Data integration for identification of important transcription factors of STAT6-mediated cell fate decisions.

Data integration has become a useful strategy for uncovering new insights into complex biological networks. We studied whether this approach can help to delineate the signal transducer and activator of transcription 6 (STAT6)-mediated transcriptional network driving T helper (Th) 2 cell fate decisions. To this end, we performed an integrative analysis of publicly available RNA-seq data of Stat6-knockout mouse studies together with STAT6 ChIP-seq data and our own gene expression time series data during Th2 cell differentiation. We focused on transcription factors (TFs), cytokines, and cytokine receptors and delineated 59 positively and 41 negatively STAT6-regulated genes, which were used to construct a transcriptional network around STAT6. The network illustrates that important and well-known TFs for Th2 cell differentiation are positively regulated by STAT6 and act either as activators for Th2 cells (e.g., Gata3, Atf3, Satb1, Nfil3, Maf, and Pparg) or as suppressors for other Th cell subpopulations such as Th1 (e.g., Ar), Th17 (e.g., Etv6), or iTreg (e.g., Stat3 and Hif1a) cells. Moreover, our approach reveals 11 TFs (e.g., Atf5, Creb3l2, and Asb2) with unknown functions in Th cell differentiation. This fact together with the observed enrichment of asthma risk genes among those regulated by STAT6 underlines the potential value of the data integration strategy used here. Thus, our results clearly support the opinion that data integration is a useful tool to delineate complex physiological processes.

Impaired T cell activation and cytokine production by calcitriol-primed human B cells.

The biologically active form of vitamin D3 , 1, 25-dihydroxyvitamin D3 (calcitriol), is a potent modulator of the immune response. We have shown previously that calcitriol modulates the immunoglobulin response in vitro and in vivo in mice and humans. To analyse the underlying molecular mechanisms we studied whether calcitriol-primed B cells modulate T cell activation and function. Human B cells were stimulated with anti-CD40 and interleukin (IL)-4 in the presence of increasing concentrations of calcitriol. After removal of calcitriol, primed B cells were co-cultured with autologous CD4(+) T cells; the B cell phenotype T cell activation and their consecutive cytokine production were also assessed. Naive T cells co-cultured with calcitriol-primed naive B cells showed a reduced expansion, nuclear factor of activated T cells, cytoplasmic 2 (NFATc2) expression and cytokine production upon restimulation. CD86 expression on B cells after calcitriol priming was identified as an underlying mechanism, as T cell activation and expansion was rescued by activating anti-CD28 antibodies. Our data indicate that calcitriol-primed B cells display an impaired capacity to activate T cells. Taken together, we identified a novel B cell-dependent vitamin D immune regulatory mechanism, namely by decreased co-stimulation of calcitriol-primed B cells.

Mechanisms and rescue strategies of calcineurin inhibitor mediated tolerance abrogation induced by anti-CD4 mAb treatment.

To ensure safety tolerance induction protocols are accompanied by conventional immunosuppressive drugs (IS). But IS such as calcineurin inhibitors (CNI), for example, cyclosporin A (CsA), can interfere with tolerance induction. We investigated the effect of an additional transient CsA treatment on anti-CD4mAb-induced tolerance induction upon rat kidney transplantation. Additional CsA treatment induced deteriorated graft function, resulting in chronic rejection characterized by glomerulosclerosis, interstitial fibrosis, tubular atrophy and vascular changes. Microarray analysis revealed enhanced intragraft expression of the B cell attracting chemokine CXCL13 early during CsA treatment. Increase in CXCL13 expression is accompanied by enhanced B cell infiltration with local and systemic IgG production and C3d deposition as early as 5 days upon CsA withdrawal. Adding different CNIs to cultures of primary mesangial cells isolated from glomeruli resulted in a concentration-dependent increase in CXCL13 transcription. CsA in synergy with TNF-α can enhance the B cell attracting and activating potential of mesangial cells. Transient B cell depletion or transfer of splenocytes from tolerant recipients 3 weeks after transplantation could rescue tolerance induction and did inhibit intragraft B cell accumulation, alloantibody production and ameliorate chronic rejection.

[Biological switches: the 'all-or-none' principle in T-cell activation]. / Biologische Schalter: Das "Alles-oder-Nichts-Prinzip" der T-Zell-Aktivierung.

T helper lymphocytes (Th cells) play a central role in cellular defence against pathogens as well as in cellular self tolerance. The activation of Th cells is a crucial process determining the course of a protective immune response. Dysregulated activation processes can lead to pathologic immune reactions and may induce autoimmune diseases. Thus, for example, chronic activation of Th cells can trigger autoimmune diseases such as rheumatoid arthritis. One fundamental feature of antigen-specific T-cell activation is the synthesis of cytokines, e.g. Interleukin- (IL-)2. Here we present results of our working group indicating for the first time that, at the level of the individual cell, IL-2 is expressed in a binary fashion, i.e. according to an all-or-none principle. The identification and characterization of such intracellular switches may provide new options to manipulate the fate of T cells and develop novel therapeutic strategies.

Low-dose cyclosporine A therapy increases the regulatory T cell population in patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a T cell dependent chronic relapsing inflammatory skin disorder successfully treated with cyclosporine A (CsA). Clinical observations indicate that even low-dose CsA therapy is successful in severely affected AD patients. We studied the impact of low-dose CsA therapy on the ability of T helper cells to be activated, and examined whether regulatory T (Treg) cells are increased in these patients. METHODS: Peripheral T cells were activated in a whole blood sample and interleukin-2 producing cells were measured by intracellular cytokine staining. Regulatory T cells were analyzed by intracellular FoxP3 staining. Regulatory T cells (CD4(+)CD25(+)CD127(low)) and effector T cells (CD4(+)CD25(-)CD127(+)) were sorted by flow cytometry and used for suppression assays. RESULTS: A group of AD patients treated with low-dose CsA had a significantly larger Treg cell population than a healthy control subject group. In individual patients, onset of low-dose CsA therapy reduced the ability of T cells to be activated to 42 +/- 18% (P < 0.005) and significantly increased Treg cells, both in absolute numbers (1.6-fold change) and frequencies (1.7-fold change). Treg cells from AD patients showed similar suppressive capacities as Treg cells from healthy donors. Furthermore, Treg cells from AD patients had skin homing properties. CONCLUSION: Our results indicate that the therapeutic effect of low-dose CsA therapy in AD patients might be not only mediated by the inhibition of T cell hyperactivity but also by an increased population of Treg cells.

The neuroprotective actions of FK506 binding protein ligands: neuronal survival is triggered by de novo RNA synthesis, but is independent of inhibition of JNK and calcineurin.

The immunosuppressant FK506 displays substantial neuroprotective and neuroregenerative effects. It is not fully understood to which extent these effects depend on the inhibition of the calcineurin phosphatase (PP2B). The present study has re-addressed this issue using Lie120, a novel highly specific inhibitor of calcineurin, which does not block the enzymatic activity of FKBPs or cyclophilins, respectively. We have determined the effect of FK506 (10-500 nM), V-10,367 (a FK506 derivative which does not block calcineurin; 1-5 microM) and Lie120 (a novel specific inhibitor of calcineurin, 0.1-5 microM) on the cellular survival and the pro-degenerative JNK activity of PC12 and Neuro2A cells following application of 200 microM H(2)O(2). FK506 and V-10,367, but not Lie120, protected both cell lines against H(2)O(2)-mediated death, whereas an increase in JNK1 activity was blocked by FK506 and Lie120, but not by V-10,367. Co-incubation of FK506 and V-10,367 with the mRNA synthesis inhibitor actinomycin D abolished the protective effect of FK506 and V-10,367. This antagonization was effective when actinomycin D was applied 30 min or 1 h, but not 2 or 4 h, after H(2)O(2) suggesting that FKBP-ligands confer their neuroprotection by rapid de novo synthesis of (functionally) anti-apoptotic proteins. The search for the corresponding effector genes revealed that the expression of FKBP25, FKBP38 and FKBP52 (analysis by reverse transcription-polymerase chain reaction (RT-PCR) did not change following H(2)O(2) or FK506, and this was also true for the expression of apoptosis-related genes caspase 3, bax, bcl-2 and bcl-xL (analysis by Multiplex-PCR). Summarizing, neuronal protection by FKBP-ligands is not mediated either by calcineurin or by JNK1 in this experimental set-up, whereas the FK506 mediated inhibition of JNK1 is realized by the inhibition of calcineurin, an effective activator of JNK1 in neurons.

Reversible inhibition of calcineurin by the polyphenolic aldehyde gossypol.

The reversible inhibition of calcineurin (CaN), which is the only Ca(2+)/calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of (33)P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC(50) value of 15 microm. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC(50) of 9 and 6 microm, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18.CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.

Improved in vitro stability of serum osteocalcin by using a new commercially available antiproteolytic compound.

The effect of a commercially available antiproteolytic compound (OSCAstabil) on the degradation in vitro of serum osteocalcin (Oc) was investigated in serum samples stored at 22, 4 or -30 degrees C (n = 20) or subjected to repeated freeze-thaw cycles (n = 8). The addition of the stabilizing agent immediately after serum preparation increased the Oc stability from < 3 to 6 h at 22 degrees C, from < 3 h to 3 days at 4 degrees C and from 1 day to more than 3 days at -30 degrees C. Up to three freeze-thaw cycles had no influence on the Oc stability, either with or without the stabilizer. The obviously prolonged Oc stability at 22 and 4 degrees C offers a more convenient and reliable Oc estimation procedure for clinical practice. The use of this antiproteolytic agent can be recommended in order to retard the in vitro degradation of serum Oc, not only for clinical routine, but also for studies on metabolic bone diseases.

Identification of peptide fragments generated by digestion of bovine and human osteocalcin with the lysosomal proteinases cathepsin B, D, L, H, and S.

We have determined the primary cleavage sites in the bone Gla protein (BGP; osteocalcin) for several of the proteases that could act on the protein during bone resorption and turnover, cathepsins B, D, L, H, and S. The time course of BGP digestion by each cathepsin was first determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We then incubated human and bovine BGP with each cathepsin for a sufficient time to reduce the level of intact protein by at least 20-fold, isolated the major cleavage peptides, and identified each by N-terminal sequence analysis and by amino acid analysis. Our results show that BGP has relatively few cathepsin-sensitive sites and that these sites are located at the N and C terminus of the 49-residue protein. Cathepsins B, L, H, and S readily cleave BGP at the G7-A8 bond; cathepsin L also cleaves at R43-R44; cathepsin B also cleaves at R44-F45; and cathepsin D cleaves only at A41-Y42. The immunoreactivity of the major peptides generated by cathepsin cleavage was evaluated using the original radioimmunoassay developed for the detection of BGP in human serum. The BGP 8-49 fragment cross-reacts identically with native BGP, while the 8-43 and the 1-44 fragments require 20- to 40-fold higher concentrations to achieve the same level of displacement as the native protein. The 1-41 and 8-41 fragments are unable to significantly displace the labeled native BGP tracer at any concentration tested. These results demonstrate the utility of peptides generated by cathepsin digestion in the mapping of the antigenic epitopes recognized by a given BGP immunoassay.

Dipeptidylpeptidase IV--inactivation with N-peptidyl-O-aroyl hydroxylamines.

Eleven N-peptidyl-O-aroyl hydroxylamines have been synthesized and their hydrolytic stability, acidity and properties during reaction with dipeptidyl peptidase IV (E.C. 3.4.14.5) investigated. N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines act as irreversible inhibitors of serine proteases. The serine enzyme, dipeptidyl peptidase IV (DP IV), is inactivated by substrate analog derivatives of this class by a suicide inactivation mechanism. During the enzyme reaction of DP IV with the suicide substrates most molecules are hydrolyzed but some irreversibly inactivate the target enzyme. In contrast to porcine pancreatic elastase and thermitase, DP IV exhibits a high ratio for hydrolysis of the compounds versus inhibition during their interaction with the enzyme. Variation of the leaving aroyl residue lowers this ratio. Variation of the substrate analog peptide moieties of the DP IV-inhibitors increases their ability to inhibit the enzyme to a remarkable extent. Possible reaction pathways are discussed.