Soluble CD163 is not increased in visceral fat and steatotic liver and is even suppressed by free fatty acids in vitro

Visceral fat differs from subcutaneous fat by higher local inflammation and increased release of IL-6 and free fatty acids (FFA) which contribute to hepatic steatosis. IL-6 has been shown to upregulate the monocyte/macrophage specific receptor CD163 whose soluble form, sCD163, is increased in inflammatory diseases. Here, it was analyzed whether CD163 and sCD163 are differentially expressed in the human fat depots and fatty liver. CD163 mRNA and protein were similarly expressed in paired samples of human visceral and subcutaneous fat, and comparable levels in portal venous and systemic venous blood of liver-healthy controls indicate that release of sCD163 from visceral adipose tissue was not increased. CD163 was also similarly expressed in steatotic liver when compared to non-steatotic tissues and sCD163 was almost equal in the respective sera. Concentrations of sCD163 were not affected when passing the liver excluding substantial hepatic removal/release of this protein. A high concentration of IL-6 upregulated CD163 protein while physiological doses had no effect. However, sCD163 was not increased by any of the IL-6 doses tested. FFA even modestly decreased CD163 and sCD163. The anti-inflammatory mediators fenofibrate, pioglitazone, and eicosapentaenoic acid (EPA) did not influence sCD163 levels while CD163 was reduced by EPA. These data suggest that in humans neither visceral fat nor fatty liver are major sources of sCD163.

PAPP-A and osteoprotegerin, together with interleukin-8 and RANTES, are elevated in the peritoneal fluid of women with endometriosis

OBJECTIVE: The purpose of this study was to evaluate whether pregnancy-associated plasma protein A, glycodelin, osteoprotegerin, and soluble CD163 are possible peritoneal fluid markers for endometriosis and to compare them with the established chemokine markers interleukin-8 and regulated on activation, normal T-cell expressed and secreted. STUDY DESIGN: Determination of the concentrations of interleukin-8, regulated on activation, normal T-cell expressed and secreted, pregnancy-associated plasma protein A, glycodelin, CD163, osteoprotegerin, and progesterone in the peritoneal fluid collected from women undergoing laparoscopy. RESULTS: From a total of 132 women, 77 women were diagnosed with endometriosis, and 55 women were free of the disease and served as control subjects. Pregnancy-associated plasma protein A and osteoprotegerin showed significantly (P < 0.05) elevated peritoneal fluid concentrations as a function of the severity of the disease, together with interleukin-8 and regulated on activation, normal T-cell expressed and secreted (P < .001). Glycodelin and CD163 did not differ between cases and control subjects. Many of these marker concentrations were intercorrelated strongly. CONCLUSION: Pregnancy-associated plasma protein A and osteoprotegerin may play a role in the inflammation process of endometriosis, but interleukin-8 and regulated on activation, normal T-cell expressed and secreted are superior peritoneal fluid markers.

Alopecia areata universalis elicited during treatment with adalimumab

Adalimumab is a fully humanized recombinant anti-tumour-necrosis-factor (TNF-alpha) monoclonal antibody which has been approved for rheumatoid arthritis, active ankylosing spondylitis, psoriatic arthritis and Crohn's disease. We report a case of alopecia areata (AA) universalis occurring 6 months after administration of adalimumab monotherapy in a patient with a long-standing history of psoriatic arthritis and psoriasis. The diagnosis was confirmed by a scalp biopsy which showed a peribulbar infiltrate of both CD4+ and CD8+ T cells, CD1a+ dendritic cells as well as CD68+ and CD163+ macrophages. In addition, immunofluorescence staining for TNF-alpha was found in the mononuclear cell infiltrate. This case suggests a complex role of TNF-alpha in the induction of AA.

Gene expression in cultured endometrium from women with different outcomes following IVF

Estradiol and progesterone are crucial for the acquisition of receptivity and the change in transcriptional activity of target genes in the implantation window. The aim of this study was to differentiate the regulation of genes in the endometrium of patients with recurrent implantation failure (IF) versus those who became pregnant after in vitro fertilization (IVF) treatment. Moreover, the effect of embryo-derived factors on endometrial transcriptional activity was studied. Nine women with known IVF outcome (IF, M, miscarriage, OP, ongoing pregnancy) and undergoing hysteroscopy with endometrial biopsy were enrolled. Biopsies were taken during the midluteal phase. After culture in the presence of embryo-conditioned IVF media, total RNA was extracted and submitted to reverse transcription, target cDNA synthesis, biotin labelling, fragmentation and hybridization using the Affymetrix Human Genome U133A 2.0 Chip. Differential expression of selected genes was re-analysed by quantitative PCR, in which the results were calculated as threshold cycle differences between the groups and normalized to Glyceraldehyde phosphate dehydrogenase and beta-actin. Differences were seen for several genes from endometrial tissue between the IF and the pregnancy groups, and when comparing OP with M, 1875 up- and 1807 down-regulated genes were returned. Real-time PCR analysis confirmed up-regulation for somatostatin, PLAP-2, mucin 4 and CD163, and down-regulation of glycodelin, IL-24, CD69, leukaemia inhibitory factor and prolactin receptor between Op and M. When the different embryo-conditioned media were compared, no significant differential regulation could be demonstrated. Although microarray profiling may currently not be sensitive enough for studying the effects of embryo-derived factors on the endometrium, the observed differences in gene expression between M and OP suggest that it will become an interesting tool for the identification of fertility-relevant markers produced by the endometrium.

Increased expression of IL-12p70 and IL-23 by multiple dendritic cell and macrophage subsets in plaque psoriasis

BACKGROUND: Psoriasis is a chronic immune-mediated skin disease, in which interleukins 12 and 23 have been postulated to play a critical role. However, the cellular source of these cytokines in psoriatic lesions are still poorly defined and their relative contribution in inducing skin inflammation has been discussed controversially. OBJECTIVES: To investigate immunoreactivity of the bioactive forms of IL-12 and IL-23 in plaque psoriasis and to characterize the dendritic cell (DC) and macrophage subsets responsible for the production of these cytokines. METHODS: Immunohistochemistry was performed on normal skin (n=11) as well as non-lesional (n=11) and lesional (n=11) skin of patients with plaque psoriasis using monoclonal antibodies targeting the bioactive forms of IL-12 (IL-12p70) and IL-23 (IL-23p19/p40) on serial cryostat sections using the alkaline phosphatase-antialkaline phosphatase. Co-localization of IL-12 and IL-23 with different dendritic cells and macrophage cell markers (CD1a, CD11c, CD14, CD32, CD68, CD163, CD208/DC-LAMP) was performed using double immunofluorescence staining. RESULTS: Immunoreactivity for IL-12 and IL-23 was significantly enhanced in lesional psoriatic skin as compared to non-lesional and normal skin. No difference was observed between IL-12 and IL-23 immunoreactivity in any skin types. Both IL-12 and IL-23 immunoreactivity was readily detected mainly in CD11c+, CD14+, CD32+, CD68+ and some CD163+, DC-LAMP+ cells. IL-12 and occasionally IL-23 were also found in some CD1a+ dendritic cells. In addition, an enhanced expression mainly of IL-23 was observed in keratinocytes. CONCLUSIONS: Bioactive forms of IL-12 and IL-23 are highly expressed in various DC and macrophage subsets and their marked in situ production suggest that both cytokines have crucial pathogenic role in psoriasis.

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

Accumulation of FOXP3+T-cells in the tumor microenvironment is associated with an epithelial-mesenchymal-transition-type tumor budding phenotype and is an independent prognostic factor in surgically resected pancreatic ductal adenocarcinoma.

Here we explore the role of the interplay between host immune response and epithelial-mesenchymal-transition (EMT)-Type tumor-budding on the outcome of pancreatic adenocarcinoma (PDAC).CD4+, CD8+, and FOXP3+T-cells as well as iNOS+ (M1) and CD163+- macrophages (M2) were assessed on multipunch tissue-microarrays containing 120 well-characterized PDACs, precursor lesions (PanINs) and corresponding normal tissue. Counts were normalized for the percentage of tumor/spot and associated with the clinico-pathological features, including peritumoral (PTB) and intratumoral (ITB) EMT-Type tumor-budding and outcome.Increased FOXP3+T-cell-counts and CD163-macrophages and decreased CD8+T-cell-counts were observed in PDACs compared with normal tissues and PanINs (p < 0.0001). Increased peritumoral FOXP3+T-cell-counts correlated significantly with venous invasion, distant metastasis, R1-status, high-grade ITB, PTB and independently with reduced survival. Increased intratumoral FOXP3+T-cells correlated with lymphatic invasion, N1-stage, PTB and marginally with adverse outcome. High peritumoral CD163-counts correlated with venous invasion, PTB and ITB. High intratumoral CD163-counts correlated with higher T-stage and PTB.PDAC-microenvironment displays a tumor-favoring immune-cell composition especially in the immediate environment of the tumor-buds that promotes further growth and indicates a close interaction of the immune response with the EMT-process. Increased peritumoral FOXP3+T-cell density is identified as an independent adverse prognostic factor in PDAC. Patients with phenotypically aggressive PDACs may profit from targeted immunotherapy against FOXP3.

Regulation of peripheral classical and non-classical monocytes on infliximab treatment in patients with rheumatoid arthritis and ankylosing spondylitis.

OBJECTIVE To investigate the regulatory effect of tumour necrosis factor (TNF) blockade with infliximab on the distribution of peripheral blood monocyte subpopulations in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). METHODS Purified CD11b+CD14+ monocytes from 5 patients with RA and 5 AS were analysed ex vivo before and after infliximab treatment by flow cytometry for CD16, CD163, CD11b, C-C chemokine receptor type 2 (CCR2) and CXC chemokine receptor 4 (CXCR4) at baseline and at days 2, 14, 84 and 168 after the first infliximab administration. Serum levels of the stromal cell-derived factor (SDF)-1 and monocyte chemotactic peptide (MCP)-1 at different time points were measured in either patient group before and on infliximab treatment. RESULTS Anti-TNF treatment with infliximab led to a significant increase of circulating CD11b+ non-classical and a concomitantly decrease of CD11b+ classical monocytes, to a decline in SDF-1 levels and reduced expression of CCR2 and CXCR4 on non-classical monocyte subpopulation. CONCLUSIONS Our study shows, that TNFα blockade by infliximab resulted in a dichotomy of the regulation of classical and non-classical monocytes that might have substantial impact on inhibition of osteoclastogenesis and of subsequent juxta-articular bone destruction and systemic bone loss in RA and AS.

Systemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation.

Besides its importance in cattle, Neospora caninum may also pose a high risk as abortifacient for small ruminants. We have recently demonstrated that the outcome of experimental infection of pregnant sheep with 10(6) Nc-Spain7 tachyzoites is strongly dependent on the time of gestation. In the current study, we assessed peripheral and local immune response in those animals. Serological analysis revealed earlier and higher IFN-γ and IgG responses in ewes infected at early (G1) and mid (G2) gestation, when abortion occurred. IL-4 was not detected in sera from any sheep. Inflammatory infiltrates in the placenta mainly consisted of CD8+ and, to a lesser extent, CD4+ T cells and macrophages (CD163+). The infiltrate was more intense in sheep infected at mid-gestation. In the foetal mesenchyme, mostly free tachyzoites were found in animals infected at G1, while those infected in G2 displayed predominantly particulate antigen, and parasitophorous vacuoles were detected in sheep infected at G3. A similar pattern of placental cytokine mRNA expression was found in all groups, displaying a strengthened upregulation of IFN-γ and IL-4 and milder increases of TNF-α and IL-10, reminiscent of a mixed Th1 and Th2 response. IL-12 and IL-6 were only slightly upregulated in G2, and TGF-β was downregulated in G1 and G2, suggestive of limited T regulatory (Treg) cell activity. No significant expression of TLR2 or TLR4 could be detected. In summary, this study confirms the pivotal role of systemic and local immune responses at different times of gestation during N. caninum infection in sheep.