Area of cartilage accessible to curettage for subsequent arthrodesis of the equine proximal interphalangeal joint. Comparison of conventional and collateral ligament sparing approaches.

This study compared the areas of cartilage accessible for curettage in arthrodesis of the equine proximal interphalangeal joint using the conventional and collateral ligament sparing approaches. For this purpose, forelimbs and hindlimbs of six equine cadavers without radiographic evidence of proximal interphalangeal joint disease were used. One limb of each pair of limbs was randomly assigned to a dissection using a standard approach to the proximal interphalangeal joint including transection of the collateral ligaments, while each contralateral limb was exposed using the same approach but leaving the collateral ligaments intact. Hohmann retractors and Spratt curettes were then used to remove as much articular cartilage as possible. Finally, proximal interphalangeal joints were photographed and image analysis was performed. Using the collateral ligament sparing procedure, the mean percentage of articular cartilage surface removed (41.2%) was significantly less than using the conventional procedure (79.6%) (p <0.01). The difference between forelimbs and hindlimbs was not significant.

Regulation of forkhead box O1 (FOXO1) by protein kinase B and glucocorticoids: different mechanisms of induction of beta cell death in vitro.

AIMS/HYPOTHESIS: In steroid diabetes insulin secretion does not adequately compensate for enhanced hepatic gluconeogenesis and peripheral insulin resistance. Previous studies suggest that activation of the transcription factor forkhead box O1 (FOXO1) contributes to glucocorticoid-induced beta cell death. This study examines the role and regulation of FOXO1 in insulin-secreting cells. METHODS: INS-1E cells and mouse islet cells were cultured in the presence of dexamethasone. Signalling pathways were modified pharmacologically or by small interfering (si)RNA-mediated inhibition of protein synthesis. Changes in protein abundance and phosphorylation were analysed by western blotting, and subcellular localisation was assessed using confocal microscopy. Transcript levels were examined by RT-PCR. RESULTS: Surprisingly, downregulation of FOXO1 by siRNA did not affect dexamethasone-induced apoptosis or Bim expression, but it prevented the effects of the pan protein kinase B (AKT) inhibitor (Akti-1/2). Indeed, dexamethasone and Akti-1/2 synergistically increased beta cell death and Bim expression. Akti-1/2 triggered dephosphorylation and nuclear translocation of FOXO1. Glucocorticoid-receptor activation stimulated Foxo1 transcription, but FOXO1 phosphorylation was unchanged and the cytosolic concentration of FOXO1 remained high in relation to its nuclear concentration. However, subcellular fractionation revealed a significant increase in both cytosolic and nuclear FOXO1 compared with untreated cells. Dexamethasone diminished Pdx1 mRNA level, an effect which was not reversed by siRNA against Foxo1. Downregulation of AKT isoforms and serum/glucocorticoid-regulated kinase 1 (SGK1) suggests that only sustained suppression of all three AKT isoforms caused dephosphorylation and nuclear accumulation of FOXO1. CONCLUSIONS/INTERPRETATION: This study reveals that FOXO1 is not the main mediator of glucocorticoid-receptor-induced beta cell apoptosis, but rather that it escalates beta cell death when AKT activity is inhibited by distinct pathways.

Involvement of ceramide in hyperosmotic shock-induced death of erythrocytes.

Erythrocytes lack nuclei and mitochondria, the organelles important for apoptosis of nucleated cells. However, following increase of cytosolic Ca(2+) activity, erythrocytes undergo cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry, all features typical for apoptosis in nucleated cells. The same events are observed following osmotic shock, an effect mediated in part by activation of Ca(2+)-permeable cation channels. However, erythrocyte death following osmotic shock is blunted but not prevented in the absence of extracellular Ca(2+) pointing to additional mechanisms. As shown in this study, osmotic shock (950 mOsm) triggers sphingomyelin breakdown and formation of ceramide. The stimulation of annexin binding following osmotic shock is mimicked by addition of ceramide or purified sphingomyelinase and significantly blunted by genetic (aSM-deficient mice) or pharmacologic (50 microM 3,4-dichloroisocoumarin) knockout of sphingomyelinase. The effect of ceramide is blunted but not abolished in the absence of Ca(2+). Conversely, osmotic shock-induced annexin binding is potentiated in the presence of sublethal concentrations of ceramide. In conclusion, ceramide and Ca(2+) entry through cation channels concert to trigger erythrocyte death during osmotic shock.

Signaling lymphocytic activation molecule is expressed on mature CD83+ dendritic cells and is up-regulated by IL-1 beta.

Signaling lymphocyte activation molecule (SLAM), a 70-kDa costimulatory molecule that mediates CD28-independent proliferation of T cells and IFN-gamma production, has been identified on human T cells, immature thymocytes, and a subset of B cells. We have found that SLAM is expressed on mature but not immature dendritic cells (DC). However, the SLAM-associated protein, is missing in DC. SLAM surface expression is strongly up-regulated by IL-1beta. Addition of IL-1beta to the DC maturation mixture also increases the stimulatory properties of DC. These findings provide a new marker for DC maturation and help to explain two areas of DC biology. First, SLAM is a receptor for the measles virus, previously shown to infect DC. Second, SLAM could possibly contribute to the enhanced immunostimulatory functions of DC that are observed following the addition of IL-1.

Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood.

It has been known for years that rodents harbor a unique population of CD4(+)CD25(+) "professional" regulatory/suppressor T cells that is crucial for the prevention of spontaneous autoimmune diseases. Here we demonstrate that CD4(+)CD25(+)CD45RO(+) T cells (mean 6% of CD4(+) T cells) are present in the blood of adult healthy volunteers. In contrast to previous reports, these CD4(+)CD25(+) T cells do not constitute conventional memory cells but rather regulatory cells exhibiting properties identical to their rodent counterparts. Cytotoxic T lymphocyte-associated antigen (CTLA)-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4(+)CD25(+) T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were nonproliferative to stimulation via their T cell receptor for antigen, but the anergic state was partially reversed by interleukin (IL)-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or platebound anti-CD3 plus anti-CD28 the CD4(+)CD25(+) T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4(+) and CD8(+) T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4(+)CD25(+) T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.

Cutting edge: resistance to apoptosis and continuous proliferation of dendritic cells deficient for TNF receptor-1.

The individual roles of the two TNFRs on dendritic cells (DC) are poorly understood. Investigating bone marrow-derived DC from TNFR-deficient mice, we found that cultures from TNFR1(-/-) mice continue to form proliferating clusters for 6-9 mo. In contrast, DC derived from wild-type, TNFR2(-/-), or TNFR1/2(-/-) mice survived for only 3-4 wk. DC obtained from these TNFR1(-/-) long term cultures (LTC) mice show an unusual mixed immature/mature phenotype. The continuous proliferation of the LTC is GM-CSF dependent and correlates with decreased protein levels of the cyclin-dependent kinase inhibitors p27(KIP1) and p21(CIP1). Prolonged survival of TNFR1(-/-) DC appears to be independent from NF-kappaB and Bcl-2 pathways and is rather enabled by the down-regulation of CD95, resulting in the resistance to CD95 ligand-induced apoptosis. These data point to proapoptotic signals mediated via TNFR1 and antiapoptotic signals mediated via TNFR2 in DC.

Regulation of the trans-activation potential of STAT5 through its DNA-binding activity and interactions with heterologous transcription factors.

Extracellular hormones, growth factors and cytokines relay their effects on the transcription of genes through the recognition of specific receptors and intracellular signalling molecules. Signal transducers and activators of transcription (STATs) have been recognized as crucial intracellular signalling molecules. The cytokine receptor-associated Janus kinases (JAKs) convert the latent monomeric form of the STAT molecules to the activated dimeric form through tyrosine phosphorylation. The dimers bind to specific DNA response elements and are able to induce transcription. This induction requires the full-length form of the STAT molecules. Negative regulatory potential is exerted by the short form of the molecule, which lacks the trans-activation domain. This short form is activated and dimerized, but dephosphorylation is impaired. The short form of STAT occupies the DNA-binding sites in a stable fashion and acts as a strong suppressor of wild-type action. Positive enhancement of STAT5 trans-activation potential is provided by the glucocorticoid receptor. Ligand activation of this receptor causes the formation of a complex with STAT5 and deviation to the STAT5 DNA-binding site. An additional regulatory loop is provided by the reactivation of the short form of STAT5 through glucocorticoid receptor association. Conversely, classical glucocorticoid-responsive genes are negatively affected by STAT5 activation.

Human dendritic cells transfected with either RNA or DNA encoding influenza matrix protein M1 differ in their ability to stimulate cytotoxic T lymphocytes.

The use of tumor antigen loaded dendritic cells (DC) is one of the most promising approaches to induce a tumor specific immune response in vivo. Several strategies have been designed to load DC with tumor antigens. In this study, we investigated the delivery of in vitro transcribed RNA and plasmid DNA into monocyte-derived, ie non-proliferating human DC, using several nonviral transfection methods including electroporation and lipofection. Green fluorescent protein (GFP) was used as a reporter gene and influenza matrix protein 1 (M1) as a model antigen for HLA class I restricted antigen presentation. Using electroporation in combination with DNA or with RNA, up to 11% of DC were GFP-positive. Using liposomes as a vehicle for DNA transport up to 10% of the DC were GFP-positive. A significant increase in transfection efficacy, of up to 20%, was observed when GFP RNA was used in combination with liposomes. Importantly, the RNA transfected DC retained their typical morphological and immunophenotypical characteristics. In addition, DC transfected with M1 RNA were able to stimulate autologous peripheral M1-specific memory cytotoxic T lymphocytes (CTL), as well as M1-specific CTL clones. Furthermore, comparison of DNA-transfected DC with RNA-transfected DC revealed the latter to be far better stimulators of antigen-specific T cells. This RNA transfection technique consequently represents a very promising tool for future immunotherapy strategies.

Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation.

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC) and represents the best marker for mature DC. Here, we report the cloning of the cDNA encoding mouse CD83 (mCD83) from a murine bone marrow-derived DC (BM-DC) cDNA library. DNA sequence analysis revealed a 196 amino acid protein including a signal peptide of 21 amino acids which shares 63% amino acid identity with hCD83. Using Northern blot analyses, mCD83 mRNA was found to be strongly expressed in mouse BM-DC and its expression was upregulated following stimulation with LPS or TNF-alpha. Transfection experiments using COS-7 cells revealed that mCD83 is glycosylated. Furthermore, the extracellular CD83 domain was recombinantly expressed in Escherichia coli and one-dimensional NMR data strongly support that the protein is structurally folded.

Human monocyte derived dendritic cells express functional P2X and P2Y receptors as well as ecto-nucleotidases.

We investigated the expression and function of P2 receptors and ecto-nucleotidases on human monocyte derived dendritic cells (DC). In addition we analyzed the effect of extracellular ATP on the maturation of DC. By RT-PCR, DC were found to express mRNA for several P2X (P2X1, P2X4, P2X5, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y11) receptors. As shown by FURA-2 measurement, triggering of P2 receptors resulted in an increase in free intracellular Ca2+. In combination with Tumor necrosis factor-alpha, ATP increased the expression of the DC surface markers CD80, CD83 and CD86 indicating a maturation promoting effect. DC expressed the ecto-apyrase CD39 and the ecto-5'-nucleotidase CD73 as demonstrated by RT-PCR. Extracellular ATP was rapidly hydrolyzed by these ecto-enzymes as shown by separation of 3H-labeled ATP metabolites using a thin layer technique. These data suggest that ATP acts as a costimulatory factor on DC maturation.

The human dendritic cell marker CD83 maps to chromosome 6p23.

The chromosomal localization of the human CD83 gene was determined using somatic cell hybrids, a radiation hybrid mapping panel and FISH analysis on human metaphase chromosomes. PCR-based analysis of a single chromosome hybrid panel identified the presence of the CD83 gene on human chromosome 6 and subsequent analysis of the Genebridge4 radiation panel located the gene between AFMa192wg9 and AFMb322wd1 with a lod score of 9.2. Finally, using FISH analysis the CD83 gene was localized to chromosome 6 band p23.

The interleukin-4 receptor activates STAT5 by a mechanism that relies upon common gamma-chain.

Interleukin (IL)-4 signaling proceeds via cytoplasmic activation of the Janus kinases JAK1 and JAK3 and the signal transducer and activator of transcription STAT6. We show that the IL-4 receptor, like other cytokine receptor systems utilizing the common receptor gamma-chain (gammac), is also connected to a signaling pathway that involves STAT5. Both STAT5a and STAT5b become tyrosine-phosphorylated and acquire specific DNA-binding properties in response to IL-4 receptor stimulation in the murine pro-B cell line Ba/F3. In preactivated human T cells, STAT5 became activated in an IL-4-dependent fashion as assayed by IL-4-induced STAT5 translocation from the cytoplasm to the cell nucleus and by binding to cognate DNA. Moreover, stimulation of preactivated human T cells by IL-4 led to specific transcriptional up-regulation of STAT5 target genes. IL-4 receptor-mediated STAT5 activation is dependent on the presence of gammac and JAK3 within the receptor complex. In COS-7 cells, the JAK/STAT pathway leading from the IL-4 receptor to STAT5-dependent regulation of a reporter gene relied largely on coexpression of JAK3. In Ba/F3 cells, studies on signal transduction evoked by directed specific receptor homo- or heterodimerization revealed that STAT5 activation can be triggered exclusively by IL-4R heterodimers containing gammac.

Dominant negative variants of the SHP-2 tyrosine phosphatase inhibit prolactin activation of Jak2 (janus kinase 2) and induction of Stat5 (signal transducer and activator of transcription 5)-dependent transcription.

PRL plays a central role in the regulation of milk protein gene expression in mammary epithelial cells and in the growth and differentiation of lymphocytes. It confers its activity through binding to a specific transmembrane, class I hematopoietic receptor. Ligand binding leads to receptor dimerization and activation of the tyrosine kinase Jak (janus kinase) 2, associated with the membrane-proximal, intracellular domain of the receptor. Jak2 phosphorylates and activates Stat5, a member of the Stat (signal transducers and activators of transcription) family. PRL receptor also activates SHP-2, a cytosolic tyrosine phosphatase. We investigated the connection between these two signaling events and derived a dominant negative mutant of SHP-2 comprising the two SH2 domains [SHP-2(SH2)2]. An analogous variant of the SHP-1 phosphatase [SHP-1(SH2)2] was used as a control. The dominant negative mutant of SHP-2 was found to inhibit the induction of tyrosine phosphorylation and DNA-binding activity of m-Stat5a, m-Stat5b, and the carboxyl-terminal deletion variant m-Stat5adelta749, as well as the transactivation potential of m-Stat5a and m-Stat5b. The dominant negative mutant SHP-1(SH2)2 had no effect. The kinase activity of Jak2 is also dependent on a functional SHP-2 phosphatase. We propose that SHP-2 relieves an inhibitory tyrosine phosphorylation event in Jak2 required for Jak2 activity, Stat5 phosphorylation, and transcriptional induction.

Activation of STAT6 is not dependent on phosphotyrosine-mediated docking to the interleukin-4 receptor and can be blocked by dominant-negative mutants of both receptor subunits.

Stimulation of susceptible cells by interleukin-4 leads to activation of signal transducer and activator of transcription (STAT6) through tyrosine phosphorylation and dimerisation, thus directing it to the cell nucleus and rendering it a sequence-specific transcription factor. We functionally reconstituted human interleukin-4 receptor complexes with intracellular truncations of either the alpha or gamma subunits and demonstrate the requirement for elements from both receptor chains for STAT6 activation induced by interleukin-4. By assaying the signalling properties of human interleukin-4-receptor alpha-chain-deletion constructs in both Ba/F3 cells and COS-7 cells, we show that all its cytoplasmic tyrosine residues can be removed without affecting the capability of the receptor complex to trigger STAT6 function with regard to tyrosine phosphorylation, DNA binding, and specific gene transcription. The activation of both STAT6 and janus kinase 1 (JAK1) by the interleukin-4 receptor was completely abolished by disruption of the membrane-proximal 'box1' motif in the interleukin-4 receptor alpha chain. Our results indicate a redundant role of the previously defined phosphotyrosine-containing STAT6 docking site and suggest a mechanism of immediate activation of STAT6 by receptor-associated janus kinase(s). In addition, we demonstrate that dominant negative versions of both interleukin-4 receptor subunits are able to block interleukin-4 induced signalling via STAT6.

Cytokine receptor-independent, constitutively active variants of STAT5.

STAT (signal transducers and activators of transcription) proteins are dual function proteins, which participate in cytokine-mediated signal transduction events at the cell surface and transcriptional regulation in the nucleus. We have exploited insights into the activation mechanism of STAT factors to derive constitutively active variants. Chimeric genes encoding fusion proteins of STAT5 and the kinase domain of JAK2 have been derived. The functional properties of the fusion proteins have been investigated in transiently transfected COS cells or in HeLa cells stably transfected with STAT5-JAK2 gene constructs regulated by a tetracycline-sensitive promoter. The STAT5-JAK2 proteins exhibit tyrosine kinase activity and are phosphorylated on tyrosine. The molecules are activated through an intramolecular or a cross-phosphorylation reaction and exhibit constitutive, STAT5-specific DNA binding activity. The transactivation potentials of three constitutively activated STAT5-JAK2 variants comprising different transactivation domains (TADs) derived from STAT5, STAT6, and VP16 were compared. The chimeric molecule containing the STAT5 TAD had no or only a very low, the molecule with the STAT6 TAD a medium, and the molecule with the VP16 TAD a very high transactivation potential. Transcription from STAT5-responsive gene promoter regions of the beta-casein, oncostatin M, and the cytokine-inducible Src homology 2 domain-containing protein genes was observed. These chimeric STAT molecules allow the study of the function of STAT5 independent of cytokine receptors and the activation of other signal transduction pathways.

Comparison of the transactivation domains of Stat5 and Stat6 in lymphoid cells and mammary epithelial cells.

Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. Stat5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates Stat5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of Stat5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of Stat5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function.

Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype.

The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.

Prolactin and interleukin-2 receptors in T lymphocytes signal through a MGF-STAT5-like transcription factor.

The cell surface receptors for PRL and interleukin-2 (IL-2) are structurally distinct, but share regulatory tasks in T lymphocytes. They can stimulate proliferation and activate transcription of over-lapping sets of genes of T cells. PRL and IL-2 receptor activation are both linked to the Jak/Stat (signal transducer and activator of transcription) pathway. We investigated the ability of PRL and IL-2 to activate Stat proteins in different T cell lines. The DNA binding specificities, the reactivities toward Stat-specific antisera, and the mol wt of IL-2- and PRL-induced DNA-binding proteins in Nb2 and C196 T cell lines were investigated. A comparison with the Stat proteins induced by interferon-gamma, PRL, and IL-6 in T47D mammary tumor cells was made. We found that these parameters were indistinguishable for one of the PRL- and IL-2-induced factors. A transcription factor closely related to mammary gland factor-Stat5 is rapidly activated upon interaction of IL-2 and PRL with their respective receptors. Activation of a second protein related to Stat1 was also observed. Our results emphasize the role of PRL as a regulator of the immune response and indicate that the Stat factors mammary gland factor-Stat5 and Stat1 play a role in the regulation of gene expression during T cell development.

The activation domain of simian immunodeficiency virus SIVmac239 Rev protein is structurally and functionally analogous to the HIV-1 Rev activation domain.

The Rev proteins of primate immunodeficiency viruses are essential transactivators for the switch from early to late phase in the viral replication cycle. By mutational analysis, a putative activation domain (AD) has been assigned to the carboxy-terminus. This leucine-rich stretch of amino acids proved to be essential for the transactivating properties of HIV-1 Rev. Some mutants in the AD transdominantly inhibit the function of wild-type Rev protein very efficiently. We identified a similar domain structure for SIVmac239 Rev by sequence comparison and in vitro mutagenesis. The leucine/isoleucine residues of the SIVmac239 Rev activation domain appeared to be of similar importance for function. The mutants of these residues in the SIV AD displayed a dominant negative phenotype on both HIV-1 and SIVmac 239 rev-responsive elements (RRE). The prokaryotically expressed wild-type and mutant proteins were analyzed for RNA-binding properties in a gel-shift assay in vitro. This assay revealed a similar binding pattern of wild-type and transdominant proteins on either RRE.