Characterization of fetal monocytes in preeclampsia and fetal growth restriction.

Background There is little available data on fetal monocyte phenotype and function. A prospective cross-sectional pilot study was conducted to describe the cord blood monocyte subset phenotype in preeclampsia (PE) and fetal growth restriction (FGR) as compared to normal pregnancy and maternal circulation. Methods Maternal and cord blood samples from 27 pregnancies were collected at delivery from normal pregnancy, PE, FGR and PE+FGR. The distribution of fetal monocyte subtypes was characterized by CD14 and CD16 expression using flow cytometry and compared for each clinical group using a classification of classical, intermediate and non-classical subsets. Results The intermediate monocytes were the dominant monocyte subset in the cord blood of PE and PE+FGR with an increase in the combined inflammatory monocyte subsets intermediate and non-classical in PE compared to normal pregnancy. The non-classical monocyte subset proportion was elevated in all pathological groups PE, FGR and PE+FGR. A significant reduction in the non-classical monocyte subset was observed in the cord blood of the normal pregnancy group as compared to the maternal circulation. Conclusion This study describes for the first time in the fetal circulation, dominant monocyte intermediate subsets and increased inflammatory subsets in PE as well as increased non-classical subsets in PE and FGR compared to normal pregnancy.

Therapeutic potential of regulatory macrophages generated from peritoneal dialysate in adriamycin nephropathy.

Cell therapy using macrophages requires large amounts of cells, which are difficult to collect from patients. Patients undergoing peritoneal dialysis (PD) discard huge numbers of peritoneal macrophages in dialysate daily. Macrophages can be modulated to become regulatory macrophages, which have shown great promise as a therapeutic strategy in experimental kidney disease and human kidney transplantation. This study aimed to examine the potential of using peritoneal macrophages (PMs) from peritoneal dialysate to treat kidney disease. Monocytes/macrophages accounted for >40% of total peritoneal leukocytes in both patients and mice undergoing PD. PMs from patients and mice undergoing PD were more mature than peripheral monocytes/macrophages, as shown by low expression of C-C motif chemokine receptor 2 (CCR2) and morphological changes during in vitro culture. PMs from patients and mice undergoing PD displayed normal macrophage function and could be modulated into a regulatory (M2) phenotype. In vivo, adoptive transfer of peritoneal M2 macrophages derived from PD mice effectively protected against kidney injury in mice with adriamycin nephropathy (AN). Importantly, the transfused peritoneal M2 macrophages maintained their M2 phenotype in kidney of AN mice. In conclusion, PMs derived from patients and mice undergoing PD exhibited conventional macrophage features. Peritoneal M2 macrophages derived from PD mice are able to reduce kidney injury in AN, suggesting that peritoneal macrophages from patients undergoing PD may have the potential for clinical therapeutic application.

Distribution of monocyte subsets and polarization in preeclampsia and intrauterine fetal growth restriction.

AIM: Monocytes are likely to play a significant role in the pathogenesis of preeclampsia (PE) and intrauterine fetal growth restriction (IUGR), given their role in homeostasis and tissue repair. Our aim was to study the gestational changes in monocytes in normal pregnancy and to determine whether monocyte subsets and phenotype are altered in pregnancy complications, such as PE and IUGR. METHODS: A prospective cross-sectional case-control study was conducted. Pregnant women between 24 and 40 weeks of gestation (n = 54) were recruited and classified into four clinical groups of normal pregnancy, PE, IUGR and PE + IUGR. The maternal monocyte subsets classical, intermediate and nonclassical were compared for each clinical group. Monocyte polarization towards M1 (inflammatory) and M2 (repair) phenotypes was assessed by surface expression of CD86 and CD163 ratio, using flow cytometry. RESULTS: The classical monocytes were reduced and intermediate monocyte elevated compared to normal pregnancy in PE, IUGR and PE + IUGR in gestations <37 weeks and IUGR in 26-40 weeks. CD163 expression was increased and CD86/CD163 ratio decreased in IUGR compared to normal pregnancy for all subsets. Nonclassical monocyte counts and CD163 expression increased with advancing gestation in normal pregnancy. CONCLUSION: These results show for the first time, a shift towards increased intermediate maternal monocyte subtype in IUGR and in preterm PE as well as skewing of maternal peripheral monocytes (all subsets) towards M2 phenotype in pregnancies complicated by IUGR.

The pro-fibrotic role of dipeptidyl peptidase 4 in carbon tetrachloride-induced experimental liver injury.

Liver fibrosis is a progressive pathological process involving inflammation and extracellular matrix deposition. Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a cell surface glycoprotein and serine protease. DPP4 binds to fibronectin, can inactivate specific chemokines, incretin hormone and neuropeptides, and influences cell adhesion and migration. Such properties suggest a pro-fibrotic role for this peptidase but this hypothesis needs in vivo examination. Experimental liver injury was induced with carbon tetrachloride (CCl4) in DPP4 gene knockout (gko) mice. DPP4 gko had less liver fibrosis and inflammation and fewer B cell clusters than wild type mice in the fibrosis model. DPP4 inhibitor-treated mice also developed less liver fibrosis. DNA microarray and PCR showed that many immunoglobulin (Ig) genes and some metabolism-associated transcripts were differentially expressed in the gko strain compared with wild type. CCl4-treated DPP4 gko livers had more IgM+ and IgG+ intrahepatic lymphocytes, and fewer CD4+, IgD+ and CD21+ intrahepatic lymphocytes. These data suggest that DPP4 is pro-fibrotic in CCl4-induced liver fibrosis and that the mechanisms of DPP4 pro-fibrotic action include energy metabolism, B cells, NK cells and CD4+ cells.

Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo.

Alternatively activated macrophages (M2) regulate immune responses and ex vivo polarized splenic M2 are able to ameliorate renal injury including models of renal disease, such as adriamycin nephropathy. Whether M2 derived from other organs have similar protective efficacy is unknown. Here, we report adoptively transferred bone marrow M2 macrophages did not improve renal function or reduce renal injury in adriamycin nephropathy, whereas splenic M2 macrophages were protective. Bone marrow and splenic M2 macrophages showed similar regulatory phenotypes and suppressive functions in vitro. Within the inflamed kidney, suppressive phenotypes in bone marrow but not in splenic M2 macrophages, were dramatically reduced. Loss of the suppressive phenotype in bone marrow M2 was related to strong proliferation of bone marrow M2. Bone marrow M2 proliferation in vivo correlated with M-CSF expression by tubular cells in the inflamed kidney. Inhibition of M-CSF in vitro limited bone marrow M2 proliferation and prevented switch of phenotype. Proliferating cells derived from transfused bone marrow M2 were inflammatory rather than regulatory in their phenotype and function. Thus bone marrow in contrast to splenic M2 macrophages do not protect against renal structural and functional injury in murine adriamycin nephropathy. The failed renoprotection of bone marrow M2 is due to the switch of transfused M2 macrophages from a regulatory to an inflammatory phenotype.

Discoidin domain receptor 1: isoform expression and potential functions in cirrhotic human liver.

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell-collagen interactions in chronic liver injury.

Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis.

The dipeptidyl peptidase IV gene family contains the four peptidases dipeptidyl peptidase IV, fibroblast activation protein, dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Dipeptidyl peptidase IV and fibroblast activation protein are involved in cell-extracellular matrix interactions and tissue remodeling. Fibroblast activation protein is upregulated and dipeptidyl peptidase IV is dysregulated in chronic liver disease. The effects of dipeptidyl peptidase 8 and dipeptidyl peptidase 9 on cell adhesion, cell migration, wound healing and apoptosis were measured by using green fluorescent protein fusion proteins to identify transfected cells. Dipeptidyl peptidase 9-overexpressing cells exhibited impaired cell adhesion, migration in transwells and monolayer wound healing on collagen I, fibronectin and Matrigel. Dipeptidyl peptidase 8-overexpressing cells exhibited impaired cell migration on collagen I and impaired wound healing on collagen I and fibronectin in comparison to the green fluorescent protein-transfected controls. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 enhanced induced apoptosis, and dipeptidyl peptidase 9 overexpression increased spontaneous apoptosis. Mechanistic investigations showed that neither the catalytic serine of dipeptidyl peptidase 8 or dipeptidyl peptidase 9 nor the Arg-Gly-Asp integrin-binding motif in dipeptidyl peptidase 9 were required for the impairment of cell survival, cell adhesion or wound healing. We have previously shown that the in vitro roles of dipeptidyl peptidase IV and fibroblast activation protein in cell-extracellular matrix interactions and apoptosis are similarly independent of catalytic activity. Dipeptidyl peptidase 9 overexpression reduced beta-catenin, tissue inhibitor of matrix metalloproteinases 2 and discoidin domain receptor 1 expression. This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell-extracellular matrix interactions, and thus may regulate tissue remodeling.

Efficacy of using cancer stem cell markers in isolating and characterizing liver cancer stem cells.

Recent evidence suggests that a subset of hepatocellular carcinomas (HCCs) are derived from liver cancer stem cells (LCSCs). In order to isolate and characterize LCSCs, reliable markers that are specific to these cells are required. We evaluated the efficacy of a range of cancer stem cell (CSC) markers in isolating and characterizing LCSCs. We show that the most widely used CSC markers are not specific to LCSCs. By western analysis, protein expression of the common markers showed no significant difference between HCC tumor tissues and adjacent non-cancerous liver. Further, isolation of LCSCs from common HCC cell lines using FACScan and microbeads showed no consistent marker expression pattern. We also show that LCSCs have unique subtypes. Immunohistochemistry of HCC tissues showed that different HCCs express unique combinations of LCSC markers. Quantitative real-time polymerase chain reaction analysis showed that LCSCs isolated using different markers in the same HCC phenotype had different expression profiles. Likewise, LCSCs isolated from different HCC phenotypes with the same marker also had unique expression profiles and displayed varying resistance profiles to Sorafenib. Thus, using a range of commonly used CSC markers in HCCs and cell lines, we demonstrate that currently available markers are not specific for LCSCs. LCSCs have unique subtypes that express distinctive combinations of LCSC markers and altered drug resistance profiles, making their identification problematic.

Regulation of dipeptidyl peptidase 8 and 9 expression in activated lymphocytes and injured liver.

AIM: To investigate the expression of dipeptidyl peptidase (DPP) 8 and DPP9 in lymphocytes and various models of liver fibrosis. METHODS: DPP8 and DPP9 expression were measured in mouse splenic CD4⁺ T-cells, CD8⁺ T-cells and B-cells (B220⁺), human lymphoma cell lines and mouse splenocytes stimulated with pokeweed mitogen (PWM) or lipopolysaccharide (LPS), and in dithiothreitol (DTT) and mitomycin-C treated Raji cells. DPP8 and DPP9 expression were measured in epidermal growth factor (EGF) treated Huh7 hepatoma cells, in fibrotic liver samples from mice treated with carbon tetrachloride (CCl4) and from multidrug resistance gene 2 (Mdr2/Abcb4) gene knockout (gko) mice with biliary fibrosis, and in human end stage primary biliary cirrhosis (PBC). RESULTS: All three lymphocyte subsets expressed DPP8 and DPP9 mRNA. DPP8 and DPP9 expression were upregulated in both PWM and LPS stimulated mouse splenocytes and in both Jurkat T- and Raji B-cell lines. DPP8 and DPP9 were downregulated in DTT treated and upregulated in mitomycin-C treated Raji cells. DPP9-transfected Raji cells exhibited more annexin V⁺ cells and associated apoptosis. DPP8 and DPP9 mRNA were upregulated in CCl4 induced fibrotic livers but not in the lymphocytes isolated from such livers, while DPP9 was upregulated in EGF stimulated Huh7 cells. In contrast, intrahepatic DPP8 and DPP9 mRNA expression levels were low in the Mdr2 gko mouse and in human PBC compared to non-diseased livers. CONCLUSION: These expression patterns point to biological roles for DPP8 and DPP9 in lymphocyte activation and apoptosis and in hepatocytes during liver disease pathogenesis.

T cell antigen receptor (TCR) transmembrane peptides colocalize with TCR, not lipid rafts, in surface membranes.

We have previously shown that a synthetic peptide termed core peptide (CP), which corresponds to a sequence within the transmembrane domain of the alpha chain of the T cell antigen receptor (TCR), can inhibit IL-2 production in antigen-stimulated T cells and can suppress inflammation in several T cell-mediated animal models of disease. As the first step in determining the mechanism of CP action, we examined the association of CP with the plasma membrane of human T cells using confocal microscopy. A homogeneous distribution of CP was observed in the plasma membrane of human T cells. This membrane localization was dependent on the presence of positive charges in the CP sequence. CP analogs, containing either neutral or negatively charged amino acids in place of the positive amino acid charges, did not localize within TCR membranes. Following antibody-induced TCR clustering, there was specific colocalization of CP with surface TCR. No association was observed with other cell surface receptors when similarly clustered. Since TCR activation leads to an increased movement of the receptor complex to cholesterol/glycosphingolipid (GSL) plasma membrane microdomains (rafts) we examined whether the association of CP with TCR was raft-driven. TCR clustering led only to a partial colocalization of TCRs with raft GSL, ganglioside GM1, and a complete colocalization of CP with TCRs. We conclude that CP associates specifically with plasma membrane TCRs and not raft lipids.

T-cell antigen receptor peptides inhibit signal transduction within the membrane bilayer.

Previous studies have shown that a synthetic peptide (core peptide, CP) corresponding to a 9-amino-acid region in the transmembrane domain of the alpha subunit of the T-cell antigen receptor (TCR) can suppress T-cell function in vitro and in vivo. The aim of these experiments was to determine the cellular site and molecular mechanism of CP inhibition in T cells. The cytochrome c-sensitive TCR-expressing hybridoma (2B4) was stimulated with pigeon cytochrome c antigen, anti-CD3 crosslinking, or PMA and ionomycin, in the presence or absence of CP, and the resulting IL-2 produced was measured in a bioassay using an IL-2-dependent cell line (CTLL-2). In the presence of CP, IL-2 production was inhibited following antigen-induced stimulation. By contrast, when stimulated with cross-linking antibodies to the CD3 complex or with PMA and ionomycin, both of which activate T cells downstream of the TCR antigen recognition site, CP had no effect on IL-2 production. These experiments suggest that CP interferes with TCR function by inhibiting T-cell activation at the transmembrane/receptor level. In addition, we show that CP inhibits early TCR signal transduction events such as TCR zeta chain phosphorylation following stimulation with either antigen or anti-CD3-crosslinking antibodies, although this is unlikely to be the mechanism leading to the reduced IL-2 production.

Gene expression profiling of alcoholic liver disease in the baboon (Papio hamadryas) and human liver.

The molecular pathogenesis of alcoholic liver disease (ALD) is not well understood. Gene expression profiling has the potential to identify new pathways and altered molecules in ALD. Gene expression profiles of ALD in a baboon model and humans were compared using DNA arrays. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used for downstream analysis of array results. cDNA array analysis revealed differential expression of several novel genes and pathways in addition to genes known to be involved in ALD pathogenesis. Overall gene expression profiles were similar in both species, with a majority of genes involved with fibrogenesis and xenobiotic metabolism, as well as inflammation, oxidant stress, and cell signaling. Genes associated with stellate cell activation (collagens, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinase) were up-regulated in humans. Decreased expression of several metallothioneins was unexpected. Fourteen molecules related to the annexin family were up-regulated, including annexin A1 and A2. Immunofluorescence revealed a marked overexpression of annexin A2 in proliferating bile duct cells, hepatocyte cell surface, and selective co-localization with CD14-positive cells in human ALD. The gene expression profile of ALD is dominated by alcohol metabolism and inflammation and differs from other liver diseases. Annexins may play a role in the progression of fibrosis in ALD.