Effect of IFN-γ and IL-4 levels on the expression of Fas and Bcl-2 in peripheral blood lymphocytes in hemodialysis patients

To study the correlation between the levels of IFN-γ and IL-4 and the expression of Fas and Bcl-2 in peripheral blood lymphocytes (PBL) in hemodialysis patients, the indirect immune fluorescein labeling method of flow cytometry and solid sandwich enzyme-linked immunosorbent assay were performed for detecting the expression of Fas and Bcl-2 in PBL and the levels of IFN-γ and IL-4 in the serum of 30 hemodialysis patients, respectively. It was found that the expression of Fas in PBL and the level of IL-4 in the serum of hemodialysis patients were significantly higher (P < 0.01), whereas Bcl-2 in PBL and IFN-γ in the serum were significantly lower (P < 0.01) than those of the normal controls. According to statistical analysis, the expression of Fas in PBL had a negative correlation with the level of IFN-γ, but a positive correlation with IL-4 in the serum of hemodialysis patients. Contrarily, the expression of Bcl-2 had a positive correlation with IFN-γ, but a negative correlation with IL-4 in the serum of hemodialysis patients. These results suggest that hemodialysis patients have a suppressed secretion of Th1-associated cytokine IFN-γ, but an increased secretion of Th2-associated cytokines IL-4, and these two aspects may play an important role in the abnormal apoptosis of PBL and its accompanying immune deficiency.     

Nestin expression in Müller glial cells in postnatal rat retina and its upregulation following optic nerve transection

This study examined the nestin immunoexpression and its specific cellular localization in the developing retina of rats and investigated its putative changes in an altered environment. At postnatal day 0, nestin immunoexpression was detected in radially oriented cells considered to be neural progenitors that were glutamine synthetase (GS) negative. With age, it was localized in differentiating and differentiated GS positive Müller glial cells. Nestin expression was down-regulated as maturation proceeded, so that by 12 weeks, it was almost completely diminished as confirmed also by real time-polymerase chain reaction analysis. Nestin expression along with that of glial fibrillary acidic protein (GFAP) was induced and upregulated in mature Müller glial cells following optic nerve transection. It is suggested that both nestin and GFAP may be useful biomarkers in retinal injuries. In view of their cytoskeletal nature, the marked expression of nestin and GFAP may provide a structural support for the framework of retina which would be disrupted as a result of loss of neurons in optic nerve lesion. It may also be neuronal protective taking into consideration the close spatial and functional links between Müller glial cells and the axotomized ganglion cells.     

Borrelia burgdorferi infection regulates CD1 expression in human cells and tissues via IL1-β

The appearance of group 1 CD1 proteins (CD1a, CD1b and CD1c) on maturing myeloid DC is a key event that converts myeloid DC to effective lipid APC. Here, we show that Borrelia burgdorferi, the causative agent of Lyme disease, triggers appearance of group 1 CD1 proteins at high density on the surface of human myeloid DC during infection. Within human skin, CD1b and CD1c expression was low or absent prior to infection, but increased significantly after experimental infections and in erythema migrans lesions from Lyme disease patients. The induction of CD1 was initiated by borrelial lipids acting through TLR-2 within minutes, but required 3 days for maximum effect. The delay in CD1 protein appearance involved a multi-step process whereby TLR-2 stimulated cells release soluble factors, which are sufficient to transfer the CD1-inducing effect in trans to other cells. Analysis of these soluble factors identified IL-1β as a previously unknown pathway leading to group 1 CD1 protein function. This study establishes that upregulation of group 1 CD1 proteins is an early event in B. burgdorferi infection and suggests a stepwise mechanism whereby bacterial cell walls, TLR activation and cytokine release cause DC precursors to express group 1 CD1 proteins.     

Diminished expression of CD59 on activated CD8+ T cells undergoing apoptosis in systemic lupus erythematosus and Sjögren's syndrome

The present study was undertaken to examine the phenotype of T cells undergoing in vitro apoptosis in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (SS). Compared with normal controls, we found diminished expression of CD59 antigen (one of the cell‐surface complement‐regulatory proteins) on CD8⁺ T cells, but not on CD4⁺ T cells, from patients with SLE and SS. Three‐colour immunofluorescence analysis revealed that these CD59^dim CD8⁺ T cells were activated T cells, expressing both human leucocyte antigen (HLA)‐DR and CD45RO antigens. In addition, these CD59^dim CD8⁺ T cells were more susceptible to in vitro apoptosis than CD59^bright CD8⁺ T cells. In two patients with active lupus, the percentage of CD59^dim CD8⁺ T cells was significantly decreased after steroid therapy. These findings suggest that decreased expression of CD59 antigen on in vivo‐activated CD8⁺ T cells may be correlated with disease activity and may be involved in activation‐induced apoptosis in patients with SLE and SS.     

The expression of CD25, CD11b, SWC1, SWC7, MHC-II, and family of CD45 molecules can be used to characterize different stages of γδ T lymphocytes in pigs

The expression of selected molecules was chosen to study porcine γδ lymphocytes and their CD2/CD8 subsets in different lymphoid organs in vivo and in vitro. Results indicate that many γδ T cells can constitutively express CD25 and MHC-II and that the frequency of γδ T cells positive for CD25, CD11b, SWC1 and SWC7 can be increased by stimulation. A diversified TCRδ repertoire was found inside CD25⁺, CD11b⁺, SWC1⁻ and CD45RA⁻ cells. Ontogenetic studies revealed various age and/or colonization dependency for expression of all studied molecules except of SWC7. Findings generally indicate that CD25 represent an activation molecule that probably marks a functionally distinct subsets, expression of CD11b is perhaps connected to early functions of naive γδ T cells in the periphery, SWC1 is lineage specific marker, SWC7 may represent an activation molecule with intrinsic or transient expression, and the expression of CD45RA/RC most likely defines naive and terminally differentiated cells.     

IL-2-mediated augmentation of NK-cell activity and activation antigen expression on NK- and T-cell subsets in patients with metastatic melanoma treated with interferon-α and DTIC

Considering that well-defined and comprehensive immunological monitoring is the basis for the evaluation of the obtained immunmodulatory effects, we evaluated NK-cell activity, the number of CD3+CD4+, CD3+CD8+ T cells and CD16+CD56+ NK cells, as well as the expression of activation antigens, CD69, CD38 and HLA-DR on CD56+ NK cells, CD8+ and CD3+ T cells, simultaneously with IL-2 and TNF-α production, during chemoimmunotherapy with dacarbazine (DTIC) and interferon-α (IFN-α) in 39 patients with metastatic melanoma. In the first cycle of therapy, there was a significant rise in NK-cell activity, CD4+ T helper cell number, CD4/CD8 T-cell ratio, and the expression of activation antigens CD69 and CD38, on NK and T cells, respectively. However, in the following cycles there was a significant increase only in activation antigens without an increase in the percent or activity of NK cells. The early, but transient, immunopotentiation, present only in the first cycle of combined DTIC and IFN-α therapy, suggests that, in spite of increased IL-2 level, associated with augmented NK-cell activity, this therapy has a limited effect probably owing to the adverse effect of persistently high level of TNF-α in metastatic disease. This revised version was published online in July 2006 with corrections to the Cover Date.     

Functional expression and characterization of grass carp IL-10: An essential mediator of TGF-β1 immune regulation in peripheral blood lymphocytes

In mammals, interleukin-10 (IL-10) is known as a potent regulatory cytokine with pleiotropic effects on various leukocytes. Although teleost IL-10 has been identified in several fish species, its immunoregulatory role is still poorly understood. In the present study, grass carp IL-10 (gcIL-10) has been identified and recombinant gcIL-10 (rgcIL-10) was prepared by using a prokaryotic expression system. Using grass carp peripheral blood lymphocytes (PBLs) as a cell model, rgcIL-10 was shown to up-regulate the cell activity, which was consistent with the regulatory tone of transforming growth factor β1 (TGF-β1). An interesting question that follows would be whether IL-10 and TGF-β1 redundantly regulate PBL activity. To address this question, we investigated the effect of IL-10 on the cell activity of grass carp PBLs challenged by TGF-β1 using immunoneutralization, showing that removal of endogenous IL-10 could abolish TGF-β1-induced cell activity, indicating an essential role of IL-10 in TGF-β1 immune regulation. Furthermore, to examine the connection between TGF-β1 and IL-10 during immunoregulatory process, effect of TGF-β1 on IL-10 expression was investigated. Results showed that TGF-β1 displayed stimulatory effects on gcIL-10 mRNA expression and protein secretion in the same cell model, suggesting that TGF-β1 may function through its effect on IL-10 production in fish immunity. These results provide clues to the potential immunoregulatory role and production of IL-10 in teleost.     

The differential effects of IL-1 and TNF-α on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells

Objective and design:Interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF- on gene expression in the human chondrosarcoma cell line, SW1353. Materials and methods:SW1353 cells were stimulated singularly with IL-1, TNF-, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. Results:Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-. The expression pattern of proIL-1, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1 is produced in the proform in IL-1-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. Conclusions:IL-1 and TNF- each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1 appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.Received 15 September 2003; returned for revision 16 October 2003; accepted by J. S. Skotnicki 11 March 2004     

Altered expression of CD44 and DKK1 in the progression of Barrett’s esophagus to esophageal adenocarcinoma

Barrett’s esophagus (BE) is an acquired condition in which the normal lining of the esophagus is replaced by intestinal metaplastic epithelium. BE can evolve to esophageal adenocarcinoma (EAC) through low-grade dysplasia (LGD) and high-grade dysplasia (HGD). The only generally accepted marker for increased risk of EAC is the presence of HGD, diagnosed on endoscopic biopsies. More specific markers for the prediction of EAC risk are needed. A tissue microarray was constructed comprising tissue samples from BE, LGD, HGD, and EAC. Marker expression was studied by immunohistochemistry using antibodies against CD44, DKK1, CDX2, COX2, SOX9, OCT1, E-cadherin, and β-catenin. Immunostaining was evaluated semi-quantitatively. CD44 expression decreased in HGD and EAC relative to BE and LGD. DKK1 expression increased in HGD and EAC relative to BE and LDG. CDX2 expression increased in HGD but decreased in EAC. COX2 expression decreased in EAC, and SOX9 expression increased only in the upper crypt epithelial cells in HGD. E-cadherin expression decreased in EAC. Nuclear β-catenin was not significantly different between BE, LGD, and HGD. Loss of CD44 and gain of DKK1 expression characterizes progression from BE and LGD to HGD and EAC, and their altered expression might indicate an increased risk for developing an EAC. This observation warrants inclusion of these immunohistochemically detectable markers in a study with a long patient follow-up.     

The effects of intensive, moderate and downhill treadmill running on human blood lymphocytes expressing the adhesion/activation molecules CD54 (ICAM-1), CD18 (β2 integrin) and CD53

This study examined the effects of intensive, moderate and downhill treadmill running on blood lymphocyte expression of adhesion/activation (AA) molecules. Trained subjects completed three treadmill-running protocols of identical duration: (1) an intensive protocol at 80% to volitional exhaustion, (2) a moderate protocol at 60% and (3) a −10% downhill (eccentric) protocol at 80% . Blood samples were taken before, immediately after, 1 and 24 h after exercise. Isolated lymphocytes were assessed for expression of the AA molecules CD54, CD18 and CD53 by flow cytometry. Lymphocyte counts increased immediately after all running protocols. Lymphocytopenia was observed 1 h after the intensive and eccentric protocols only. Plasma creatine kinase increased 24 h after the downhill protocol only. Increases in the number and percentage of CD54⁺, CD18^bright and CD53^bright lymphocytes were observed immediately after the intensive and eccentric protocols, with the numbers falling below pre-exercise values at 1 h post-exercise for all protocols. No differences were found between the intensive protocol and the eccentric protocol at the same relative intensity. Analysis of lymphocyte subsets showed that the total number of CD3⁺, CD4⁺, CD8⁺ and CD56⁺ lymphocytes increased after the intensive protocol before falling below pre-exercise values at 1 h post-exercise. A relatively greater mobilisation of CD56⁺ and CD8⁺ cells accounts for the changes in CD54⁺, CD18^bright and CD53^bright cell populations. Lymphocytes that enter and exit the circulation following exercise express high levels of AA molecules, which may mediate extravasation and post-exercise lymphocytopenia. This effect appears to be influenced by exercise intensity and not muscle damage.     

Effects of myostatin deficiency on PGC-1α and FNDC5 expression in three different murine muscle types

Myostatin (MSTN) is a key negative regulator of muscle growth and development. Skeletal, cardiac, and smooth muscles were isolated from MSTN knockout (MSTN?∕?) and control mice to investigate the effect of knocking out MSTN on peroxisome proliferator-activated receptor 1 coactivator (PGC-1α)-III and fibronectin domain 5 (FNDC5) expression. Various molecular biology techniques were used to analyze the changes in PGC-1α-FNDC5 in different muscle types from MSTN?∕? mice. The expression levels of PGC-1α and FNDC5 in the skeletal, cardiac, and smooth muscles of MSTN?∕? mice differed from those in the skeletal, cardiac, and smooth muscles of normal mice. This study revealed that knocking out MSTN resulted in inconsistent PGC-1α and FNDC5 expression in specific muscles. It proved for the first time that MSTN deletion attenuated the expression of PGC-1α and FNDC5 in three different murine muscle types. MSTN deletion may have additional effects on the status ofFNDC5 expression. Further research, however, is needed to confirm this conclusion.     

Effects of interleukin-1β and tumor necrosis factor-α on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro

Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1) and tumor necrosis factor-alpha (TNF-) for their effects on osteoblastic proliferation and development and expression of alkaline phosphatase and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1 and TNF- were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1 and TNF- were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)₂-vitamin D₃, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF- stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1 and TNF- failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1 and l nM TNF-) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.     

Function and expression of CD1d and invariant natural killer T‐cell receptor in the cotton rat (Sigmodon hispidus)

The cotton rat (Sigmodon hispidus) belongs to the rodent family of Cricetidae and provides a powerful model to study the pathogenesis of human respiratory viruses and measles virus. Recent studies in other rodent models have suggested a role for invariant natural killer T (iNKT) cells in antiviral immunity and vaccination against respiratory virus infections. Using new experimental tools, we provide the first evidence for a functional CD1d cell molecule (crCD1d) and iNKT T-cell receptor in cotton rats. The crCD1d cDNA sequence was identified and crCD1d transductants showed that monoclonal antibody WTH-2 stains crCD1d as efficiently as mouse or rat CD1d. The expression of crCD1d was clearly weaker for thymocytes and B cells, and higher for T cells, which is different to what is found in murine species. The antigen-presenting capacity of crCD1d was demonstrated with crCD1d–immunoglobulin dimers loaded with the glycolipid PBS57, which bound iNKT T-cell receptors. Evidence for functional cotton rat iNKT cells was provided by detection of interferon-γ and interleukin-4 in cultures of splenocytes stimulated with PBS57 and α-galactosylceramide and by specific staining of about 0·2% of splenocytes with PBS57-loaded crCD1d dimers. Canonical AV14/AJ18 rearrangements were identified and found to contain multiple members of the AV14 (AV11) family. One of them was expressed and found to bind CD1d dimers. In summary, these data provide the first evidence for functional CD1d molecules and iNKT T-cell receptors in cotton rats and provide the tools to analyse them both in the cotton rat model of infectious diseases.     

Anastrozole and RU486: Effects on estrogen receptor α and Mucin 1 expression and correlation in the MCF-7 breast cancer cell line

Anastrozole and RU486 are shown to reduce hormone-responsive breast cancer progression when used as adjuvant treatments to surgical intervention, however, a high incidence of cancer recurrence remains. Estrogen receptor alpha (ERα) and Mucin 1 (MUC1), a glycoprotein, are both implicated in breast cancer progression. We assessed whether Anastrozole and RU486 treatment affects the expression of, and relationship between, ERα and MUC1 in the ERα⁺ MUC1⁺ MCF-7 breast cancer cell line. MCF-7 cells, treated with physiological concentrations of either Anastrozole or RU486 for 18 h or 72 h, were subjected to immunolocalization of both markers. CellProfiler software was used to quantify intensity for statistical analyses. ERα expression increased at both time periods following treatment. MUC1 expression increased with RU486-treatment at both times, whereas Anastrozole induced increased MUC1 expression at 72 h only. The biomarkers demonstrated increased point association at 72 h within treatment groups despite MUC1 diverging from correlation with ERα. We propose that tumor progression is independent of MUC1 and ERα correlation. These preliminary results indicate that withdrawal of adjuvant treatment may result in residual cell populations expressing increased ERα and MUC1. This phenotype may allow enhanced estrogenic and metastatic capacity influencing cancer recurrence, a hypothesis we are investigating further.     

Inhibition of histone deacetylases antagonized FGF2 and IL-1β effects on MMP expression in human articular chondrocytes

Fibroblast growth factor-2 (FGF2) and interleukin-1β (IL-1β) stimulate the expression of matrix metalloproteinases (MMPs) in articular chondrocytes, which may contribute to cartilage degradation and development of osteoarthritis. Histone deacetylases (HDACs) have recently been implicated in the regulation of MMP gene expression. To investigate the functional involvement of HDACs in the signaling pathway of FGF2 and IL-1β, we examined the effects of HDAC inhibition on activities of FGF2 or IL-1β on gene expression of MMP-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5), collagen type II, and aggrecan. Human articular chondrocyte cultures were treated with FGF2 or IL-1β in the presence or absence of HDAC inhibitor (trichostatin A, TSA). Gene expression levels after treatments were assessed using quantitative real time PCR. Results showed that FGF2 and IL-1β both increased MMP-1 and -13 expression, while IL-1β also increased MMP-3 mRNA levels. These effects were attenuated in the presence of TSA in a dose dependent manner. In contrast to the effects on MMPs, FGF2 decreased mRNA levels of ADAMTS-5, which was not affected by HDAC inhibition. FGF2, IL-1β, and TSA inhibited expression of aggrecan, while TSA also decreased mRNA levels of collagen type II. These findings showed that HDAC inhibition antagonized FGF2 and IL-1β induced MMP expression. Combination of FGF2 and the HDAC inhibitor decreases both anabolic and catabolic genes, which may slow the cartilage turnover and be beneficial for maintaining cartilage integrity.     

Differential effects of TGF-β1 and FGF-2 on SDF-1α expression in human periodontal ligament cells derived from deciduous teeth in vitro

Stromal cell-derived factor (SDF)-1α has been reported to play a crucial role in stem cell homing and recruitment to injured sites. However, no information is available about its role in periodontal tissues. The aim of this in vitro study was to investigate the effects of basic fibroblast growth factor (FGF-2) and transforming growth factor (TGF)-β1 on SDF-1α expression in immortalized periodontal ligament (PDL) cells derived from deciduous teeth (SH9 cells). Real-time PCR and western blot analyses showed that SDF-1α mRNA expression in SH9 cells was markedly inhibited by FGF-2 treatment for 48 h. SU5402, which directly interacts with the catalytic domain of the FGF receptor 1 (FGFR1) and suppresses its phosphorylation, inhibited the FGF-2-related decrease in SDF-1α expression. These results suggest that FGF-2 signaling via the FGFR1 pathway inhibits SDF-1α expression. Conversely, SDF-1α expression in SH9 cells was increased by TGF-β1 treatment for 12 h. Western blot analysis showed that this treatment induced Smad2/3 phosphorylation. A time-course experiment showed that SDF-1α expression levels reached a maximum 12 h after the TGF-β1 treatment and returned to basal levels by 48 h. Real-time PCR analysis showed that Smad7 mRNA expression peaked by 6 h after TGF-β1 treatment. Since Smad7 siRNA downregulated Smad7 expression by approximately 2.5-fold compared with the negative control siRNA, the induction of SDF-1α expression was prolonged. Furthermore, treatment of SH9 cells with TGF-β1 for 12 h induced transwell migration of UE7T-13 cells, which are mesenchymal stem cells derived from human bone marrow. Therefore, SDF-1α may play an important role in stem and progenitor cell recruitment and homing to injured sites in the periodontal ligament, and regulation of SDF-1α expression may be a useful tool in cell-based therapy for periodontal tissue regeneration.