Characterization of cells containing factor XIII subunita in benign and malignant buccal lesions

Summary In the present study, the distribution pattern and characteristics of cells containing Factor XIII subunita (FXIII A) have been studied in benign and malignant lesions of human buccal mucosa. Tissues from four irritation fibromas and three squamous cell carcinomas were studied by means of double immunofluorescent staining techniques in which the detection of FXIII A was combined with a reaction with CD14 (recognizing a monocyte/macrophage differentiation marker antigen), Mac 387 (reacting with a special subset of macrophages), anti-HLA-DR, Ki-M7 (labelling phagocytosing macrophages) or Ki-67 (visualizing a nuclear antigen associated with cell proliferation) monoclonal antibodies. FXIII A was detected in cells of the connective tissue stroma in both benign and malignant buccal lesions. The number of these FXIII A-reactive cells (FXIII A⁺ cells) increased considerably in the tumour tissues, in particular in those surrounding tumour cell clusters. FXIII A⁺ cells scattered in the fibromatous tissues were spindle-shaped, whereas in the tumour stroma, large stellate cells predominated, and round cells were likewise labelled around blood vessels. FXIII A⁺ cells were labelled with CD14 and Ki-M7 in both fibromatous and tumoural buccal mucosa; however, they failed to show any reaction with Ki-67. FXIII A⁺ cells accumulated in the tumour stroma reacted for HLA-DR as well. These results indicate that in both the benign and malignant buccal lesions FXIII A is contained in a subpopulation of tissue macrophages, which represents a monocyte-derived (CD14⁺) and phagocytosing (KiM7⁺) cell population. The accumulation of the FXIII A⁺ cells in the tumour stroma is believed to be a result of direct migration from the circulating blood. The FXIII A⁺ cells of the tumour stroma may be actively involved in both antigen presentation and matrix remodelling during tumour progression.     

The Distribution of Macrophages with a M1 or M2 Phenotype in Relation to Prognosis and the Molecular Characteristics of Colorectal Cancer

High macrophage infiltration has been correlated to improved survival in colorectal cancer (CRC). Tumor associated macrophages (TAMs) play complex roles in tumorigenesis since they are believed to hold both tumor preventing (M1 macrophages) and tumor promoting (M2 macrophages) activities. Here we have applied an immunohistochemical approach to determine the degree of infiltrating macrophages with a M1 or M2 phenotype in clinical specimens of CRC in relation to prognosis, both in CRC in general but also in subgroups of CRC defined by microsatellite instability (MSI) screening status and the CpG island methylator phenotype (CIMP). A total of 485 consecutive CRC specimens were stained for nitric oxide synthase 2 (NOS2) (also denoted iNOS) as a marker for the M1 macrophage phenotype and the scavenger receptor CD163 as a marker for the M2 macrophage phenotype. The average infiltration of NOS2 and CD163 expressing macrophages along the invasive tumor front was semi-quantitatively evaluated using a four-graded scale. Two subtypes of macrophages, displaying M1 (NOS2⁺) or M2 (CD163⁺) phenotypes, were recognized. We observed a significant correlation between the amount of NOS2⁺ and CD163⁺ cells (P<0.0001). A strong inverse correlation to tumor stage was found for both NOS2 (P<0.0001) and CD163 (P<0.0001) infiltration. Furthermore, patients harbouring tumors highly infiltrated by NOS2⁺ cells had a significantly better prognosis than those infiltrated by few NOS2⁺ cells, and this was found to be independent of MSI screening status and CIMP status. No significant difference was found on cancer-specific survival in groups of CRC with different NOS2/CD163 ratios. In conclusion, an increased infiltration of macrophages with a M1 phenotype at the tumor front is accompanied by a concomitant increase in macrophages with a M2 phenotype, and in a stage dependent manner correlated to a better prognosis in patients with CRC.     

CD4+ T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

CD4⁺ T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4⁺ T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4⁺ T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV⁺) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV⁺ plasma neutralization. However, plasma from SIV-infected CD4⁺ T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4⁺ T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4⁺ target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.     

M2 differentiation of MonoMac-1 cell line induced by M-CSF and glucocorticoid pathways

Models of macrophage subtypes require molecular characterization to reliably facilitate their differentiation. Although CD16⁺ (Fc-gamma III receptor) monocytes that express CD163 (a hemoglobin/haptoglobin receptor) have been implicated in a variety of disease states, the conditions necessary to establish lineages of these cell subtypes remains unknown. The current investigations utilize a cell line derived from acute myelogenous leukemia lineage, MonoMac-1, to interrogate the factors that promote the development of CD16⁺ macrophages that express CD163. Results implicate the glucocorticoid pathway as well as c-fms signaling based on the action of dexamethasone and macrophage colony-stimulating factor-1 in promoting CD16⁺ expression, in addition to phorbol myristate acetate and lipopolysaccharides treatment. The ability of glucocorticoid and c-fms receptor antagonists to inhibit CD16⁺ cell formation further establishes the role of these pathways in CD16 expression in this cell line. In view of the inherent difficulty in working with primary cells as well as donor variation, cell lines may be preferable to primary cells for their consistency. We envision that the process we use to induce CD16 expression in this cell type will be useful for screening and identification of drug candidates potentially useful for the treatment of diseases where the etiology involves the expansion of the CD16⁺ monocytes subset or the accumulation of CD163⁺ tissue macrophages.     

Overexpression of IL-10 in C2D Macrophages Promotes a Macrophage Phenotypic Switch in Adipose Tissue Environments

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1β and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206⁺, CD301⁺, CD11c⁻CD206⁺ (M2) and CD11c⁺CD206⁺ (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.     

Impact of CD68/(CD3+CD20) Ratio at the Invasive Front of Primary Tumors on Distant Metastasis Development in Breast Cancer

Tumors are infiltrated by macrophages, T and B-lymphocytes, which may favor tumor development by promoting angiogenesis, growth and invasion. The aim of this study was to investigate the clinical relevance of the relative amount of macrophages (CD68⁺), T-cells (CD3⁺) and B-cells (CD20⁺) at the invasive front of breast carcinomas, and the expression of matrix metalloproteases (MMPs) and their inhibitors (TIMPs) either at the invasive front or at the tumor center. We performed an immunohistochemical study counting CD3, CD20 and CD68 positive cells at the invasive front, in 102 breast carcinomas. Also, tissue sections were stained with MMP-2, -9, -11, -14 and TIMP-2 antibodies, and immunoreactivity location, percentage of reactive area and intensity were determined at the invasive front and at the tumor center. The results showed that an increased CD68 count and CD68/(CD3+CD20) ratio were directly associated with both MMP-11 and TIMP-2 expression by mononuclear inflammatory cells at the tumor center (p = 0.041 and p = 0.025 for CD68 count and p = 0.001 and p = 0.045 for ratio, respectively for MMP-11 and TIMP-2). In addition, a high CD68/(CD3+CD20) ratio (>0.05) was directly associated with a higher probability of shortened relapse-free survival. Multivariate analysis revealed that CD68/(CD3+CD20) ratio was an independent factor associated with distant relapse-free survival (RR: 2.54, CI: (1.23–5.24), p<0.01). Therefore, CD68/(CD3+CD20) ratio at the invasive front could be used as an important prognostic marker.     

Accumulation of cells containing factor XIII subunit a around the foci of intense fibrosis in human epulides

Summary On the basis of clinical and biochemical findings, Factor XIII subunita (FXIII A) has been conjectured to play an important role in fibrotic processes. Epulis samples at different stages of fibrotic tissue formation were used as a model system for studying the localization and tissue distribution of FXIII A during the course of connective tissue generation. Marker characteristics of cells containing FXIII A (FXIII A⁺ cells) were determined as well. In double immunofluorescent labelling systems, FXIII A was localized in monocyte-derived (CD-14⁺), activated (HLA-DR⁺), and phagocytosing (Ki-M7⁺) tissue macrophages, which are widely distributed homogeneously in granulation tissues, but start to accumulate around foci of fibrosis as soon as the foci appear. During the relatively long process of fibrosis, FXIII A⁺ macrophages continuously decrease in number, and their morphological appearance changes from stellate to spindle-shaped. The nuclei of these cells were not labelled by Ki-67 monoclonal antibody; this indicating that they represent a non-proliferating cell population in the connective tissue stroma. The present findings may help to link theories concerning the role of FXIII A and those of macrophages in the connective tissue formation so far found separately in the literature.     

Characterization of the inflammatory cell populations in normal colon and colonic carcinomas

Little is known about the nature of the mucosa-associated immune system within the normal colon, or about the immune response to colon carcinoma. In this study inflammatory cells (ICs) in 14 normal colons and 14 carcinomas were characterized. Overall inflammation, lymphocytes, plasma cells, neutrophils, and eosinophils were graded in routine H & E sections. Frozen sections were stained by an immunoperoxidase technique using antibodies to the T cell associated antigens CD2, CD7, CD4, CD8, and T cell receptors αβ and γδ. B cells were identified with CD20, macrophages with CD68, and Class II antigen with anti-HLA DR. Each cell type was semiquantitatively graded in 10 high power fields (HPFs) in the lumenal half (LH) or basal half (BH) of the normal mucosae, and in epithelium or stroma of the carcinomas. In normal colons, ICs were more frequent in LH than in BH. Plasma cells, lymphocytes and monocytes predominated. Subtyping of lymphocytes showed that CD4⁺ TCR αβ⁺ T lymphocytes were most numerous in the lamina propria. Lymphocytes within the epithelium were CD8⁺ T cells. Around carcinomas the overall grade of ICs was 1+ in the majority of cases. Plasma cells, CD4⁺ and CD8⁺ cells with the TCR αβ receptor, and macrophages were most frequent. Lymphoid aggregates of both T and B cells were frequent. Conclusions: 1. Normal colon contains a diffuse lumenally oriented population of TCR αβ⁺ CD4+ T cells, plasma cells, macrophages and class II antigen-expressing cells in the lamina propria. Intraepithelial lymphocytes are of the T suppressor phenotype. CD4+ T cells, macrophages and HLG-DR⁺ cells predominate in the response to colon carcinomas.     

The relationship between non-HDL cholesterol and macrophage phenotypes in human adipose tissue

Data from experimental animal models and in vitro studies suggest that both hyperlipoproteinemia and obesity predispose to development of proinflammatory pathways of macrophages within adipose tissue. The aim of this study was to analyze whether non-HDL cholesterol concentration in healthy living kidney donors (LKDs) is related to the number and phenotype of proinflammatory macrophages in visceral and subcutaneous adipose tissue. Adipose tissue samples were collected by cleansing the kidney grafts of LKDs obtained peroperatively. The stromal vascular fractions of these tissues were analyzed by flow cytometry. Proinflammatory macrophages were defined as CD14⁺ cells coexpressing CD16⁺ and high-expression CD36 as well (CD14⁺CD16⁺CD36⁺⁺⁺), while CD16 negativity and CD163 positivity identified alternatively stimulated, anti-inflammatory macrophages. Non-HDL cholesterol concentration positively correlated to proinflammatory macrophages within visceral adipose tissue, with increased strength with more precise phenotype determination. On the contrary, the proportion of alternatively stimulated macrophages correlated negatively with non-HDL cholesterol. The present study suggests a relationship of non-HDL cholesterol concentration to the number and phenotype proportion of macrophages in visceral adipose tissue of healthy humans.     

Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages

 Macrophages are key players in many aspects of human physiology and disease. It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions. In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages. The present technique does not select for monocyte subpopulations prior to the onset of differentiation. Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively. They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity. Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis. While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36⁺CD14^–LDL-R^– (58±12%), CD36⁺CD14⁺LDL-R⁺(18±5%), the remaining cells being CD36^–CD14^–LDL-R^–. The first two subsets decreased in size during further differentiation (51±12% and 8±3%, respectively). Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis. Accepted: 13 February 1998     

Phenotypic characterization of chronic inflammation in a rare case of endobronchial carcinoma

This report presents a phenotypical characterization of the immune cell infiltrate in a rare case of endobronchial carcinoma. A patient initially treated for an adenocarcinoma of the esophagus developed an endobronchial carcinoma surrounded by gastric metaplasia distal to a suspected gastrobronchial fistula, 11 years after esophagectomy. Our hypothesis is that the sustained exposure of the bronchial mucosa to a mixed acid and pancreatobiliary refluxate led to chronic inflammation and promoted malignant transformation. We performed an immunohistochemical study of the tumor microenvironment evaluating the density of CD3⁺, CD8⁺ T lymphocytes, CD20⁺ B lymphocytes, CD68⁺ macrophages and FoxP3⁺ regulatory T cells. Quantification of immune cell density was completed using a novel software-based analysis method. Our results suggest that, within all the tissues analyzed, FoxP3⁺ regulatory T cells were present at their highest density in the malignant and metaplastic tissues. The endobronchial metaplasia biopsied several years prior to the detection of the endobronchial adenocarcinoma was already densely infiltrated by B cells and macrophages, when compared to the immune cell infiltrate of the endobronchial carcinoma. Altogether, these observations support the current understanding of carcinogenesis promoted by chronic inflammation.     

CD44及CD24在乳腺癌组织中的表达及与临床病理特征的关系研究

目的:探讨肿瘤标志因子CD44及CD24在乳腺癌组织中的表达及与临床病理特征的关系。方法:选择从2015年1月到2017年1月在我院接受手术治疗的乳腺癌患者80例纳入本次研究,另选同期在我院治疗的导管原位癌患者30例,小叶增生患者20例及导管单纯增生患者20例的组织提取标本进行对照,分析CD44及CD24在乳腺癌组织和不同病变类型中的表达,并分析CD44~+/CD24~-细胞在癌症免疫分型中的表达以及CD44~+/CD24~-细胞与乳腺浸润导管癌相关病理特征的关系。结果:乳腺癌组织内的CD44阳性率为52.50%,CD24的阳性率为57.50%,均显著高于癌旁组织的11.25%和15.00%,差异均有统计学意义(均P0.05)。CD44及CD24在导管原位癌及乳腺浸润导管癌中的阳性率高于小叶增生和导管单纯增生,导管原位癌的阳性率高于乳腺浸润导管癌,差异均有统计学意义(均P0.05),且CD44在乳腺浸润导管癌不同分化类型中的阳性率差异有统计学意义(P0.05)。CD24在乳腺浸润导管癌不同分化类型中的阳性率差异不显著(P0.05)。CD44~+/CD24~-细胞在不同癌症免疫分型以及不同分化中的阳性率比较差异均有统计学意义(P0.05)。CD44~+/CD24~-细胞与乳腺浸润导管癌患者的年龄、月经状态、肿瘤直径、淋巴结转移以及远处转移之间均无明显关系(均P0.05)。结论:CD44及CD24在乳腺癌组织内存在较高的阳性率,且CD44~+/CD24~-在乳腺原位癌及低分化的乳腺癌组织内具有更高的阳性率,临床上可尝试通过监测CD44~+/CD24~-的阳性表达情况评价患者的病情及预后。     

Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1⁺ microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32⁺ microglia or macrophages, but an increased CD206⁺ phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1⁺ microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1⁺ microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.     

Histone deacetylase 9 deficiency exaggerates uterine M2 macrophage polarization

The maternal-foetal interface is an immune-privileged site where the semi-allogeneic embryo is protected from attacks by the maternal immune system. Uterine macrophages are key players in establishing and maintaining pregnancy, and the dysregulation of the M1-M2 subpopulation balance causes abortion. We separated two distinct mouse uterine macrophage subpopulations during early pregnancy, CD45⁺F4/80⁺CD206⁻ M1-like (M1) and CD45⁺F4/80⁺CD206⁺ M2-like (M2) cells. The M1 preponderance was significantly exaggerated at 6 hours after lipopolysaccharide (LPS) treatment, and adoptive transfer of M2 macrophages partially rescued LPS-induced abortion. RNA sequencing analysis of mouse uterine M2 versus M1 revealed 1837 differentially expressed genes (DEGs), among which 629 was up-regulated and 1208 was down-regulated. Histone deacetylase 9 (Hdac9) was one of the DEGs and validated to be significantly up-regulated in uterine M2 as compared with M1. Remarkably, this differential expression profile between M1 and M2 was also evident in primary splenic macrophages and in vitro polarized murine peritoneal, bone marrow–derived and RAW 264.7 macrophages. In Hdac9/HDAC9 knockout RAW 264.7 and human THP-1–derived macrophages, the expression of M1 differentiation markers was unchanged or decreased whereas M2 markers were increased compared with the wild-type cells, and these effects were unrelated to compromised proliferation. Furthermore, Hdac9/HDAC9 ablation significantly enhanced the phagocytosis of fluorescent microspheres in M2 Raw 264.7 cells yet decreased the capacity of THP-1-derived M1 macrophages. The above results demonstrate that Hdac9/HDAC9 deficiency exaggerates M2 macrophage polarization in mouse and human macrophages, which may provide clues for our understanding of the epigenetic regulation on macrophage M1/M2 polarization in maternal-foetal tolerance.     

Class II MHC-expressing cells in the rat adrenal gland defined by monoclonal antibodies

 The detailed distribution and heterogeneity of various immunocompetent cells were characterized in the normal adrenal gland of the rat, with special emphasis on major histocompatibility complex (MHC) class II-expressing cells and macrophages. All adrenals contained at least two different populations of cells reactive with the dendritic cell or the macrophage antibodies. These cells were clearly distinguished from adrenal parenchymal cells by their morphology and location. The majority of dendritic cells were immunoreactive for the MHC class II (Ia) antigen (MRC OX6) and/or the dendritic cell antibodies (MRC OX62), and negative for the macrophage antibodies (ED1, ED2, and/or MRC OX42), whereas the main population of macrophages was immunonegative for the former antibodies and positive for the latter. The OX62-positive cells and the OX42-labeled cells occurred exclusively throughout the medulla. The cellular density of dendritic cells in the adrenal cortex was significantly higher than that of macrophages. Double-immunoperoxidase staining for ED1 and OX6 revealed that positively stained cells could be classified into the following categories: ED1⁺OX6⁺, ED1⁺OX6⁻, and ED1⁻OX6⁺. More then 40% of OX6⁺ cells were immunoreactive for ED1 in the zona glomerulosa, while approximately 15%, 20%, and 30% of OX6⁺ cells were positive for ED1 in the zona fasciculata, zona reticularis and medulla, respectively. ED1⁺ED2⁻ cells were more frequently detected in the zona glomerulosa than in other adrenal zones. Only a few ED1⁻ED2⁺ cells were located in the zona glomerulosa, whereas a large number of them were found in the zona fasciculata. In the zona reticularis and medulla, ED1⁺ED2⁺, ED1⁺ED2⁻, and ED1⁻ED2⁺ cells were detected in the ratio 2:1:3. Our rsults suggest that dendritic cells and macrophages mature during their migration within the adrenal gland. These immunocompetent cells may contribute to a paracrine regulation of adrenal function under physiological conditions. Accepted: 3 November 1997     

CD204-positive monocytes and macrophages ameliorate septic shock by suppressing proinflammatory cytokine production in mice

Sepsis is defined as a life-threatening multiorgan dysfunction caused by dysregulated inflammatory response to infection. It remains the primary cause of death from infection if not diagnosed and treated promptly. Therefore, a better understanding of the mechanism for resolving inflammation is needed. Monocytes and macrophages play a pivotal role not only in the induction but also in the suppression of inflammation. However, a tissue-resident macrophage subset that regulates a hyperinflammatory state during sepsis has not been explored. Here we show that CD204⁺ monocytes and/or macrophages rescued mice from endotoxin-induced septic shock. Serum and tissue proinflammatory cytokine levels were significantly upregulated in the absence of these cells. This study provided evidence that CD204⁺ monocytes and/or macrophages ameliorate septic shock by suppressing proinflammatory cytokine production.     

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44^high) and a main population which was either weakly positive or negative for CD44 (CD44^low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44^highCD90⁺ sub-population. Moreover, these CD44^highCD90⁺ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44^highCD90⁺ population a good candidate for the lung CSCs. Both CD44^highCD90⁺ and CD44^highCD90⁻ cells in the PLCCL derived from SCC formed spheroids, whereas the CD44^low/− cells were lacking this potential. These results indicate that CD44^highCD90⁺ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44^high sub-population.     

A multiparameter approach to monitor disease activity in collagen-induced arthritis

Introduction

Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.

Methods

Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.

Results

In the skin from SSc patients, the number of CD163⁺ cells or CD204⁺ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14⁺ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14⁺ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163⁺ cells belong to CD14^brightCD204⁺ population.

Conclusions

This is the first report indicating CD163⁺ or CD204⁺ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14^brightCD163⁺CD204⁺ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163⁺ or CD204⁺ macrophages in the skin.     

T Cell-Dependent Activation of Macrophages and Enhancement of Their Phagocytic Activity in the Lungs of Mice Inoculated with Heat-Killed Cryptococcus neoformans: Involvement of IFN-γ and Its Protective Effect against Cryptococcal Infection

Previous investigations have demonstrated that macrophages play a critical role in the first-line cellular defense mechanism against infection with Cryptococcus neoformans. In the present study, to elucidate the way in which anticryptococcal activity of macrophages is regulated at the site of infection, pulmonary intraparenchymal macrophages were directly analyzed for expression of their surface molecules and their phagocytic activities against the organism, and the effects of depletion of T cells and endogenous IFN-γ in vivo on these parameters were examined. In the lungs of mice intratracheally inoculated with heat-killed C. neoformans, macrophages were activated, as indicated by augmented expression of MHC class II, intercellular adhesion molecule-1 (ICAM-1) and Fc receptor (FcR), and about two-thirds of macrophages were found to have ingested an average of 3.77 ± 0.12 yeast cells per macrophage. In mice depleted of both CD4⁺ and CD8⁺ T cells by injecting the specific monoclonal antibodies (mAbs) or anti-IFN-γ mAb, not only augmentation of the expression of macrophage activation markers but also phagocytosis of C. neoformans was significantly reduced. These results suggest that anticryptococcal activity of macrophages is regulated by IFN-γ endogenously produced by T cells. Additionally, treatment with IFN-γ were shown to significantly prolong the survival time of mice infected with viable C. neoformans. Additionally, preimmunization with heat-killed C. neoformans significantly prolonged the survival time of mice which received the following infection.     

Characterization of monocyte/macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis

Introduction

Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.

Methods

Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.

Results

In the skin from SSc patients, the number of CD163⁺ cells or CD204⁺ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14⁺ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14⁺ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163⁺ cells belong to CD14^brightCD204⁺ population.

Conclusions

This is the first report indicating CD163⁺ or CD204⁺ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14^brightCD163⁺CD204⁺ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163⁺ or CD204⁺ macrophages in the skin.