Jose Zamorano

Kaempferol Inhibits IL-4-Induced STAT6 Activation by Specifically Targeting JAK3

IL-4 is involved in several human diseases including allergies, autoimmunity, and cancer. Its effects are mainly mediated through the transcription factor STAT6. Therefore, investigation of compounds that regulate STAT6 activation is of great interest for these diseases. Natural polyphenols are compounds reported to have therapeutic properties in diseases involving IL-4 and STAT6. The aim of this study was to investigate the effect of these compounds in the activation of this transcription factor. We found that in hemopoietic cells from human and mouse origin, some flavonoids were able to inhibit the activation of STAT6 by IL-4. To identify molecular mechanisms, we focused on kaempferol, the compound that showed the greatest inhibitory effect with the lowest cell toxicity. Treatment of cells with kaempferol did not affect activation of Src kinase by IL-4 but did prevent the phosphorylation of JAK1 and JAK3. Further enzymatic analysis demonstrated that kaempferol blocked the in vitro phosphorylation activity of JAK3 without affecting JAK1, suggesting that it specifically targeted JAK3 activity. Accordingly, kaempferol had no effect on STAT6 activation in nonhemopoietic cell lines lacking JAK3, supporting its selective inhibition of IL-4 responses through type I receptors expressing JAK3 but not type II lacking this kinase. The inhibitory effect of kaempferol was also observed in IL-2 but not IL-3-mediated responses and correlated with the inhibition of MLC proliferation. These findings reveal the potential use of kaempferol as a tool for selectively controlling cell responses to IL-4 and, in general, JAK3-dependent responses.

Omeprazole inhibits IL-4 and IL-13 signaling signal transducer and activator of transcription 6 activation and reduces lung inflammation in murine asthma

interaction was lacking. Of 10 SNPs evaluated, only rs8079416 was nominally significant at a P value of .01. There was no interaction with age or smoke exposure. This continues to suggest that the tagging SNPs are not linked strongly to the causal variant or that the causal variant is rare in this population. In conclusion, we confirm the interaction among the 17q12-21 variants, childhood smoke exposure, and pediatric asthma in a white population. However, our data show no age-of-onset effect. Although this study does not refute earlier findings suggesting such an effect, further prospective exploration of interaction between the 17q12-21 variants and age of onset is warranted. James H. Flory, MD, MS Patrick M. Sleiman, PhD Jason D. Christie, MD, MS Kiran Annaiah, MSc Jonathan Bradfield, BS Cecilia E. Kim, BA Joseph Glessner, MSc Marcin Imielinski, MD, PhD Hongzhe Li, PhD Edward C. Frackelton, BA Hou Cuiping, PhD George Otieno, MSc Kelly Thomas, BA Ryan Smith, BA Wendy Glaberson, BA Maria Garris, BA Rosetta Chiavacci, MSc Julian Allen, MD Jonathan Spergel, MD, PhD Robert Grundmeier, MD, PhD Michael Grunstein, MD, PhD Michael Magnusson, MD, PhD Struan F. A. Grant, PhD Klaus Bønnelykke, MD Hans Bisgaard, MD Hakon Hakonarson, MD, PhD From the Center for Applied Genomics, the Division of Pulmonary Medicine, the Division of Allergy and Immunology, the Department of Bioinformatics, and the Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, Pa; the Center for Clinical Epidemiology and Biostatistics, Department of Medicine, University of Pennsylvania, Philadelphia, Pa; and the Department of Health Sciences of the University of Copenhagen, Copenhagen, Denmark. E-mail: hakonarson@ CHOP.EDU. Disclosure of potential conflict of interest: J. H. Flory has received nonprofit grants from FOCUS, the University of Pennsylvania, and the National Institutes of Health. J. D. Christie receives grant support from the National Institutes of Health and has provided legal consultation/expert witness testimony in cases related to mesothelioma in brake workers. J. Allen receives grant support from the National Institutes of Health. J. Spergel receives grant support from Ception and is on the speakers’ bureau for Schering-Plough and AstraZeneca. R. Grundmeier receives grant support from the Agency for Healthcare Research and Quality. M. Magnusson has provided legal consultation/expert witness testimony in cases related to medical malpractice. S. F. A. Grant receives grant support from the National Institutes of Health. H. Bisgaard has been a consultant to and paid lecturer for and holds sponsored grants from Aerocrine, Altana, GlaxoSmithKline, Merck, MedImmune, NeoLab, and Pfizer and has provided legal consultation/expert witness testimony on behalf of NeoLab. The rest of the authors have declared that they have no conflict of interest.

HMG-I(Y) Phosphorylation Status as a Nuclear Target Regulated through Insulin Receptor Substrate-1 and the I4R Motif of the Interleukin-4 Receptor

Interleukin (IL)-4 is a cytokine that regulates both the growth and differentiation of hematopoietic cells. Its ligand binding specificity and important signal transduction mechanisms are conferred by the IL-4 receptor α chain (IL-4Rα). The I4R is a tyrosine-containing motif within IL-4Rα that is critical for proliferative responses to IL-4. Although the I4R also contributes to gene regulation, nuclear targets directly regulated by this motif have not been described. It is shown here that the tyrosine at position 497 in the I4R is critical for regulation of the phosphorylation status of a set of nuclear proteins that includes HMG-I(Y), small non-histone chromosomal proteins involved in the control of gene expression in hematopoietic cell lines. Moreover, IL-4 is unable to induce HMG-I(Y) phosphorylation in insulin receptor substrate-1-deficient cells, and the inhibitor wortmannin completely blocks IL-4 regulation of HMG-I(Y) phosphorylation status but not activation of an IL-4 Stat protein. Taken together, these data indicate that HMG-I(Y) is a nuclear target whose phosphorylation status is regulated through the I4R motif via insulin receptor substrate proteins, independent of activation of the Stat pathway.

Proteolytic Regulation of Activated STAT6 by Calpains

The transcription factor STAT6 plays an important role in cell responses to IL-4. Its activation is tightly regulated. STAT6 phosphorylation is associated with JAKs, whereas dephosphorylation is associated with specific phosphatases. Several studies indicate that proteases can also regulate STAT6. The aim of this study was to investigate the nature of these proteases in mouse T cell lines. We found that STAT6 was degraded in cell extracts by calcium-dependent proteases. This degradation was specifically prevented by calpain inhibitors, suggesting that STAT6 was a target for these proteases. This was supported by the cleavage of STAT6 by recombinant calpains. The proteolytic regulation of STAT6 was more complex in vivo. Calcium signaling was not sufficient to induce STAT6 degradation. However, treatment of IL-4-stimulated cells with calcium ionophores resulted in the absence of phosphorylated STAT6. This effect correlated with the loss of STAT6 protein and was prevented by calpain inhibitors. Cytoplasmic calpains seemed to be responsible for STAT6 degradation. Calpains can target signaling proteins; in this study we found that they can negatively regulate activated STAT6.

Costimulation of resting B lymphocytes alters the IL-4-activated IRS2 signaling pathway in a STAT6 independent manner: implications for cell survival and proliferation

ABSTRACTIL-4 is an important B cell survival and growth factor. IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts. Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3′ kinase in both resting and activated cells. By contrast, association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed. However, IL-4 induced association of IRS2 with GRB2 in B cell blasts. The pattern of IL-4-induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice. While IL-4 alone does not induce activation of MEK, a MEK1 inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts. These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation. Furthermore, proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway.

Phosphatidylcholine-Specific Phospholipase C Activity Is Necessary for the Activation of STAT6

It is well established that Janus kinase (JAK) tyrosine kinases play a key role in the activation of STAT6 by IL-4. In this study, we investigated additional molecules involved in this process. We previously found that IL-4 and TNF-α cooperate in the activation of STAT6 and NF-κB, suggesting that these transcription factors are regulated by common intracellular signaling pathways. To test this hypothesis, we analyzed the effect of known inhibitors of NF-κB on the activation of STAT6. We discovered that inhibitors of phosphatidylcholine-specific phospholipase C (PC-PLC), but not other lipases, blocked the activation of STAT6 by IL-4. The activation of PC-PLC seems to be an early event in IL-4 signaling, because its inhibition abrogated JAK activation and STAT6 tyrosine phosphorylation. Interestingly, we found that the effects of pervanadate and sodium orthovanadate on STAT6 activation correspond to their effect on PC-PLC. Thus, pervanadate by itself activated PC-PLC, JAK, and STAT6, whereas sodium orthovanadate suppressed PC-PLC, JAK, and STAT6 activation by IL-4. We further found that PC-PLC activation is necessary but not sufficient to promote STAT6 activation, and therefore, additional intracellular pathways regulated by IL-4 and pervanadate may collaborate with PC-PLC to signal STAT6 activation. It has been reported that IL-4 signals PC-PLC activation; in this study, we provide evidence that this phospholipase plays a key role in IL-4 signaling.

Su1828 Remission in Proton Pump Inhibitors-Responsive Esophageal Eosinophilia Correlates With Downregulation of Eotaxin-3 and TH2 Cytokines, Similarly to Eosinophilic Esophagitis After Steroids

Background: In eosinophilic esophagitis (EoE), eosinophils invade the esophagus where they degranulate and release very cytotoxic proteins, including major basic protein (MBP-1). Currently the diagnosis of EoE necessitates endoscopy with biopsy that characterizes less than 2% of the esophagus. Furthermore, the inflammation is patchy, necessitating numerous biopsies to ensure that the involved area has been adequately sampled. Improved methods are needed to characterize the eosinophil density throughout the esophagus and to thoroughly understand this disease. Aim: To prepare the first MBP-1 specific ultrasound contrast agents to facilitate the detection of EoE associated inflammation by tethering EoE specific antibodies to their surfaces and demonstrating the binding of these agents to eosinophil proteins on ex vivo monkey esophagi. Method: The contrast agent was synthesized by preparing 2 mg/ mL of insulin in HCl (pH 1.6) at 65C for 12 hours. The resulting particles were incubated withmaleimide activated streptavidin. Purified antibodies specific to MBP-1 were biotinylated and reacted with the streptavidin functionalized fibers to form an insulin aggregate-steptavidin-biotin antibody complex. This complex comprises the MBP-1 specific ultrasound contrast agent. A Macaca monkey esophagus, incubated overnight in MBP-1, was filled with this solution. Ultrasound images were taken before and after washing at 15 MHz. Results: Insulin aggregates were observed using ultrasound. Incubating the insulin complex in monkey esophagi coated with MBP-1 yields an extra contrast layer at the inner wall of the esophagus, demonstrating the specific binding of these agents to eosinophil granule proteins. No such layer is visualized in control samples (not treated with MBP-1). Discussion: This is the first reported use of insulin particles as contrast agents for ultrasound. These findings indicate that insulin aggregates provide clear ultrasound contrast in liquid environments. When coupled with disease specific antibodies, these novel agents have the potential to be used for detection, minimizing the need for anesthesia or radiation associated with other diagnostic or imaging modalities. This finding is important because it provides a new avenue for clinical detection of EoE.

Treatment of cells with n-alpha-tosyl-l-phenylalanine-chloromethyl ketone induces the proteolytic loss of STAT6 transcription factor

The implication of the STAT6 transcription factor in several human diseases makes the regulation of its activity a topic of great biological interest. The activation of this transcription factor is tightly regulated by kinases, phosphatases, and proteases. The initial aim of this study was to investigate the utility of protease inhibitors in controlling STAT6 activation. Among all inhibitors analyzed, n-alpha-tosyl-L-phenylalanine-chloromethyl ketone (TPCK) was found to inhibit the IL-4-induced STAT6 activation. Unexpectedly, this inhibition was accompanied by a loss of STAT6 protein. Thus, TPCK promoted the loss of STAT6 by a mechanism sensitive to the serine-protease inhibitor 4-(2-aminoetyl)-benzenesulfonyl fluoride. However, the effects of TPCK seemed not to be mediated by its protease inhibitory activity since multiple protease inhibitors tested had no effect on STAT6 expression. The results found suggest that the effect of TPCK was mediated by its alkylating activity. Thus, cysteine reactive and thiol antioxidant compounds prevented the loss of STAT6 induced by TPCK. The reactivity of thiol groups on STAT6 was moreover demonstrated with biotinylated sulfhydryl-reactive compounds. Analysis of other signaling molecules indicated that STAT5, but not other STATs, Shc, or c-Rel, was also affected by TPCK, suggesting a common downregulatory mechanism for STAT6 and STAT5. These results reveal a novel mechanism of action of TPCK in inducing a selective loss of STAT proteins. These findings may have implications for diseases in which STAT proteins are involved.

Significance of Including a Surrogate Arousal for Sleep Apnea-Hypopnea Syndrome Diagnosis by Respiratory Polygraphy

RATIONALE Respiratory polygraphy is an accepted alternative to polysomnography (PSG) for sleep apnea/hypopnea syndrome (SAHS) diagnosis, although it underestimates the apnea-hypopnea index (AHI) because respiratory polygraphy cannot identify arousals. OBJECTIVES We performed a multicentric, randomized, blinded crossover study to determine the agreement between home respiratory polygraphy (HRP) and PSG, and between simultaneous respiratory polygraphy (respiratory polygraphy with PSG) (SimultRP) and PSG by means of 2 AHI scoring protocols with or without hyperventilation following flow reduction considered as a surrogate arousal. METHODS We included suspected SAHS patients from 8 hospitals. They were assigned to home and hospital protocols at random. We determined the agreement between respiratory polygraphy AHI and PSG AHI scorings using Bland and Altman plots and diagnostic agreement using receiver operating characteristic (ROC) curves. The agreement in therapeutic decisions (continuous positive airway pressure treatment or not) between HRP and PSG scorings was done with likelihood ratios and post-test probability calculations. RESULTS Of 366 randomized patients, 342 completed the protocol. AHI from HRP scorings (with and without surrogate arousal) had similar agreement with PSG. AHI from SimultRP with surrogate arousal scoring had better agreement with PSG than AHI from SimultRP without surrogate arousal. HRP with surrogate arousal scoring had slightly worse ROC curves than HRP without surrogate arousal, and the opposite was true for SimultRP scorings. HRP with surrogate arousal showed slightly better agreement with PSG in therapeutic decisions than for HRP without surrogate arousal. CONCLUSION Incorporating a surrogate arousal measure into HRP did not substantially increase its agreement with PSG when compared with the usual procedure (HRP without surrogate arousal).

Distinguishing Eosinophilic Esophagitis from Gastroesophageal Reflux Disease upon PPI Refractoriness: What about PPI-Responsive Esophageal Eosinophilia?

on PPI therapy [3–7] , leading to the description of a new potential disease phenotype in the 2011 updated guidelines, i.e. PPI-responsive esophageal eosinophilia (PPIRee) [8] . Whether these patients represent a sub-phenotype of GERD, EoE or a combined mechanism of both disorders remains unknown. In the aforementioned reports neither histological specific features [6] , pH esophageal monitoring [4, 6] nor quantitative immunohistochemistry for mast cells [7] were capable to discern between PPIRee and EoE. In addition, a recent investigation has demonstrated that the secretion of eotaxin-3 could be blocked by PPI therapy in esophageal squamous epithelial cell lines from patients with EoE stimulated with either IL-13 or IL-4 [9] . These findings suggest that PPI therapy can have anti-inflammaDear Sir, We read with great interest the article by von Arnim et al. [1] recently published in your journal. The authors show that eosinophilic esophagitis (EoE) can be easily suspected before upper endoscopy on the basis of some laboratory and clinical markers. The authors conclude that their set of markers ‘allow physicians to distinguish EoE from gastroesophageal reflux disease (GERD) even before upper gastrointestinal endoscopy’, but things may not be as simple as they seem. According to the 2007 first consensus EoE guidelines [2] , it was established that GERD and EoE could be easily distinguished upon responsiveness to PPI therapy. However, recent reports since 2006 have shown that up to 40–50% of pediatric and adult patients with dense esophageal eosinophilia achieve complete remission Published online: February 17, 2012