsets mediating the progression or resolution of CNS inflammation due to HIV and SIV infection are not well defined.Current research suggests that virus enters the CNS via trafficking of infected monocytes and macrophages, although other mechanisms, including infection of or transcytosis through endothelial cells and direct transmission of free virus from the blood to the cerebrospinal fluid, have been suggested.^9,20,38 Productive infection of the CNS can be detected within days to weeks of initial infection, primarily within perivascular macrophages, but then resolves until the development of AIDS (pre-ART) or chronic infection with HIVE lesion formation (post-ART).^{23,32,38–42} Productive infection of the CNS at end-stage disease may represent recrudescence of virus seeded in the CNS with primary/acute infection, reintroduction of virus into the CNS terminally with AIDS, or both. Regardless, productive infection of macrophages in the perivascular space, encephalitis lesions, and meninges and choroid plexus likely results in production of toxic factors, including cytokines and viral proteins, which contribute metabolic encephalopathy with resultant neuronal and glial cell aberrations.^8,43We studied macrophage recruitment to the CNS in a rapid simian model of neuro-AIDS by 5-bromo-2′-deoxyuridine (BrdU) labeling of myeloid progenitors in the bone marrow, which traffic to the CNS, and serial labeling perivascular macrophages, within the CNS, by intracisternal injection of dextran conjugates in the cerebrospinal fluid. We compared macrophage recruitment rates between early/acute and terminal disease, or between animals with SIVE and animals with SIV but no encephalitis (SIVnoE). Early CNS inflammation was characterized by an influx of MAC387⁺ macrophages in acute infection. Later, recruitment of both MAC387⁺ and CD163⁺ macrophages was ongoing and was greater terminally in animals that developed AIDS and SIVE. BrdU⁺ macrophages present in CNS tissues were primarily MAC387⁺, but CD163⁺BrdU⁺ macrophages were present in the meninges and choroid plexus terminally with AIDS. Overall, few BrdU⁺ macrophages were present in the perivascular space and SIVE lesions. The ratio of CD163⁺/MAC387⁺ macrophages in the CNS was greater in animals with SIVE compared with SIV-infected animals without SIVE. Recruitment of CD163⁺ macrophages in the CNS correlated with plasma soluble CD163 (sCD163) in animals with SIVE. In all animals, a greater rate of CD163⁺ macrophage recruitment correlated with shorter time to death. Terminally with AIDS, CD163⁺ macrophages accumulated in the perivascular space and SIVE lesions, and not in the meninges or choroid plexus. Interestingly, SIVE lesions were composed primarily of CD163⁺ macrophages that were present in the CNS early in infection, by 20 dpi, before SIVE lesions are typically present. In SIVE lesions, the percentage of productively infected CD163⁺ macrophages was 2.9 times higher in macrophages that entered the CNS terminally with AIDS compared with macrophages present in the CNS at 20 dpi. These data indicate a role for resident perivascular macrophages that line CNS blood vessels and migrate to form SIVE lesions and suggest that virus is reintroduced to the CNS terminally with AIDS.     

Galectin-9 expands unique macrophages exhibiting plasmacytoid dendritic cell-like phenotypes that activate NK cells in tumor-bearing mice

Galectin-9 (Gal-9) inhibits the metastasis of tumor cells by blocking their adhesion to endothelium and the extracellular matrix. In this study, we addressed the involvement of Gal-9 in anti-tumor activity. Gal-9 significantly prolonged the survival of B16F10 melanoma-bearing mice. Gal-9 increased the numbers of NK cells, CD8 T cells and macrophages in tumor-bearing mice. Gal-9-mediated anti-tumor activity was not induced in NK cell-, macrophage- and CD8 T cell-depleted mice. NK cells from Gal-9-treated mice, compared to PBS-treated mice, exhibited significantly higher cytolytic activity. Co-culture of naïve NK cells with macrophages from Gal-9-treated mice resulted in enhanced NK activity, although Gal-9 itself did not enhance the NK activity. We also found that Ly-6C⁺CD11b⁺F4/80⁺ macrophages with plasmacytoid cell (pDC)-like phenotypes (PDCA-1 and B220) were responsible for the enhanced NK activity. These results provide evidence that Gal-9 promotes NK cell-mediated anti-tumor activity by expanding unique macrophages with a pDC-like phenotype.     

Phenotypic and functional heterogeneity of CD169 and CD163 macrophages from porcine lymph nodes and spleen

Secondary lymphoid organ macrophages are involved in the establishment of innate and acquired immunity. Here, we have isolated and characterized porcine lymph node and spleen CD169⁺ and spleen CD163⁺ macrophages. Lymph node and spleen CD169⁺ macrophages can be both identified as CD172a⁺SLA-DR^hiCD80/86^hiCD14^intTLR2⁺TLR4⁺. On the other side, spleen CD163⁺ macrophages are CD172a⁺SLA-DR^intCD80/86^intCD14⁻/^loTLR2^intTLR4^int. In addition, these macrophages can be subdivided based on the expression of CD11R1 or CD11R3. Lymph node CD169⁺ macrophages phagocytozed polystyrene microspheres more efficiently than spleen CD163⁺ and CD169⁺ macrophages. All macrophages exhibited low capacity to take up and process the soluble antigen DQ-OVA. Finally, spleen CD163⁺ macrophages displayed the highest ability to present lysozyme to CD4⁺ T cells in a secondary in vitro response, followed by lymph node and spleen CD169⁺ macrophages.     

Antibody combinations for optimized staining of macrophages in human lung tumours

The analysis of tumour-associated macrophages (TAMs) has a high potential to predict cancer recurrence and response to immunotherapy. However, the heterogeneity of TAMs poses a challenge for quantitative and qualitative measurements. Here, we critically evaluated by immunohistochemistry and flow cytometry two commonly used pan-macrophage markers (CD14 and CD68) as well as some suggested markers for tumour-promoting M2 macrophages (CD163, CD204, CD206 and CD209) in human non–small cell lung cancer (NSCLC). Tumour, non-cancerous lung tissue and blood were investigated. For immunohistochemistry, CD68 was confirmed to be a useful pan-macrophage marker although careful selection of antibody was found to be critical. The widely used anti-CD68 antibody clone KP-1 stains both macrophages and neutrophils, which is problematic for TAM quantification because lung tumours contain many neutrophils. For TAM counting in tumour sections, we recommend combined labelling of CD68 with a cell membrane marker such as CD14, CD163 or CD206. In flow cytometry, the commonly used combination of CD14 and HLA-DR was found to not be optimal because some TAMs do not express CD14. Instead, combined staining of CD68 and HLA-DR is preferable to gate all TAMs. Concerning macrophage phenotypic markers, the scavenger receptor CD163 was found to be expressed by a substantial fraction (50%-86%) of TAMs with a large patient-to-patient variation. Approximately 50% of TAMs were positive for CD206. Surprisingly, there was no clear overlap between CD163 and CD206 positivity, and three distinct TAM sub-populations were identified in NSCLC tumours: CD163⁺CD206⁺, CD163⁺CD206⁻ and CD163⁻CD206⁻. This work should help develop macrophage-based prognostic tools for cancer.     

Tumor‐Associated Macrophages Associate with Cerebrospinal Fluid Interleukin‐10 and Survival in Primary Central Nervous System Lymphoma (PCNSL)

Increased tumor‐associated macrophages (TAMs) have been reported to be associated with poor prognosis in various tumors; however, the importance of TAMs in primary central nervous system lymphoma (PCNSL) has not been clarified. In 47 patients with PCNSL who were treated with high‐dose methotrexate (MTX) and radiotherapy, the relationships between the infiltration levels of TAMs and the clinicopathological parameters were analyzed. Univariate analysis of the Cox proportional hazards model using continuous scales revealed that increased CD68 positive (+) TAMs was significantly associated with inferior progression‐free survival (PFS) (P = 0.04), and trends were observed for the increased CD163⁺ TAMs and having shorter PFS (P = 0.05). However, increased TAMs were not associated with overall survival. Because TAMs are known to produce various cytokines, we examined the relationships between cerebrospinal fluid (CSF) cytokines and TAMs. CSF interleukin‐6 (IL‐6) and soluble IL‐2 receptor were not correlated with the infiltration rate of TAMs; however, CSF IL‐10 level was correlated with infiltration levels of CD68 and CD163⁺ TAMs. We also confirmed the expression of IL‐10 in CD68⁺ and CD163⁺ TAMs by double immunostaining analysis. Our results indicate that a high level of IL‐10 in CSF may be positively associated with the infiltration level of TAMs in PCNSLs.     

Localization of IL-17+Foxp3+ T Cells in Esophageal Cancer

The etiology of cancer is unclear. Recent studies indicate that some cytokines, such as interleukin (IL)-17, and regulatory T cells are involved in the development of cancer. This study aims to detect a subset of T cell, IL17+Foxp3+ T cell, in the pathogenesis of esophageal cancer (Eca). Twelve patients with squamous Eca were recruited in this study. The surgically removed Eca tissue was collected. Cells isolated from Eca tissue were analyzed by flow cytometry. The results showed that 2–10% Eca tissue-derived CD4⁺ T cells expressed Foxp3; only 0.2–0.8% non-ca tissue-derived CD4⁺ T cells expressed Foxp3. Further analysis showed that 3–15% Eca-isolated CD4⁺ T cells were also IL-17 positive whereas only 0.4–1.5% non-ca tissue-isolated CD4⁺ T cells were IL-17 positive. We also found that about 4.8–11.2% Foxp3⁺ IL-17⁺ T cells in isolated CD4⁺ T cells from Eca tissue that were significantly less than in non-ca tissue derived CD4⁺ T cells. Less than 1% Foxp3⁺ IL-17⁺ T cells in isolated CD4⁺ T cells in both Eca patients and healthy controls. Treatment with hypoxia markedly increased the expression of IL-6 in peripheral CD68+ cells. Coculturing CD68+ cells and Foxp3+ T cells under hypoxic environment resulted in abundant expression of IL-17 in Foxp3+ T cells that could be blocked by pretreatment with either anti-IL-17 or anti-transforming growth factor beta antibodies. We conclude that IL-17+Foxp3+ T cells may contribute to the development of Eca.     

Ligands of CD4 inhibit the association of phospholipase Cγ1 with phosphoinositide 3 kinase in T cells: regulation of this association by the phosphoinositide 3 kinase activity

We have previously shown that CD4 ligands inhibit interleukin-2 (IL-2) production and T cell proliferation in human peripheral CD4⁺ T lymphocytes, in an MHC-independent way. Two major pathways implicated in T cell activation are inhibited by binding of CD4 ligands to the CD4 molecule, i. e. Ca²⁺ signaling by phospholipase Cγ1 (PLCγ1), and ERK-2 activation, suggesting a p21^ras inhibition. We have correlated these inhibitions with the disruption of multifunctional complexes containing PLCγ1, p120GAP and Sam68, induced by T cell activation. We report here that T cell activation through the TCR/CD3 induces an association of the phosphoinositide 3 kinase (PI3 kinase) with PLCγ1, both in peripheral CD4⁺ T lymphocytes and the HUT-78 CD4⁺ T cell line. PI3 kinase is present in the multifunctional complexes that we have described previously. Preincubation of human peripheral CD4⁺ T cells and HUT-78 CD4⁺ T cells with gp160 or a peptide analogue of the HLA class II DR molecule precludes the association of PLCγ1 with PI3 kinase. We also demonstrate, using two specific inhibitors of PI3 kinase activity (LY294002 and wortmannin), that this activity plays a key role in the association of PLCγ1 with PI3 kinase. Moreover, we demonstrate the implication of the PI3 kinase activity in the negative signal mediated by HIV gp160 binding to CD4 molecules. We propose that the products of the PI3 kinase are important mediators of the negative signaling induced by the binding of CD4 ligands to the CD4 molecule implicated in the regulation of the formation of multifunctional complexes.     

Pentoxifylline attenuates nitrogen mustard-induced acute lung injury,oxidative stress and inflammation

Nitrogen mustard (NM) is a toxic alkylating agent that causes damage to the respiratory tract. Evidence suggests that macrophages and inflammatory mediators including tumor necrosis factor (TNF)α contribute to pulmonary injury. Pentoxifylline is a TNFα inhibitor known to suppress inflammation. In these studies, we analyzed the ability of pentoxifylline to mitigate NM-induced lung injury and inflammation. Exposure of male Wistar rats (150–174 g; 8–10 weeks) to NM (0.125 mg/kg, i.t.) resulted in severe histopathological changes in the lung within 3 d of exposure, along with increases in bronchoalveolar lavage (BAL) cell number and protein, indicating inflammation and alveolar-epithelial barrier dysfunction. This was associated with increases in oxidative stress proteins including lipocalin (Lcn)2 and heme oxygenase (HO)-1 in the lung, along with pro-inflammatory/cytotoxic (COX-2⁺ and MMP-9⁺), and anti-inflammatory/wound repair (CD163⁺ and Gal-3⁺) macrophages. Treatment of rats with pentoxifylline (46.7 mg/kg, i.p.) daily for 3 d beginning 15 min after NM significantly reduced NM-induced lung injury, inflammation, and oxidative stress, as measured histologically and by decreases in BAL cell and protein content, and levels of HO-1 and Lcn2. Macrophages expressing COX-2 and MMP-9 also decreased after pentoxifylline, while CD163⁺ and Gal-3⁺ macrophages increased. This was correlated with persistent upregulation of markers of wound repair including pro-surfactant protein-C and proliferating nuclear cell antigen by Type II cells. NM-induced lung injury and inflammation were associated with alterations in the elastic properties of the lung, however these were largely unaltered by pentoxifylline. These data suggest that pentoxifylline may be useful in treating acute lung injury, inflammation and oxidative stress induced by vesicants.     

Deletion of galectin-3 in the host attenuates metastasis of murine melanoma by modulating tumor adhesion and NK cell activity

Galectin-3, a β galactoside–binding lectin, plays an important role in the processes relevant to tumorigenesis such as malignant cell transformation, invasion and metastasis. We have investigated whether deletion of Galectin-3 in the host affects the metastasis of B16F1 malignant melanoma. Galectin-3-deficient (Gal-3^−/−) mice are more resistant to metastatic malignant melanoma as evaluated by number and size of metastatic colonies in the lung. In vitro assays showed lower number of attached malignant cells in the tissue section derived from Gal-3^−/− mice. Furthermore, lack of Galectin-3 correlates with higher serum levels of IFN-γ and IL-17 in tumor bearing hosts. Interestingly, spleens of Gal-3^−/− mice have lower number of Foxp3⁺ T cells after injection of B16F1 melanoma cells. Finally, we found that while CD8⁺ T cell and adherent cell cytotoxicity were similar, there was greater cytotoxic activity of splenic NK cells of Gal-3^−/− mice compared with “wild-type” (Gal-3 ^+/+ ) mice. Despite the reduction in total number of CD3ε⁻NK1.1⁺, Gal-3^−/− mice constitutively have a significantly higher percentage of effective cytotoxic CD27^highCD11b^high NK cells as well as the percentage of immature CD27^highCD11b^low NK cells. In contrast, CD27^lowCD11b^high less functionally exhausted NK cells and NK cells bearing inhibitory KLRG1 receptor were more numerous in Gal-3 ^+/+ mice. It appears that lack of Galectin-3 affects tumor metastasis by at least two independent mechanisms: by a decrease in binding of melanoma cells onto target tissue and by enhanced NK-mediated anti-tumor response suggesting that Galectin-3 may be considered as therapeutic target.     

The class D scavenger receptor CD68 contributes to mouse chronic liver injury

Scavenger receptors, which are expressed on monocyte/macrophages, play a central role in many pathogenic processes. Here, we examined the role of the class D scavenger receptor (CD68) in bone marrow-derived monocyte/macrophages (BMMs) in chronic liver injury. The expression pattern of multiple scavenger receptors in two liver injury models (methionine-choline-deficient and high fat (MCDHF), carbon tetrachloride (CCl₄)) were analyzed by qRT-PCR. CD68 expression was characterized by flow cytometric analysis, immunofluorescence, and qRT-PCR. A selective monocyte/macrophage toxicant, gadolinium chloride (GdCl₃) was applied to analyze the function of CD68 in vitro and in vivo. Among the seven examined scavenger receptors (CD68, CD36, CD204, MARCO, LOX1, SREC, and CD163), the mRNA expression of CD68 first got uppermost and continuously increased throughout the entire stage of chronic liver injury, thus attracting our attention. In the injured liver, the percentage of recruited CD68⁺ BMM increased notably, aligning along the developing fibrotic septa, while the proportion of CD68⁺ KC stayed the same compared with that of control mice. In vitro CD68 was highly expressed in primary cultured BMM, and CD68 reduction was triggered by macrophage phagocytosis and apoptosis in the presence of GdCl₃. In the damaged liver, the recruitment of CD68⁺ BMM and CD68 mRNA expression were reduced by GdCl₃ administration, leading to the attenuation of liver inflammation and fibrosis. Altogether, scavenger receptor CD68 plays a key role in mouse chronic liver injury, which has important implications for the design of anti-fibrotic therapies.     

Macrophages participate in lymphangiogenesis in idiopathic diffuse alveolar damage through CCL19-CCR7 signal

An abnormal healing process is believed to be involved in lung tissue damage in pulmonary fibrosis. Lymphangiogenesis has been determined to play a role in structural remodeling in diffuse alveolar damage. However, the mechanism of lymphangiogenesis remains unclear. The aim of this study is to investigate the cellular mechanisms of lymphangiogenesis in diffuse alveolar damage, focusing on the roles of macrophages. Formalin-fixed and paraffin-embedded lung tissues from 13 autopsy cases with idiopathic diffuse alveolar damage were used. Antibodies specific for D2-40 and CD68 were mainly used as the markers for lymphatics and macrophages, respectively, and immunohistochemical examinations and morphometric analyses were performed. Immunohistochemistry showed the aggregation of numerous CD68⁺ macrophages around newly formed lymphatics in the intraalveolar fibrotic lesions in the proliferative stage, and some of the CD68⁺ macrophages were colocalized with the lymphatic endothelium. These macrophages were characterized by the expression of vascular endothelial growth factor-C. Among the 3 stages of diffuse alveolar damage, the number of Ki-67⁺ cells on the lymphatic endothelium tended to increase in newly formed lymphatics of the proliferative stage. In addition, the number of CD68⁺ macrophages on the lymphatic endothelium was significantly increased in the newly formed lymphatics of the proliferative stage more than those of the fibrotic stage. The CD68⁺ macrophages around the newly formed lymphatics coexpressed CCR7. Dual immunostaining showed the coexpression of CCL19—a ligand for CCR7—on the lymphatic endothelium. Thus, macrophages may participate in lymphangiogenesis in diffuse alveolar damage, which is facilitated by CCL19 and CCR7.     

Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection

Summary. Monocytes-macrophages, the target cells of African swine fever virus (ASFV) are highly heterogeneous in phenotype and function. In this study, we have investigated the correlation between the phenotype of specific populations of porcine macrophages and their permissiveness to ASFV infection. Bone marrow cells and fresh blood monocytes were less susceptible to in vitro infection by ASFV than more mature cells, such as alveolar macrophages. FACS analyses of monocytes using a panel of mAbs specific for porcine monocyte/macrophages showed that infected cells had a more mature phenotype, expressing higher levels of several macrophage specific markers and SLA II antigens. Maturation of monocytes led to an increase in the percentage of infected cells, which correlated with an enhanced expression of CD163. Separation of CD163⁺ and CD163^– monocytes demonstrated the specific sensitivity of the CD163⁺ subset to ASFV infection. In vivo experiments also showed a close correlation between CD163 expression and virus infection. Finally, mAb 2A10 and, in a lower extent, mAb 4E9 were able to inhibit, in a dose-dependent manner, both ASFV infection and viral particle binding to alveolar macrophages. Altogether, these results strongly suggest a role of CD163 in the process of infection of porcine monocytes/macrophages by ASFV.     

Effects of cold stress and Salmonella Heidelberg infection on bacterial load and immunity of chickens

We analysed the effects of cold stress (19?±?1°C, 6?h /day, from the first to the seventh day of life) applied to specific pathogen free (SPF) chickens. On experimental Day 1 (ED1), chicks were divided into four groups: C (not infected and kept under thermoneutral condition); CS (not infected and cold stressed); PC (Salmonella Heidelberg (SH) infected and kept under thermoneutral condition) and PCS (SH infected and cold stressed). High concentrations of corticosterone were found in the cold stressed birds on ED7 and ED21, with a greater increase in birds of the PCS group. Stress or non-stressed SH-infected birds had high levels of norepinephrine on ED21. On ED21, an increased percentage and number of SH were found in birds of the PCS group. On ED7, a decrease in macrophages presenting MHCII, CD8⁺ and CD8⁺ γδ cells was observed in the chickens of the CS group. Decrease was observed in CD3⁺ cells in the birds of the PCS group and increase in macrophages presenting MHCII cells and of the CD4⁺/CD8⁺ ratio in chickens of the CS group on ED21. There was a decrease in CD8⁺ γδ cells in birds of the CS group on ED21 and in the CD3⁺ and CD8⁺cell numbers in chickens of the PCS group on ED21. Our results suggest that cold stress applied to chickens in the first 7 days of life increases both the hypothalamus pituitary adrenal axis and the sympathetic nervous system activities, leading to long-term immune cell dysfunction, thus allowing increased SH invasion and persistence within the birds’ body.