Assignment of the CD163 antigen (CD163) to mouse chromosome 6 band F2 by radiation hybrid mapping.
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The aim of this immunohistochemical and cytochemical study was to select specific antibodies to establish an efficient purification protocol for first trimester trophoblast and for subsequent purity screening of isolated trophoblast cells. The reactivity of antibodies to various cytokeratin filaments, glycoprotein CD9, fibroblast specific antigen (FSA), common leukocyte antigen CD45RB and macrophage antigens CD163, CD68 and CD14 were studied on cryosections of placental tissue. Among the cytokeratins tested, cytokeratin 7 was the only keratin filament type, which was not expressed in placental mesenchymal cells, but in all trophoblast subpopulations. Since anti-CD9, in addition to mesenchymal cells, also strongly labels extravillous cytotrophoblast cells, whereas the antibody to FSA only reacts with mesenchymal cells, anti-FSA is suitable as a depletion antibody for mesenchymal cells. Among the macrophage markers anti-CD163 was the most specific for Hofbauer cells. CD45RB was expressed on maternal and fetal leukocytes as well as on Hofbauer cells. Isolated first trimester placental cell preparations that have been collected from a density gradient contained up to 45 per cent non-trophoblast cells. Immunocytochemistry using antibodies to CK7, FSA, vimentin, CD45RB and CD163 demonstrated that subsequent immunodepletion with antibodies to CD45RB and FSA increased the purity of the trophoblast preparation to greater than 98 per cent. According to this study trophoblasts from first trimester placentae should be identified by cytokeratin antibodies specific for the isoform 7. Purification of isolated trophoblasts by density gradient alone does not result in a sufficient degree of purity.
An inflammatory process may be involved in nitric oxide production in skeletal muscle of type 2 diabetic patients. Nitric oxide generation in skeletal muscle was assessed in 14 non-complicated type 2 diabetic patients and in 12 healthy subjects. In samples of quadriceps femoris muscle, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nitrite, nitrate and nitrotyrosine were determined. The macrophage-specific antigen CD163, the T-cell membrane factor CD154 and tumour necrosis factor-alpha (TNF-alpha) were also assayed. In six patients, ultrastructural analysis of muscle was performed. Nitrites and nitrates were increased in patients as compared to controls (22.7+/-4.5 and 32.7+/-7.0 vs 16.0+/-2.9 and 22.8+/-4.0 micromol/mg protein; P<0.001, Mann-Whitney U test). Endothelial NOS was similar in diabetic and control subjects (36.4+/-13.8 vs 36.3+/-6.8 ng/mg protein), contrasting with the significant increase of iNOS recorded in patients (34.3+/-13.0 vs 8.5+/-2.8 ng/mg protein, P<0.00002). Nitrotyrosine levels were higher in the patient than in the control group (42.1+/-24.4 vs 10.3+/-2.5 ng/mg protein, P<0.00002), as were CD163 (10-fold) and TNF-alpha (fourfold) levels. Furthermore, CD154 levels were detectable only in the patient samples (10.2+/-5.3 ng/mg protein). By multiple-regression analysis, changes in glycated haemoglobin values could predict 96% variation in nitrotyrosine. Macrophages were present in all muscle samples analysed by electromicroscopy. The increased levels of CD163, CD154 and TNF-alpha indicate that an inflammatory process occurs in skeletal muscle of type 2 diabetic patients. This may contribute to iNOS induction, muscle damage and insulin resistance.
CD163, also referred to as M130, a member of the scavenger receptor cysteine-rich family (SRCR) is exclusively expressed on cells of the monocyte lineage. In freshly isolated monocytes the CD14bright CD16+ monocyte subset revealed the highest expression of CD163 among all monocyte subsets. CD163 mRNA and protein expression is up-regulated during macrophage colony-stimulating factor (M-CSF)-dependent phagocytic differentiation of human blood monocytes. In contrast, monocytic cells treated with GM-CSF and interleukin-4 (IL-4) for dendritic differentiation down-regulate this antigen. CD163 expression is also suppressed by proinflammatory mediators like lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and tumor necrosis factor alpha, whereas IL-6 and the antiinflammatory cytokine interleukin-10 (IL-10) strongly up-regulate CD163 mRNA in monocytes and macrophages. The effects of the immunosuppressants dexamethasone, cyclosporin A (CA), and cortisol differ in their capacity to influence CD163 mRNA levels. Our results demonstrate that CD163 expression in monocytes/macrophages is regulated by proinflammatory and antiinflammatory mediators. This expression pattern implies a functional role of CD 163 in the antiinflammatory response of monocytes.
BACKGROUND: Although several immunological abnormalities have been demonstrated in Behçet's disease (BD), the exact mechanism of the inflammatory changes occurring is still unknown. Antigen-presenting cells, such as mononuclear phagocytes, play a major role in the regulation of immune-mediated as well as of non-specific inflammation. OBJECTIVE: To investigate the serum activity of patients with BD on antigen and chemokine expression of human macrophages in vitro. METHODS: Serum of 15 patients (8 women, 7 men; mean age 33 +/- 10 years) with BD was incubated with cultured macrophages isolated from peripheral blood of healthy volunteers. Macrophages maintained in patients' serum, fetal calf serum with/without dexamethasone and interleukin (IL)-4 or gamma-interferon were investigated for alternative macrophage-activation-associated CC-chemokine 1 (AMAC-1) and IL-8 mRNA expression by Northern blotting. In addition, cytocentrifuge macrophage preparations were stained with monoclonal antibodies against proteins indicating alternative (anti-inflammatory) macrophage activation, such as MS-1 high-molecular-weight protein (MS-1-HMWP), RM3/1 antigen (CD163) and 25F9, as well as classical (pro-inflammatory) macrophage activation, such as CD11c, class I receptor binding the Fc part of IgG (FcgammaRI: CD64) and class III receptor binding the Fc part of IgG (FcgammaRIII: CD16). RESULTS: Macrophages treated with patients' serum showed neither AMAC-1 expression nor staining with monoclonal antibodies for MS-1-HMWP, CD163 or 25F9. Concomitant treatment with IL-4/dexamethasone up-regulated significantly the expression of CD163. In contrast, IL-8 mRNA expression and staining for CD11c and CD64 were strongly positive after treatment with serum of patients with BD. CD64 positivity and IL-8 mRNA expression were more prominent in patients with active BD than in patients with inactive disease. CONCLUSION: Taken together, our results indicate that serum of patients with BD induces classical (pro-inflammatory) activation of human peripheral blood macrophages in vitro. Our findings suggest that serum factor(s) may be responsible for inflammatory changes in BD.
The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5-50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10- monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10- monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10- cells. LPS-stimulated 2A10+ and 2A10- monocytes both produce proinflammatory cytokines (TNF-alpha and IL-1alpha), but antiinflammatory IL-10 is only detected on the latter population. When 2A10- monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10- circulating monocytes.
The extracellular moiety of the hemoglobin/haptoglobin scavenger receptor CD163 (RM3/1 antigen) can be shed from monocytes and is a normal plasma component. We found that in a dose-dependent manner soluble CD163 induces a decrease in CD69 expression, a reduced [(3)H]thymidine uptake and a down-regulated matrix metalloproteinase-9 RNA expression in phorbol myristate acetate-stimulated T-cells. Co-culturing T-cells on transgenic fibroblasts, expressing membrane-bound CD163, yielded no differences compared to culture on non-transfected cells. We conclude that CD163 has at least two distinct functions: the clearance of hemoglobin in its cell-bound form and participation in anti-inflammation as a soluble factor, exhibiting cytokine-like functions.
Swine monocytes constitute a heterogeneous population of cells which can be divided into four subsets based on the expression of SWC3, CD14, CD163 and swine leucocyte antigen (SLA) DR markers. These subsets appear to represent different maturation stages in a pathway along which these cells up-regulate the expression of SLA DR and CD163 antigens and reduce that of CD14. Differences in the expression of adhesion and costimulatory molecules are also patent, with a progressive increase in the expression of CD11a, wCD11R1, CD29, CD49d, CD61, CD1a and CD80/86, and a concomitant decrease in that of wCD11R2. Besides, these subsets differ in their capacity for tumour necrosis factor-alpha (TNF-alpha) production in response to lipopolysaccharide + interferon-gamma. The CD163(+) CD14(-) SLA DR(+) subset produces higher amounts of TNF-alpha than the CD163(-) CD14(+) SLA DR(-) subset, whereas CD163(+) CD14(+) SLA DR(+) and CD163(-) CD14(+) SLA DR(+) subsets show intermediate values. CD163(+) monocytes also display a higher ability to present soluble antigens to T cells than CD163(-) monocytes.
We have recently described the existence of two subsets of porcine monocytes based on the expression of CD163. In this study we compare the expression of a number of cell surface antigens in CD163+ and CD163- monocyte subsets using three-color flow cytometry. These monocyte subsets show differences with respect to the expression of MHC class II antigens (SLA-DR and DQ) and a variety of adhesion molecules (CD11a, wCD11c, wCD29, CD49d) that are expressed at higher levels on CD163+ monocytes, and of CD14 that is higher expressed by CD 163- cells. These differences on phenotype could reflect differences in the ability of these two subsets to migrate to tissues and may account for the higher allostimulatory capacity of CD163+ cells. In some aspects, the phenotype of CD163+ monocytes resembles that of mature macrophages. In vitro serum-induced maturation of monocytes into macrophages lead to the expression of SWC9 together with an increase in the expression of CD163 and a reduction in that of CD14. These results delineate a maturation pathway where CD14hiCD163-SWC9- monocytes develop into CD14loCD163+SWC9- monocytes and these cells into CD14loCD163+SWC9+ macrophages.
BACKGROUND: The immunomodulating effects of both immunosuppressive and nifedipine medication have been associated with drug-induced gingival overgrowth. The aim of the study reported here was to evaluate the presence of macrophage subpopulations in normal human gingiva and in gingival overgrowth induced by nifedipine and immunosuppressive medication. METHODS: Gingival samples were taken from 11 nifedipine-medicated cardiac outpatients (nifedipine group), 11 triple-medicated organ-transplant recipients also taking nifedipine (immunosuppression plus nifedipine group), 12 triple-medicated organ-transplant recipients (immunosuppression group), and 20 generally healthy individuals (control group). Cryostat sections were stained with mAbs for inflammatory 27E10, reparative RM3/1, and resident 25F9 macrophages using an avidin-biotin enzyme complex method. Total numbers of mAb-labeled cells were determined in connective tissue beneath sulcular epithelium, connective tissue beneath oral epithelium, and middle connective tissue. Expression of 27E10 was determined in keratinocytes in the oral epithelium. Statistics analyses were undertaken using the chi-square test, the Mann-Whitney U test, the independent samples t test, analysis of variance, and analysis of covariance. RESULTS: Greater numbers of inflammatory 27E10-positive macrophages were found in all 3 medicated groups and counting zones than in the control group except in connective tissue beneath sulcular epithelium in the immunosuppression group. The incidence of specimens expressing 27E10 antigen throughout the oral epithelium was significantly higher in the immunosuppression group (8 of 12) than in the control group (4 of 20) and the nifedipine group (2 of 11). Numbers of reparative RM3/1-positive macrophages were significantly greater in the immunosuppression group in connective tissue beneath oral epithelium than in the control group. The effect was markedly associated with degree of inflammation. Numbers of resident 25F9-positive macrophages were lower in connective tissue beneath sulcular epithelium in the immunosuppression group, and higher in middle connective tissue in the nifedipine group than in the control group. CONCLUSION: Our results show that the nature of drug-induced gingival overgrowth differs somewhat between immunosuppressive and nifedipine medications.
Bovine milk and meat factors (BMMFs) are plasmid-like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co-markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor-adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n = 246)], low-/high-grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co-immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor-adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68+/CD163+ macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD-adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC-specific death were increased for higher Rep expression (TMA), with high tumor-adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF-specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC.
PURPOSE: Upon glucocorticoid stimulation, human mononuclear leucocytes express an antigen, RM3/1, which characterizes a subpopulation of human monocytes and macrophages evolving in late phase of inflammation. We investigated biochemical properties of the RM3/1 antigen and correlations between antigen expression and glucocorticoid potency. METHODS: Biochemical properties were analyzed after solubilization by immunoaffinity methods and SDS-PAGE. RESULTS: Induction of the RM3/1 antigen is a glucocorticoid receptor mediated process, in contrast, inflammatory mediators such as LPS or TPA were not able to upregulate RM3/1 expression. After SDS-PAGE, the antigen appeared as a 130 kDa (nonreduced)/150 kDa (reduced) glycoprotein with a 25 kDa N-linked glycoportion. The interdependence between antigen density and glucocorticoid efficacy was assessed by calculation of relative antigen expression induced by dexamethasone, fluticasone propionate, budesonide, triamcinolone acetonide, flunisolide, beclomethasone, prednisolone and triamcinolone. Relative antigen expression was significantly correlated with the relative receptor affinity of the glucocorticoid. CONCLUSIONS: We described biochemical properties of the glucocorticoid-induced protein RM3/1. Though the precise role of the RM3/1 antigen in the antiinflammatory process is not fully understood yet, an useful application of the induced expression could already be demonstrated for pre-clinical screening of glucocorticoid potency.
AIMS: Rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVNS) are aggressive diseases with progressive joint destruction. The present study aims to define cell cycle phases, polyploidy and the immunophenotype of proliferating synovial cells in both diseases. METHODS AND RESULTS: Synovial tissues from patients with proliferative-active RA, localized and diffuse PVNS were analysed by DNA flow cytometry, immunohistochemistry and double immunofluorescence with confocal laser scan microscopy. Expression of macrophage markers (CD68/CD163), fibroblast markers (h4Ph/CD55) and Ki67 antigen was examined. Synovial cells positive for either macrophage or fibroblast markers as well as double-labelled cells were found in both RA and PVNS. In RA, CD68/CD163+ synoviocytes were preferentially located in the vicinity of the synovial lining layer, while they were more randomly distributed in PVNS. Of cases with diffuse PVNS, 20% showed an aneuploid cell pattern. All samples of localized PVNS and RA were diploid. Proliferative activity was significantly higher in aneuploid PVNS. CONCLUSIONS: In spite of their histologically homogeneous appearance, proliferating synovial cells display a heterogeneous immunophenotype in both RA and PVNS, indicating functional properties of both macrophages and fibroblasts. Aneuploidy seems to be a special feature of diffuse PVNS.
The functions of different populations of peripheral blood monocytes in the course of an inflammatory reaction are presently not fully understood. In particular, the mechanisms for their specific recruitment to an inflammatory site are not yet known. We investigated a dexamethasone (Dex)-inducible monocyte subtype and its adhesion to either unstimulated or lipopolysaccharide (LPS)- or interferon (IFN)-gamma-stimulated human umbilical vein endothelial cells (HUVEC). The Dex-induced monocytes were characterized by the expression of the surface glycoprotein RM3/1. It was found that pretreatment of monocytes with Dex increased their adhesion to unstimulated and stimulated HUVEC. This increase in adhesion was paralleled by the expression of the RM3/1 surface molecule. Treatment with cyclosporin A (CsA) caused a down-regulation of the RM3/1 density per cell by 67% and also decreased adhesion to HUVEC. In contrast, the expression of other adhesion molecules remained unaffected by Dex or CsA treatment. Treatment of Dex-induced monocytes with antibodies against RM3/1, CD11a, CD11b, CD11c, CD14 and CD18 resulted in suppression of adhesion to stimulated HUVEC by 46%, 18%, 17%, 12%, 25% and 15%, respectively. Antibodies to the known adhesion molecules on endothelial cells (vascular cell adhesion molecule-1, E-selectin) did not block adhesion of Dex-induced monocytes. However, the combination of antibodies to RM3/1 and CD14 inhibited adhesion to LPS-stimulated HUVEC by 74%. These effects were also seen on IFN-gamma-stimulated HUVEC, where adhesion of Dex-induced monocytes was blocked with antibodies to RM3/1 + CD14 by 63%. From this, it is concluded that the RM3/1-molecule is a novel surface molecule that contributes to the adhesion of cortisone-induced monocytes to LPS or cytokine-stimulated endothelial cells.
Mollaret's meningitis is a rare disease with a characteristic clinical course and distinctive cerebrospinal fluid (CSF) cytology. Although Mollaret originally described the large mononuclear cells seen in the CSF as endothelial, subsequent ultrastructural and immunocytochemical studies support a monocyte/macrophage lineage for these cells. To data, the pathogenesis of this entity remains uncertain, although an association with herpes simplex virus (HSV) has been reported in rare cases. In the current case study, immunocytochemistry for factor VIII-related antigen, leukocyte common antigen, and macrophage-specific antigen were performed and provide additional evidence of a monocyte/macrophage lineage for Mollaret cells. Polymerase chain reaction amplification for HSV DNA was done to further explore one possible etiology for this disease, but was negative.
The primary purpose of this study was to describe the expression pattern of the surface antigens CD68, CD36, 27E10, G16/1, and RM3/1 on bronchoalveolar lavage (BAL) cells of smokers and nonsmokers. We found a cell type, morphologically similar to lymphocytes, which showed a strong expression of the monocyte markers CD68 and CD36. We therefore recognized these cells as plasmacytoid monocytes (PM). Hereby we report the appearance of PM in the BAL and its dependency of smoking. The study was conducted in 40 patients with various lung diseases and normal subjects. Sixteen patients and normal subjects were smokers and 24 were nonsmokers. There was a significant increase of PM in the BAL of smokers compared to nonsmokers. However we could not find a disease-related increase of PM in the BAL. This is the first report demonstrating the occurrence of PM in the BAL. Our data support the monocytic origin of PM and suggest an involvement in T cell-mediated responses of the lung.
OBJECTIVE: To investigate the number, location, and secretory products of macrophages in human testes showing normal and abnormal spermatogenesis. DESIGN: Evaluation of testicular biopsies with the use of immunohistochemistry, laser capture microdissection, and reverse transcriptase polymerase chain reaction. SETTING: University research and clinical institutes. PATIENT(S): Infertile men with germ cell arrest (n = 10), Sertoli cell only (n = 8), or mixed atrophy (n = 7) syndromes, and with cases of idiopathic infertility showing normal spermatogenesis (n = 8). INTERVENTION(S): Diagnostic testicular biopsy was performed on participants. MAIN OUTCOME MEASURE(S): We recorded the location, number, distribution, and cytokine expression of human testicular macrophages. RESULT(S): CD68-positive macrophages were found in the testes of all groups analyzed. These macrophages expressed the genes for interleukin 1 and tumor necrosis factor-alpha, and were located in the interstitium, tubular wall, and tubular lumen. In Sertoli cell only and germ cell arrest syndromes, the overall macrophage number was increased over twofold. In all pathologic states, there was a significant shift of these cells from the interstitium to the tubules. CONCLUSION(S): Our study suggests that increased numbers of CD68-positive macrophages directly (via phagocytosis) or indirectly (via paracrine actions exerted through their secretory products) are involved in the regulation of steroidogenesis, Sertoli cell activity, germ cell survival, and, in consequence, in the pathogenesis or maintenance of infertility states in the human testes.