Abstract: The common γ-chain (γ(c)) cytokines signal through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and play pivotal roles in lymphocyte activation. We investigated the effect of immunosuppressive drugs targeting this pathway, the JAK inhibitor tofacitinib (CP-690,550) and the anti-interleukin (IL)-2R antibody basiliximab, as part of a phase 2 study.
Abstract: The Jak inhibitor CP-690,550 inhibits alloreactivity and is currently being investigated for prevention of allograft rejection after transplantation. In this study, we examined the effect of CP-690,550 on IL-2-mediated Jak/STAT5 phosphorylation by CD4(+)CD25(bright)FoxP3(+)CD127(-/low) T cells (Treg) and CD4(+)CD25(neg) effector T cells (Teff) in kidney transplant (KTx) patients. Phosphospecific flow cytometry was used to study the effect of CP-690,550 on IL-2-induced intracellular STAT5-phosphorylation. IL-2-induced phosphorylation of STAT5 (P-STAT5) in both Treg and Teff, which was significantly higher for CD4(+)CD25(bright) Treg (increased by 71%, mean) than for CD4(+)CD25(neg) Teff (increased by 42%). In the presence of 100 ng/mL CP-690,550, a clinically relevant exposure, IL-2-induced P-STAT5 was partially inhibited in CD4(+)CD25(bright)Treg (% inhibition; 51%), while almost completely blocked in Teff (%inhibition; 84%, p = 0.03). The IC(50) was 2-3 times higher for Treg (104 ng/mL) than for Teff (40 ng/mL, p = 0.02). In the presence of CP-690,550, Treg exhibited additional suppressive activities on the alloactivated proliferation of Teff (56%, mean). In addition, CD4(+)CD25(bright) Treg from KTx-patients receiving CP-690,550 vigorously suppressed the proliferation of Teff (87%, mean). Our findings show that CP-690,550 effectively inhibits Teff function but preserves the suppressive activity of CD4(+)CD25(bright) regulatory T cells.
Abstract: Measurement of the pharmacodynamic biomarker inosine monophosphate dehydrogenase (IMPDH) activity in renal transplant recipients has been proposed to reflect the biological effect better than using pharmacokinetic parameters to monitor mycophenolate mofetil therapy. The IMPDH assays are however labor intensive and this complicates implementation into patient care. Quantification of IMPDH messenger RNA (mRNA) could form an attractive alternative. This study was designed to correlate IMPDH mRNA levels with IMPDH activity and clinical outcome in renal transplant recipients. From a cohort of 101 renal transplant patients, blood samples were drawn pre transplantation and at 4 times after transplantation. IMPDH activity, IMPDH type 1 and type 2 mRNA levels, and mycophenolic acid concentrations were measured and correlated to clinical outcomes. No correlation was found between IMPDH type 1 and type 2 mRNA levels and IMPDH activity in pre- and posttransplant samples. A significant increase in IMPDH mRNA levels was found between day 6 and day 140 after transplantation. IMPDH type 1 and type 2 mRNA levels before transplant showed a trend toward statistically significant higher levels in patients with an acute rejection (P = 0.052 and P = 0.058). After transplant, the IMPDH type 1 and type 2 mRNA levels were significantly lower in patients with an acute rejection (P = 0.026 and P = 0.007). We conclude that IMPDH mRNA levels do not correlate with IMPDH activity but are nevertheless correlated with acute rejections. Furthermore, although the regulation of the expression of the 2 isoforms is presumed to be different, in this study, the changes in the expression of type 1 mRNA closely paralleled those of type 2.
Abstract: BACKGROUND.: The small molecule drug CP-690,550 inhibits Janus kinase 3 at nanomolar concentrations and has recently been shown to prevent allograft rejection in rodents and nonhuman primates. METHODS.: As part of a phase 1 clinical trial, we investigated the effect of CP-690,550 after 29 days of 30 mg twice daily treatment at the cellular level in eight kidney transplant patients by studying ex vivo phosphorylation of STAT5 (P-STAT5), the key substrate of JAK3. RESULTS.: As determined by quantitative fluorescent western blotting, interleukin-2-induced P-STAT5 in YT cells was reduced by a median of 73% (P<0.01) in the presence of serum collected on day 29 compared with pretreatment baseline. When evaluated by phosphospecific flow cytometry, CP-690,550 also reduced interleukin-2-induced P-STAT5 in CD3 (median 20%; P<0.05), CD3CD4 (median 37%; P<0.05), and CD3CD8 (median 34%; P<0.01) populations in patient-derived peripheral blood mononuclear cells. At the functional level, the inhibitory effect of CP-690,550 was confirmed by determining the expression of several STAT5 targets genes. CONCLUSION.: Analysis of P-STAT5 may, therefore, be used to determine the immunomodulatory effect of CP-690,550 at the cellular level in transplant patients.
Abstract: Mesenchymal stromal cells regulate immune cell function via the secretion of soluble factors. Cell membrane interactions between these cell types may play an additional role. Here, we demonstrate that subpopulations of allo-activated T cells are capable of binding to human adipose-derived stromal cells (ASC). The bound T-cell population contained CD8+ T cells and was enriched for CD4-CD8- T cells, whereas the proportion of CD4+ T cells was decreased compared with the non-bound T-cell population. Bound CD4+ T cells had high proliferative activity and increased CD25 and FoxP3 expression. However, they also expressed CD127, excluding regulatory T-cell function. In CD8+ T cells, IL-2 sensitivity, as determined by the analysis of phosphorylated STAT5, was lower in the presence of ASC and even lower in bound cells. In contrast, IL-2-induced phosphorylated STAT5 levels were higher in bound CD4+ T cells than in non-bound CD4+ T cells. Additionally, pro-proliferative TGF-beta signalling via endoglin and SMAD1/5/8 phosphorylation was detected in bound CD4+ T cells. Even after prolonged co-culture with ASC, the activated phenotype of bound CD4+ T cells persisted. In conclusion, these results demonstrate that the binding of lymphocytes to ASC represents an immunomodulatory mechanism in which CD8+ T cells are inhibited in their responsiveness to pro-inflammatory stimuli and reactive CD4+ T cells are depleted from the immune response.
Abstract: Inhibition of NF-kappaB transcriptional activity by steroid receptors is the basis for the antiinflammatory actions of steroid hormones and the molecular mechanism underlying this cross-talk is thought to involve direct protein-protein interactions. In this study, we show that estrogen receptor (ER)alpha and NF-kappaB interact in vivo by using fluorescence resonance energy transfer (FRET) and co-immunoprecipitation. U2-OS cells were used to study direct interactions between fluorescent fusion proteins of ERalpha and the NF-kappaB subunits p50 and p65. Interactions were observed only in the nucleus and maximal FRET signal was detected when ERalpha is co-expressed with both NF-kappaB subunits and cells were stimulated with estrogen. This is in agreement with the induction of nuclear co-localization of the proteins under this condition. Moreover, in a U2-OS clone stably expressing ERalpha, interaction with NF-kappaB was confirmed. A p65 deletion mutant lacking the Rel homology domain was strongly impaired in its interaction with ERalpha showing the importance of this domain. Taken together, these findings provide a strong basis for the direct protein-protein interaction model for cross-talk between ERalpha and NF-kappaB.
Abstract: In the past decade the list of chemicals in the environment that are able to mimic the natural hormone estrogen, thereby disrupting endocrine function, has grown rapidly. These chemicals are able to bind to estrogen receptors (ERs) and influence estrogen signaling pathways, although several of them have structures that differ substantially from the endogenous hormone 17beta-estradiol. Because of their polycyclic nature, the polycyclic musks AHTN (6-acetyl-1,1,2,4,4,7-hexamethyltetraline) and HHCB (1,2,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran) were assessed for interaction with estrogen receptors. These compounds are ubiquitously present in surface waters and have been identified in human milk fat and blood. Using sensitive in vitro reporter gene assays, we found that AHTN and HHCB act as selective estrogen receptor modulators (SERMs), inducing both estrogenic and antiestrogenic activity dependent on the cell line and the ER subtype targeted. Weak estrogenic effects were observed only at relatively high concentrations (10 microM). Antiestrogenic effects were observed in various cell lines starting at concentrations of 0.1 microM. In comparison with the well-known SERM, 4-hydroxytamoxifen, AHTN and HHCB have a much lower potency in suppressing estradiol-induced transactivation. Results with various mutant ER receptor types suggest that binding sites of the musk compounds differ from 17beta-estradiol and 4-hydroxytamoxifen. The cell type dependency of ER transactivation shows that caution should be exerted when interpreting effects of estrogenic compounds using in vitro systems.
Abstract: Estrogens are important mediators of bone homeostasis, and postmenopausal estrogen replacement therapy is extensively used to prevent osteoporosis. The biological effects of estrogen are mediated by receptors belonging to the superfamily of steroid/thyroid nuclear receptors, estrogen receptor (ER)alpha and ER beta. ER alpha, not only trans-activates target genes in a hormone-specific fashion, but it can also neutralize other transcriptional activators, such as nuclear factor (NF)-kappa B, causing repression of their target genes. A major mechanism by which estrogens prevent osteoporosis seems to be repression of transcription of NF-kappa B target genes, such as the osteoclast-activating cytokines interleukin-6 and interleukin-1. To study the capacity of both ERs in repression of NF-kappa B signaling in bone cells, we first carried out transient transfections with ER alpha or ER beta of the human osteoblastic U2-OS cell line, in which endogenous NF-kappa B was stimulated by tumor necrosis factor alpha. Repression by ER alpha was already observed without 17 beta-estradiol, whereas addition of the ligand increased repression to 90%. ER beta, however, was able to repress NF-kappa B activity only in the presence of ligand. Because it is known that some antiestrogens can also display tissue-specific agonistic properties, 4-hydroxytamoxifen was tested for its capacity in repressing NF-kappa B activity and was found to be active (albeit less efficient than 17 beta-estradiol) and, interestingly, only with ER alpha. The pure antagonist ICI 164,384 was incapable of repressing through any ER subtypes. Deletion analysis and the use of receptor ER alpha/ER beta-chimeras showed that the A/B domain, containing activation function-1, is essential for this suppressive action. Next, we developed stable transfectants of the human osteoblastic U2-OS cell line containing ER alpha or ER beta in combination with an NF-kappa B luciferase reporter construct. In these cell lines, repression of NF-kappa B activity was only mediated through ER alpha and not through ER beta. These findings offer new insights into the specific role of both ER subtypes in bone homeostasis and could eventually help in developing more specific medical intervention strategies for osteoporosis.
