Search

The integrity of the blood-labyrinth barrier (BLB) is essential for inner ear homeostasis, regulating the ionic composition of endolymph and perilymph and preventing harmful substance entry. Endothelial hyperpermeability, central in inflammatory and immune responses, is managed through complex intercellular communication and molecular signaling pathways. Recent studies link BLB permeability dysregulation to auditory pathologies like acoustic trauma, autoimmune inner ear diseases, and presbycusis. Polymorphonuclear granulocytes (PMNs), or neutrophils, significantly modulate vascular permeability, impacting endothelial barrier properties. Neutrophil extracellular traps (NETs) are involved in diseases with autoimmune and autoinflammatory bases. The present study evaluated the impact of NETs on a BLB cellular model using a Transwell ® setup. Our findings revealed a concentration-dependent impact of NETs on human inner ear-derived endothelial cells. In particular, endothelial permeability markers increased, as indicated by reduced transepithelial electrical resistance, enhanced dextran permeability, and downregulated junctional gene expression ( ZO1 , OCL , and CDH5 ). Changes in cytoskeletal architecture were also observed. These preliminary results pave the way for further research into the potential involvement of NETs in BLB impairment and implications for auditory disorders.

Hearing loss is one of the 10 leading causes of disability worldwide. No drug therapies are currently available to protect or restore hearing. Inner ear auditory hair cells and the blood-labyrinth barrier (BLB) are critical for normal hearing, and the BLB between the systemic circulation and stria vascularis is crucial for maintaining cochlear and vestibular homeostasis. BLB defects are associated with inner ear diseases that lead to hearing loss, including vascular malformations, inflammation, and Meniere's disease (MD). Antibodies against proteins in the inner ear and cytokines in the cochlea, including IL-1α, TNF-α, and NF-kβ, are detected in the blood of more than half of MD patients. There is also emerging evidence of inner ear inflammation in some diseases, including MD, progressive sensorineural hearing loss, otosclerosis, and sudden deafness. Here, we examined the effects of TNF-α, IL6, and LPS on human stria vascularis-derived primary endothelial cells cultured together with pericytes in a Transwell system. By measuring trans-endothelial electrical resistance, we found that TNF-α causes the most significant disruption of the endothelial barrier. IL6 had a moderate influence on the barrier, whereas LPS had a minimal impact on barrier integrity. The prominent effect of TNF-α on the barrier was confirmed in the expression of the major junctional genes responsible for forming the tight endothelial monolayer, the decreased expression of ZO1 and OCL . We further tested permeability using 2 μg of daptomycin (1,619 Da), which does not pass the BLB under normal conditions, by measuring its passage through the barrier by HPLC. Treatment with TNF-α resulted in higher permeability in treated samples compared to controls. LPS-treated cells behaved similarly to the untreated cells and did not show differences in permeability compared to control. The endothelial damage caused by TNF-α was confirmed by decreased expression of an essential endothelial proteoglycan, syndecan1. These results allowed us to create an inflammatory environment model that increased BLB permeability in culture and mimicked an inflammatory state within the stria vascularis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sekulic, Puche, Bodmer and Petkovic.)

Hearing loss is one of the leading causes of disability worldwide, usually as a result of hair cell damage in the inner ear due to aging, acoustic trauma, or exposure to antibiotics or chemotherapy. No drug therapies can protect or restore hearing and current in vitro and animal models used in drug discovery have a very low success rate, mostly due to major differences in anatomy and accessibility of the inner ear environment between species. The blood-labyrinth barrier (BLB) in the stria vascularis is a highly specialized capillary network that controls exchanges between the blood and interstitial space in the cochlea. The BLB is critical for normal hearing, functioning as a physical, transport, and metabolic barrier. To address its complexity and accessibility, we created the first micro-engineered human model of BLB on a chip using autogenous progenitor cells from adult temporal bones. We successfully isolated the BLB from post-mortem human tissue and established an endothelial cell and pericyte culture system on a BLB chip. Using biocompatible materials, we fabricated sustainable two chamber chips. We validated the size-dependent permeability limits of our BLB model by measuring the permeability to daptomycin (molecular weight 1.6 kDa) and midazolam (molecular weight 325.78 Da). Daptomycin did not pass through the BLB layer, whereas midazolam readily passed through the BLB in our system. Thus, our BLB-chip mimicked the integrity and permeability of human stria vascularis capillaries. This represents a major step towards establishing a reliable model for the development of hearing loss treatments., Competing Interests: The authors have no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Acoustic trauma, autoimmune inner ear disease, and presbycusis feature loss of the integrity of the blood-labyrinth barrier (BLB). Normal BLB function depends on endothelial structural integrity, which is supported and maintained by tight junctions and adherens junctions within the microvascular endothelial layer. When these junctions are disrupted, vascular leakage occurs. Tight junctions and adherens junctions are functionally and structurally linked, but the exact signaling pathways underlying their interaction remain unknown. In addition, solute carriers (SC) are essential for optimal exchange through BLB. Previously, we found that SC family member, the sodium-hydrogen exchanger NHE6, was expressed in all wildtype cochlear tissues, and that Nhe6 -knockout mice displayed moderate hearing loss. Moreover, NHE6 depletion affected Trk protein turnover and endosomal signaling. Here, we investigated whether NHE6 might impact BLB integrity. We found that Nhe6 -knockout, BLB-derived endothelial cells showed reduced expression of major junctional genes: Tjp1 , F11r , Ocln , Cdh5 , and Cldn5 . Co-culturing BLB-derived endothelial cells with pericytes and/or perivascular resident macrophage-like melanocytes in a transwell system showed that monolayers of Nhe6 -knockout BLB-derived cells had lower electrical resistance and higher permeability, compared to wildtype endothelial monolayers. Additionally, another SC, NKCC1, which was previously linked to congenital deafness, was downregulated in our Nhe6 -knockout mouse model. Blocking NKCC1 with a NKCC1-specific inhibitor, bumetanide, in wildtype BLB-derived endothelial cells also caused the downregulation of major junctional proteins, particularly Tjp1 and F11r , which encode the zonula occludens and junctional adhesion molecule-1 proteins, respectively. Moreover, bumetanide treatment increased cell permeability. In conclusion, we showed that the lack or inhibition of NHE6 or NKCC1 affected the permeability of endothelial BLB-derived cells. These findings suggested that NHE6 and NKCC1 could serve as potential targets for modifying BLB permeability to facilitate drug delivery across the BLB to the cochlea or to protect the cochlea from ototoxic insults., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sekulic-Jablanovic, Paproth, Sgambato, Albano, Fuster, Bodmer and Petkovic.)

This study focuses on the ability of the Global Precipitation Measurement (GPM) passive microwave sensors to detect and provide quantitative precipitation estimates (QPE) for extreme lake-effect snowfall events over the U.S. lower Great Lakes region. GPM Microwave Imager (GMI) high-frequency channels can clearly detect intense shallow convective snowfall events. However, GMI Goddard Profiling (GPROF) QPE retrievals produce inconsistent results when compared with the Multi-Radar Multi-Sensor (MRMS) ground-based radar reference dataset. While GPROF retrievals adequately capture intense snowfall rates and spatial patterns of one event, GPROF systematically underestimates intense snowfall rates in another event. Furthermore, GPROF produces abundant light snowfall rates that do not accord with MRMS observations. Ad hoc precipitation-rate thresholds are suggested to partially mitigate GPROF's overproduction of light snowfall rates. The sensitivity and retrieval efficiency of GPROF to key parameters (2-m temperature, total precipitable water, and background surface type) used to constrain the GPROF a priori retrieval database are investigated. Results demonstrate that typical lake-effect snow environmental and surface conditions, especially coastal surfaces, are underpopulated in the database and adversely affect GPROF retrievals. For the two presented case studies, using a snow-cover a priori database in the locations originally deemed as coastline improves retrieval. This study suggests that it is particularly important to have more accurate GPROF surface classifications and better representativeness of the a priori databases to improve intense lake-effect snow detection and retrieval performance.

Noise trauma, infection, and ototoxic drugs are frequent external causes of hearing loss. With no pharmacological treatments currently available, understanding the mechanisms and pathways leading to auditory hair cell (HC) damage and repair is crucial for identifying potential pharmacological targets. Prior research has implicated increased reactive oxygen species (ROS) and inflammation as general mechanisms of hearing loss common to diverse causes. Novel targets of these two key mechanisms of auditory damage may provide new paths toward the prevention and treatment of hearing loss. Pioglitazone, an oral antidiabetic drug from the class of thiazolidinediones, acts as an agonist of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) and is involved in the regulation of lipid and glucose metabolism. PPAR-γ is an important player in repressing the expression of inflammatory cytokines and signaling molecules. We evaluated the effects of pioglitazone in the mouse Organ of Corti (OC) explants to characterize its influence on signaling pathways involved in auditory HC damage. The OC explants was cultured with pioglitazone, gentamicin, or a combination of both agents. Pioglitazone treatment resulted in significant repression of interferon (IFN)-α and -gamma pathways and downstream cytokines, as assessed by RNA sequencing and quantitative PCR gene expression assays. More detailed investigation at the single gene and protein level showed that pioglitazone mediated its anti-inflammatory effects through alterations of the Toll-like receptor (TLR) and STAT pathways. Together, these results indicate that pioglitazone significantly represses IFN and TLR in the cochlea, dampening the activity of gentamicin-induced pathways. These data support our previous results demonstrating significant protection of auditory HCs in the OC explants exposed to pioglitazone and other PPAR-targeted agents., (Copyright © 2020 Sekulic-Jablanovic, Wright, Petkovic and Bodmer.)

Hair cell (HC) degeneration causes hearing loss in millions of people worldwide. Aminoglycoside exposure is one major cause of sensory HC damage. Aminoglycosides generate free radicals within the inner ear, permanently damaging sensory cells, and thus causing hearing loss. Hearing protection requires strategies to overcome the apparently irreversible loss of HCs in mammals. The nuclear factor of activated T cells (NFAT) inhibitor 11R-VIVIT reportedly protects HCs from gentamicin toxicity. Here we investigated whether the combination of 11R-VIVIT with the antioxidant L-carnitine or N-acetylcysteine could protect mouse cochlear HCs from gentamicin damage. Compared to single-component treatment, combined treatment with 11R-VIVIT plus L-carnitine yielded significant protection from gentamicin, and 11R-VIVIT plus N-acetylcysteine provided almost complete protection of HCs from gentamicin. Caspase activity in organ of Corti was significantly reduced by combined treatment with 11R-VIVIT + N-acetylcysteine + gentamicin, compared to 11R-VIVIT + gentamicin or gentamicin alone. Analysis of relative gene expression by qPCR revealed down-regulation of the pro-apoptotic genes Fasl and Casp9, and up-regulation of the antioxidant genes Hmox1 and Nrf2 after treatment with 11R-VIVIT + N-acetylcysteine + gentamicin, compared to single-compound treatment or gentamicin alone in cultures. Selective NFAT inhibition by 11R-VIVIT may be a good strategy for preventing gentamicin-induced HC damage. L-carnitine and N-acetylcysteine, with their ROS-reducing properties, contribute to the synergistic effectiveness with 11R-VIVIT by decreasing ROS-induced NFAT translocation. Our data suggest that a combined approach of NFAT inhibition together with an antioxidant, like N-acetylcysteine, could be useful for hearing loss treatment and/or prevention. Cover Image for this issue: https://doi.org/10.1111/jnc.14759., (© 2019 International Society for Neurochemistry.)

Aim: Newly synthesized platinum(IV) complexes with ethylenediamine-N,N'-diacetate ligands (EDDA-type) (butyl-Pt and pentyl-Pt) were investigated against two cancer (A549 lung, and HTB 140 melanoma) and one non-cancerous (MRC-5 embryonic lung fibroblast) human cell lines., Materials and Methods: The effects of these agents were compared with those of cisplatin after 6-, 24- and 48-h treatment. Sulforhodamine-B (SRB) assay was performed to estimate the cytotoxic effect, while the inhibitory effect on cell proliferation was measured using 5-bromo-2,-deoxyuridine (BrdU) incorporation assay. Cell cycle analysis was performed by flow cytometry. Type of cell death induced by these agents was determined by electrophoretic analysis of DNA, flow cytometry and by western blot analysis of proteins involved in induction of apoptosis. The effects of gamma irradiation, alone and in combination with platinum-based compounds, were examined by clonogenic and SRB assays., Results: All examined platinum-based compounds had inhibitory and antiproliferative effects on A549 cells, but not on HTB140 and MRC-5 cells. Butyl-Pt, pentyl-Pt and cisplatin arrested the cell cycle in the S-phase and induced apoptotic cell death via regulation of expression of B-cell lymphoma 2 (BCL2) and BCL2-associated X (BAX) proteins. Platinum-based compounds increased the sensitivity of A549 cells to gamma irradiation. Butyl-Pt and pentyl-Pt showed better antitumour effects against A549 cells than did cisplatin, by interfering in cell proliferation and the cell cycle, and by triggering apoptosis., Conclusion: The effects of gamma irradiation on tumour cells may be amplified by pre-treatment of cells with platinum-based compounds., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Acid-base homeostasis is critical for normal growth, development, and hearing function. The sodium-hydrogen exchanger 6 (NHE6), a protein mainly expressed in early and recycling endosomes, plays an important role in regulating organellar pH. Mutations in NHE6 cause complex, slowly progressive neurodegeneration. Little is known about NHE6 function in the mouse cochlea. Here, we found that all NHE isoforms were expressed in wild-type (WT) mouse cochlea. Nhe6 knockout (KO) mice showed significant hearing loss compared to WT littermates. Immunohistochemistry in WT mouse cochlea showed that Nhe6 was localized in the organ of Corti (OC), spiral ganglion (SG), stria vascularis (SV), and afferent nerve fibres. The middle and the inner ears of WT and Nhe6 KO mice were not different morphologically. Given the putative role of NHE6 in early endosomal function, we examined Rab GTPase expression in early and late endosomes. We found no change in Rab5, significantly lower Rab7, and higher Rab11 levels in the Nhe6 KO OC, compared to WT littermates. Because Rabs mediate TrkB endosomal signalling, we evaluated TrkB phosphorylation in the OCs of both strains. Nhe6 KO mice showed significant reductions in TrkB and Akt phosphorylation in the OC. In addition, we examined genes used as markers of SG type I (Slc17a7, Calb1, Pou4f1, Cal2) and type II neurons (Prph, Plk5, Cacna1g). We found that all marker gene expression levels were significantly elevated in the SG of Nhe6 KO mice, compared to WT littermates. Anti-neurofilament factor staining showed axon loss in the cochlear nerves of Nhe6 KO mice compared to WT mice. These findings indicated that BDNF/TrkB signalling was disrupted in the OC of Nhe6 KO mice, probably due to TrkB reduction, caused by over acidification in the absence of NHE6. Thus, our findings demonstrated that NHEs play important roles in normal hearing in the mammalian cochlea.

Gentamicin is a widely used antibiotic for the treatment of gram-negative bacterial infections; however, its use often results in significant and permanent hearing loss. Hearing loss resulting from hair cell (HC) degeneration affects millions of people worldwide, and one major cause is the loss of sensory HCs in the inner ear due to aminoglycoside exposure. Strategies to overcome the apparently irreversible loss of HCs in mammals are crucial for hearing protection. Here, we report that the somatostatin analog pasireotide protects mouse cochlear HCs from gentamicin damage using a well-established in vitro gentamicin-induced HC loss model and that the otoprotective effects of pasireotide are due to Akt up-regulation via the PI3K-Akt signal pathway activation. We demonstrate active caspase signal in organ of Corti (OC) explants exposed to gentamicin and show that pasireotide treatment activates survival genes, reduces caspase signal, and increases HC survival. The neuropeptide somatostatin and its selective analogs have provided neuroprotection by activating five somatostatin receptor (SSTR1-SSTR5) subtypes. Pasireotide has a high affinity for SSTR2 and SSTR5, and the addition of SSTR2- and SSTR5-specific antagonists leads to a loss of protection. The otoprotective effects of pasireotide were also observed in a gentamicin-injured animal model. In vivo studies have shown that 13 days of subcutaneous pasireotide application prevents gentamicin-induced HC death and permanent hearing loss in mice. Auditory brainstem response analysis confirmed the protective effect of pasireotide, and we found a significant threshold shift at all measured frequencies (4, 8, 16, 24, and 32 kHz). Together, these findings indicate that pasireotide is a novel otoprotective peptide acting via the PI3K-Akt pathway and may be of therapeutic value for HC protection from ototoxic insults.

Insulin receptors are expressed on nerve cells in the mammalian brain, but little is known about insulin signaling and the expression of the insulin receptor (IR) and glucose transporters in the cochlea. We performed immunohistochemistry and gene/protein expression analysis to characterize the expression pattern of the IR and glucose transporters in the mouse organ of Corti (OC). We also performed glucose uptake assays to explore the action of insulin and the effects of pioglitazone, an insulin sensitizer, on glucose transport in the OC. Western blots of protein extracts from OCs showed high expression of IR and glucose transporter 3 (GLUT3). Immunohistochemistry demonstrated that the IR is specifically expressed in the supporting cells of the OC. GLUT3 was found in outer and inner hair cells, in the basilar membrane (BM), the stria vascularis (SV), Reissner's membrane and spiral ganglion neurons (SGN). Glucose transporter 1 (GLUT1) was detected at low levels in the BM, SV and Reissner's membrane, and showed high expression in the SGN. Fluorescence glucose uptake assays revealed that hair cells take up glucose and that addition of insulin (10 nM or 1 µM) approximately doubled the rate of uptake. Pioglitazone conferred a small but nonsignificant potentiation of glucose uptake at the highest concentration of insulin. Gene expression analysis confirmed expression of IR, GLUT1 and GLUT3 mRNA in the OC. Pioglitazone significantly upregulated IR and GLUT1 mRNA expression, which was further increased by insulin. Together, these data show that insulin-stimulated glucose uptake occurs in the OC and may be associated with upregulation of both the IR and GLUT1., (© 2019 S. Karger AG, Basel.)

Various insults cause ototoxicity in mammals by increasing oxidative stress leading to apoptosis of auditory hair cells (HCs). The thiazolidinediones (TZDs; e.g., pioglitazone) and fibrate (e.g., fenofibrate) drugs are used for the treatment of diabetes and dyslipidemia. These agents target the peroxisome proliferator-activated receptors, PPARγ and PPARα, which are transcription factors that influence glucose and lipid metabolism, inflammation, and organ protection. In this study, we explored the effects of pioglitazone and other PPAR agonists to prevent gentamicin-induced oxidative stress and apoptosis in mouse organ of Corti (OC) explants. Western blots showed high levels of PPARγ and PPARα proteins in mouse OC lysates. Immunofluorescence assays indicated that PPARγ and PPARα proteins are present in auditory HCs and other cell types in the mouse cochlea. Gentamicin treatment induced production of reactive oxygen species (ROS), lipid peroxidation, caspase activation, PARP-1 cleavage, and HC apoptosis in cultured OCs. Pioglitazone mediated its anti-apoptotic effects by opposing the increase in ROS induced by gentamicin, which inhibited the subsequent formation of 4-hydroxy-2-nonenal (4-HNE) and activation of pro-apoptotic mediators. Pioglitazone mediated its effects by upregulating genes that control ROS production and detoxification pathways leading to restoration of the reduced:oxidized glutathione ratio. Structurally diverse PPAR agonists were protective of HCs. Pioglitazone (PPARγ-specific), tesaglitazar (PPARγ/α-specific), and fenofibric acid (PPARα-specific) all provided >90% protection from gentamicin toxicity by regulation of overlapping subsets of genes controlling ROS detoxification. This study revealed that PPARs play important roles in the cochlea, and that PPAR-targeting drugs possess therapeutic potential as treatment for hearing loss.

Hearing impairment is a global health problem with a high socioeconomic impact. Damage to auditory hair cells (HCs) in the inner ear as a result of aging, disease, trauma, or toxicity, underlies the majority of cases of sensorineural hearing loss. Previously we demonstrated that the Ca 2+ -sensitive neuropeptide, somatostatin (SST), and an analog, octreotide, protect HCs from gentamicin-induced cell death in vitro. Aminoglycosides such as gentamicin trigger a calcium ion influx (Ca 2+ ) that activates pro-apoptotic signaling cascades in HCs. SST binding to the G-protein-coupled receptors (SSTR1-SSTR5) that are directly linked to voltage-dependent Ca 2+ channels inhibits Ca 2+ channel activity and associated downstream events. Here, we report that the SST analog pasireotide, a high affinity ligand to SSTRs 1-3, and 5, with a longer half-life than octreotide, prevents gentamicin-induced HC death in the mouse organ of Corti (OC). Explant experiments using OCs derived from SSTR1 and SSTR1and 2 knockout mice, revealed that SSTR2 mediates pasireotide's anti-apoptotic effects. Mechanistically, pasireotide prevented a nuclear translocation of the Ca 2+ -sensitive transcription factor, nuclear factor of activated T cells (NFAT), which is ordinarily provoked by gentamicin in OC explants. Direct inhibition of NFAT with 11R-VIVIT also prevented the gentamicin-dependent nuclear translocation of NFAT and apoptosis. Both pasireotide and 11R-VIVIT partially reversed the effects of gentamicin on the expression of downstream survival targets (NMDA receptor and the regulatory subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase, PI3K). These data suggest that SST analogs antagonize aminoglycoside-induced cell death in an NFAT-dependent fashion. SST analogs and NFAT inhibitors may therefore offer new therapeutic possibilities for the treatment of hearing loss., (© 2016 International Society for Neurochemistry.)

Isolated GH deficiency (IGHD) type II, the autosomal dominant form of GHD, is mainly caused by mutations that affect splicing of GH-1. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH isoform that reduces the accumulation and secretion of wild-type-human GH (wt-hGH). Usually, isolated GHD type II patients are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the 17.5-kDa GH isoform on the pituitary gland, which can eventually lead to other hormonal deficiencies. Here, we tested the possibility to restore the constitutive splicing pattern of GH-1 by using butyrate, a drug that mainly acts as histone deacetylase inhibitor. To this aim, wt-hGH and/or different hGH-splice site mutants (GH-IVS3+2, GH-IVS3+6, and GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GHRHR) (GC-GHRHR). Upon butyrate treatment, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed increased GH transcript level, intracellular GH content, and GH secretion when compared with the corresponding untreated condition. The effect of butyrate was most likely mediated by the alternative splicing factor/splicing factor 2. Overexpression of alternative ASF/SF2 in the same experimental setting, indeed, promoted the amount of full-length transcripts thus increasing synthesis and secretion of the 22-kDa GH isoform. In conclusion, our results support the hypothesis that modulation of GH-1 splicing pattern to increase the 22-kDa GH isoform levels can be clinically beneficial and hence a crucial challenge in GHD research.

Butyrate is a short-chain fatty acid (SCFA) closely related to the ketone body ß-hydroxybutyrate (BHB), which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH) rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR), GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH)-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

Several monogenic defects have been reported to be associated with idiopathic short stature. Focusing on growth hormone receptor (GHR)-gene alterations, the heterozygosity of the same gene defect may be associated with a range of growth deficits. We found a heterozygous mutation (V144I) within exon 6 of the GHR gene in a patient with a low level of insulin-like growth factor I (IGF-I), normal level of GH, and severe short stature. Despite the lack of statistical difference, an overall tendency for reduced wt-GH-induction of GHR activation and Jak/Stat signalling in cells transiently expressing GHR-V144I alone or co-expressing wt-GHR compared to cells expressing only wt-GHR was found when GH doses were increased. Our results suggest that, although GHR sequence variants are responsible for some functional alterations commonly observed in children with idiopathic short stature, these changes may not explain all the height deficits observed in these subjects.

Background: Aggregation of growth hormone (GH) required for its proper storage in granules is facilitated by zinc (Zn(2+)) transported by specific zinc transporters in and out of the regulated secretory pathway. Slc30a5 (ZnT5) was reported to have the highest gene expression among all zinc transporters in primary mouse pituitary cells while ZnT5-null mice presented with abnormal bone development and impaired growth compared to wild-type counterparts., Methods: In vitro studies performed in GH3 cells, a rat pituitary cell line that endogenously produces rat GH (rGH), included analysis of: cytoplasmic Zn(2+) pool changes after altering rSlc30a5 expression (luciferase assay), rZnT5 association with different compartments of the regulated secretory pathway (confocal microscopy), and the rGH secretion after rSlc30a5 knock-down (Western blot)., Results: Confocal microscopy demonstrated high co-localization of rZnT5 with ER and Golgi (early secretory pathway) while siRNA-mediated knock-down of rSlc30a5 gene expression led to a significant reduction in rGH secretion. Furthermore, altered expression of rSlc30a5 (knock-down/overexpression) evoked changes in the cytoplasmic Zn(2+) pool indicating its important role in mediating Zn(2+) influx into intracellular compartments of the regulated secretory pathway., Conclusion: Taken together, these results suggest that ZnT5 might play an important role in regulated GH secretion that is much greater than previously anticipated., (© 2014 S. Karger AG, Basel.)

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn(2+) binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn(2+) from culture medium using N,N,N',N'-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn(2+) chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn(2+) to culture medium increased intracellular free Zn(2+) levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn(2+) levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn(2+) as well as altering intracellular free Zn(2+) content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.

Objective: Short stature caused by biologically inactive GH is clinically characterized by lack of GH action despite normal-high secretion of GH, pathologically low IGF1 concentrations and marked catch-up growth on GH replacement therapy., Design and Methods: Adopted siblings (girl and a boy) of unknown family history were referred for assessment of short stature (-4.5 and -5.6 SDS) at the age of 10 and 8.1 years respectively. They had delayed bone ages (6.8 and 4.5 years), normal GH peaks at stimulation tests, and severely reduced IGF1 concentrations (-3.5 and -4.0 SDS). Genetic analysis of the GH1 gene showed a heterozygous P59S mutation at position involved in binding to GH receptor (GHR)., Results: Isoelectric focusing analysis of secreted GH in patient serum revealed the presence of higher GH-P59S peak compared with that of wt-GH. Furthermore, computational simulation of GH-P59S binding to GHR suggested problems in correct binding of the mutant to the GHR. In vitro GHR binding studies revealed reduced binding affinity of GH-P59S for GHR (IC₅₀, 30  ng/ml) when compared with the wt-GH (IC₅₀, 11.8  ng/ml) while a significantly decreased ability of the mutant to activate the Jak2/Stat5 signaling pathway was observed at physiological concentrations of 25-100  ng/ml., Conclusions: The clinical and biochemical data of our patients support the diagnosis of partial bioinactive GH syndrome. The higher amount of GH-P59S secreted in their circulation combined with its impact on the wt-GH function on GHR binding and signaling may alter GHR responsiveness to wt-GH and could ultimately explain severe short stature found in our patients.

Suboptimal dietary zinc (Zn(2+)) intake is increasingly appreciated as an important public health issue. Zn(2+) is an essential mineral, and infants are particularly vulnerable to Zn(2+) deficiency, as they require large amounts of Zn(2+) for their normal growth and development. Although term infants are born with an important hepatic Zn(2+) storage, adequate Zn(2+) nutrition of infants mostly depends on breast milk or formula feeding, which contains an adequate amount of Zn(2+) to meet the infants' requirements. An exclusively breast-fed 6 months old infant suffering from Zn(2+) deficiency caused by an autosomal dominant negative G87R mutation in the Slc30a2 gene (encoding for the zinc transporter 2 (ZnT-2)) in the mother is reported. More than 20 zinc transporters characterized up to date, classified into two families (Slc30a/ZnT and Slc39a/Zip), reflect the complexity and importance of maintaining cellular Zn(2+) homeostasis and dynamics. The role of ZnTs is to reduce intracellular Zn(2+) by transporting it from the cytoplasm into various intracellular organelles and by moving Zn(2+) into extracellular space. Zips increase intracellular Zn(2+) by transporting it in the opposite direction. Thus the coordinated action of both is essential for the maintenance of Zn(2+) homeostasis in the cytoplasm, and accumulating evidence suggests that this is also true for the secretory pathway of growth hormone.

Human growth hormone (GH) causes a variety of physiological and metabolic effects in humans and plays a pivotal role in postnatal growth. In somatotroph cells of the anterior pituitary, GH is stored in concentrated forms in secretory granules to be rapidly released upon GH-releasing hormone stimulation. During the process of secretory granule biogenesis, self-association of GH occurs in the compartments of the early secretory pathway (endoplasmic reticulum and Golgi complex). Since this process is greatly facilitated by the presence of zinc ions, it is of importance to understand the potential role of zinc transporters that participate in the fine-tuning of zinc homeostasis and dynamics, particularly in the early secretory pathway. Thus, the role of zinc transporters in supplying the secretory pathway with the sufficient amount of zinc required for the biogenesis of GH-containing secretory granules is essential for normal secretion. This report, illustrated by a clinical case report on transient neonatal zinc deficiency, focuses on the role of zinc in GH storage in the secretory granules and highlights the role of specific zinc transporters in the early secretory pathway. © 2013 S. Karger AG, Basel.

Objective: A number of mathematical models for predicting growth and final height outcome have been proposed to enable the clinician to 'individualize' growth-promoting treatment. However, despite optimizing these models, many patients with isolated growth hormone deficiency (IGHD) do not reach their target height. The aim of this study was to analyse the impact of polymorphic genotypes [CA repeat promoter polymorphism of insulin-like growth factor-I (IGF-I) and the -202 A/C promoter polymorphism of IGF-Binding Protein-3 (IGFBP-3)] on variable growth factors as well as final height in severe IGHD following GH treatment. DESIGN, PATIENTS AND CONTROLS: One hundred seventy eight (IGF-I) and 167 (IGFBP-3) subjects with severe growth retardation because of IGHD were studied. In addition, the various genotypes were also studied in a healthy control group of 211 subjects., Results: The frequency of the individual IGF-I (CA)(n) repeats ranging from 10 to 24, with the most frequent allele containing CA(19), was similar in controls and in IGHD subjects. However, in controls, the pooled CA(19) and CA(20) as well as -202 A IGFBP-3 alleles were significantly (P < 0·01 and P < 0·001) more common in the taller [≥2 to 0 standard deviation score (SDS)] when compared with the shorter subgroup (<0 to ≤-2 SDS). Overall, the effect of recombinant human growth hormone (rhGH) replacement did not reveal any difference between the various genotypes in terms of final height. Independent of their genotype, all subjects showed a slightly lower adult height SDS compared with midparental height SDS., Conclusion: Our results indicate that in patients with severe IGHD, although the various IGF-I and IGFBP-3 genotypes may play a role in GH responsiveness, there was no effect on final height., (© 2012 Blackwell Publishing Ltd.)

Endocrine and neuroendocrine cells differ from cells which rapidly release all their secreted proteins in that they store some secretory proteins in concentrated forms in secretory granules to be rapidly released when cells are stimulated. Protein aggregation is considered as the first step in the secretory granule biosynthesis and, at least in the case of prolactin and growth hormone, greatly depends on zinc ions that facilitate this process. Hence, regulation of cellular zinc transport especially that within the regulated secretory pathway is of importance to understand. Various zinc transporters of Slc30a/ZnT and Slc39a/Zip families have been reported to fulfil this role and to participate in fine tuning of zinc transport in and out of the endoplasmic reticulum, Golgi complex and secretory granules, the main cellular compartments of the regulated secretory pathway. In this review, we will focus on the role of zinc in the formation of hormone-containing secretory granules with special emphasis on conditions required for growth hormone dimerization/aggregation. In addition, we highlight the role of zinc transporters that govern the process of zinc homeostasis in the regulated hormone secretion., (Copyright © 2012 S. Karger AG, Basel.)

Context and Objective: Despite the differences in the main characteristics between the autosomal dominant form of GH deficiency (IGHD II) and the bioinactive GH syndrome, a common feature of both is their impact on linear growth leading to short stature in all affected patients., Design: The index patient, a boy, was referred for assessment of his short stature (-2.54 SD score) and a delayed bone age of 5.9 yrs at the chronological age of 7.7 yrs. The GHD was confirmed by standard GH provocation tests, which revealed modestly reduced GH and IGF-I concentrations. Further genetic analysis of GH-1 gene identified heterozygosity for GH-P59L mutation. The secretion of the GH-P59L following stimulation with forskolin was investigated and compared to that of the wt-GH after expression of both GH variants in AtT-20 cells. Based on the position of P59L mutation that lies within a patch of residues composing the GH binding site 1 for GHR, we performed the analysis of GH-P59L binding to GHR by in silico mutagenesis and molecular dynamics simulations, which suggested possible problems in correct binding of GH-P59L to the GHR. Therefore, the functional characterization of this GH mutant was assessed through studies of GHR binding and activation of Jak2/Stat5 signaling pathway., Results: In line with the clinical data of the patient GH deficiency is suggested, underlined by GH-secretion studies revealing a moderate difference in secretion between GH-P59L and wt-GH. In addition, further functional characterization of the GH-P59L by studies of GH-receptor binding and activation of Jak2/Stat5 pathway presented with a reduced binding affinity of GH-P59L for GHR and decreased bioactivity compared to the wt-GH., Conclusions: The clinical data of the patient combined with the laboratory data support the diagnosis of partial IGHD type II. Since the GH deficiency was not total, additional binding and signaling studies were performed, which revealed that the GH-P59L variant displays some of the common features of bioinactive GH syndrome. Taken together, in this study we report a patient suffering from the combination of two growth disorders (alteration of secretion as well as bioactivity) caused by a GH-1 gene alteration highlighting the necessity of functional analysis of any GH variant, despite the presence of obvious clinical features of IGHD type II., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Delta3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Delta3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Delta3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Delta3GH mRNA of human (shRNAmir-Delta3). Rat pituitary tumor GC cells expressing Delta3GH upon doxycycline induction were transduced with shRNAmir-Delta3 lentiviral vectors, which significantly reduced Delta3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Delta3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients.

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Context and Objective: Main features of the autosomal dominant form of GH deficiency (IGHD II) include markedly reduced secretion of GH combined with low concentrations of IGF-I leading to short stature., Design, Setting, and Patients: A female patient presented with short stature (height -6.0 sd score) and a delayed bone age of 2 yr at the chronological age of 5 yr. Later, at the age of 9 yr, GHD was confirmed by standard GH provocation test, which revealed subnormal concentrations of GH and a very low IGF-I. Genetic analysis of the GH-1 gene revealed the presence of a heterozygous R178H mutation., Interventions and Results: AtT-20 cells coexpressing both wt-GH and GH-R178H showed a reduced GH secretion after forskolin stimulation compared with the cells expressing only wt-GH, supporting the diagnosis of IGHD II. Because reduced GH concentrations found in the circulation of our untreated patient could not totally explain her severe short stature, functional characterization of the GH-R178H performed by studies of GH receptor binding and activation of the Janus kinase-2/signal transducer and activator of transcription-5 pathway revealed a reduced binding affinity of GH-R178H for GH receptor and signaling compared with the wt-GH., Conclusion: This is the first report of a patient suffering from short stature caused by a GH-1 gene alteration affecting not only GH secretion (IGHD II) but also GH binding and signaling, highlighting the necessity of functional analysis of any GH variant, even in the alleged situation of IGHD II.

Context: A polymorphism of the GH receptor (GHR) gene resulting in genomic deletion of exon 3 (GHR-d3) has been associated with responsiveness to GH therapy. However, the data reported so far do vary according to the underlying condition, replacement dose, and duration of the treatment. OBJECTIVE, DESIGN: The aim of this study was to analyze the impact of the GHR genotypes in terms of the initial height velocity (HV) resulting from treatment and the impact upon adult height in patients suffering from severe isolated GH deficiency. CONTROLS, PATIENTS, SETTING: A total of 181 subjects (peak stimulated GH

Context and Objective: Alteration of exon splice enhancers (ESE) may cause autosomal dominant GH deficiency (IGHD II). Disruption analysis of a (GAA) (n) ESE motif within exon 3 by introducing single-base mutations has shown that single nucleotide mutations within ESE1 affect pre-mRNA splicing., Design, Setting, and Patients: Confirming the laboratory-derived data, a heterozygous splice enhancer mutation in exon 3 (exon 3 + 2 A-->C) coding for GH-E32A mutation of the GH-1 gene was found in two independent pedigrees, causing familial IGHD II. Because different ESE mutations have a variable impact on splicing of exon 3 of GH and therefore on the expression of the 17.5-kDa GH mutant form, the GH-E32A was studied at the cellular level., Interventions and Results: The splicing of GH-E32A, assessed at the protein level, produced significantly increased amounts of 17.5-kDa GH isoform (55% of total GH protein) when compared with the wt-GH. AtT-20 cells coexpressing both wt-GH and GH-E32A presented a significant reduction in cell proliferation as well as GH production after forskolin stimulation when compared with the cells expressing wt-GH. These results were complemented with confocal microscopy analysis, which revealed a significant reduction of the GH-E32A-derived isoform colocalized with secretory granules, compared with wt-GH., Conclusion: GH-E32A mutation found within ESE1 weakens recognition of exon 3 directly, and therefore, an increased production of the exon 3-skipped 17.5-kDa GH isoform in relation to the 22-kDa, wt-GH isoform was found. The GH-E32A mutant altered stimulated GH production as well as cell proliferation, causing IGHD II.

A heterozygous missense mutation in the GH-1 gene converting codon 77 from arginine (R) to cysteine (C), which was previously reported to have some GH antagonistic effect, was identified in a Syrian family. The index patient, a boy, was referred for assessment of his short stature (-2.5 SDS) at the age of 6 years. His mother and grandfather were also carrying the same mutation, but did not differ in adult height from the other unaffected family members. Hormonal examination in all affected subjects revealed increased basal GH, low IGF-I concentrations, and subnormal IGF-I response in generation test leading to the diagnosis of partial GH insensitivity. However, GH receptor gene (GHR) sequencing demonstrated no abnormalities. As other family members carrying the GH-R77C form showed similar alterations at the hormonal level, but presented with normal final height, no GH therapy was given to the boy, but he was followed through his pubertal development which was delayed. At the age of 20 years he reached his final height, which was normal within his parental target height. Functional characterization of the GH-R77C, assessed through activation of Jak2/Stat5 pathway, revealed no differences in the bioactivity between wild-type-GH (wt-GH) and GH-R77C. Detailed structural analysis indicated that the structure of GH-R77C, in terms of disulfide bond formation, is almost identical to that of the wt-GH despite the introduced mutation (Cys77). Previous studies from our group demonstrated a reduced capability of GH-R77C to induce GHR/GH-binding protein (GHBP) gene transcription rate when compared with wt-GH. Therefore, reduced GHR/GHBP expression might well be the possible cause for the partial GH insensitivity found in our patients. In addition, this group of patients deserve further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may also have a direct impact on GHR/GHBP gene expression causing partial GH insensitivity. This might be responsible for the delay of growth and pubertal development. Finally, we clearly demonstrate that GH-R77C is not invariably associated with short stature, but that great care needs to be taken in ascribing growth failure to various heterozygous mutations affecting the GH-IGF axis and that careful functional studies are mandatory.

Context and Objective: A single missense mutation in the GH-1 gene converting codon 77 from arginine (R) to cysteine (C) yields a mutant GH-R77C peptide, which was described as natural GH antagonist., Design, Setting, and Patients: Heterozygosity for GH-R77C/wt-GH was identified in a Syrian family. The index patient, a boy, was referred for assessment of his short stature (-2.5 SD score) and partial GH insensitivity was diagnosed. His mother and grandfather were also carrying the same mutation and showed partial GH insensitivity with modest short stature., Interventions and Results: Functional characterization of the GH-R77C was performed through studies of GH receptor binding and activation of Janus kinase 2/Stat5 pathway. No differences in the binding affinity and bioactivity between wt-GH and GH-R77C were found. Similarly, cell viability and proliferation after expression of both GH peptides in AtT-20 cells were identical. Quantitative confocal microscopy analysis revealed no significant difference in the extent of subcellular colocalization between wt-GH and GH-R77C with endoplasmic reticulum, Golgi, or secretory vesicles. Furthermore studies demonstrated a reduced capability of GH-R77C to induce GHR/GHBP gene transcription rate when compared with wt-GH., Conclusion: Reduced GH receptor/GH-binding protein expression might be a possible cause for the partial GH insensitivity with delay in growth and pubertal development found in our patients. In addition, this group of patients deserves further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may also have a direct impact on GHR/GHBP gene expression causing partial GH insensitivity.

Background: Testicular tumours are relatively uncommon in infants and children, accounting for only 1-2% of all paediatric solid tumours. Of these approximately 1.5% are Leydig-cell tumours. Further, activating mutations of the luteinizing hormone receptor gene (LHR), as well as of the G protein genes, such as Gsalpha (gsp) and Gialpha (gip2) subunits, and cyclin-dependent kinase gene 4(CDK4) have been associated with the development of several endocrine neoplasms., Aims/methods: In this report, the clinical variability of Leydig-cell tumours in four children is described. The LHR-, gsp-, gip2- and CDK4 genes were investigated to establish the possible molecular pathogenesis of the variable phenotype of the Leydig-cell tumours., Results: No activating mutations in these genes were found in the four Leydig-cell tumours studied. Therefore, the absence of activating mutations in LHR, as well as in both the 'hot spot' regions for activating mutations within the G-alpha subunits and in the regulatory 'hot spot' on the CDK4 genes in these tumours indicates molecular heterogeneity among Leydig-cell tumours., Conclusion: Four children with a variable phenotype caused by Leydig-cell tumours are described. A molecular analysis of all the 'activating' genes and mutational regions known so far was performed, but no abnormalities were found. The lessons learnt from these clinically variable cases are: perform ultrasound early and most importantly, consider discrepancies between testicular swelling, tumour size and androgen production., (Copyright (c) 2007 S. Karger AG, Basel.)

Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

The migration of leukocytes in immune surveillance and inflammation is largely determined by their response to chemokines. While the chemokine specificities and expression patterns of chemokine receptors are well defined, it is still a matter of debate how leukocytes integrate the messages provided by different chemokines that are concomitantly produced in physiologic or pathologic situations in vivo. We present evidence for a novel regulatory mechanism of leukocyte trafficking. Our data are consistent with a mode of action where CC-chemokine receptor 7 (CCR7) agonists and unrelated, nonagonist chemokines first form a heteromeric complex, in the presence of which the triggering of CCR7 can occur at a much lower agonist concentration. The increase is synergistic and can be evoked by many but not all chemokines. Chemokine-induced synergism might provide an amplification system in "chemokine-rich" tissues, rendering leukocytes more competent to respond to migratory cues.

The selective CXC chemokine receptor 3 (CXCR3) agonists, monokine induced by interferon-gamma (IFN- gamma)/CXC chemokine ligand 9 (CXCL9), IFN-inducible protein 10/CXCL10, and IFN-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, attract CXCR3+ cells such as CD45RO+ T lymphocytes, B cells, and natural killer cells. Further, all three chemokines are potent, natural antagonists for chemokine receptor 3 (CCR3) and feature defensin-like, antimicrobial activities. In this study, we show that I-TAC, in addition to these effects, acts as an antagonist for CCR5. I-TAC inhibited the binding of macrophage-inflammatory protein-1alpha (MIP-1alpha)/CC chemokine ligand 3 (CCL3) to cells transfected with CCR5 and to monocytes. Furthermore, cell migration evoked by regulated on activation, normal T expressed and secreted (RANTES)/CCL5 and MIP-1beta/CCL4, the selective agonist of CCR5, was inhibited in transfected cells and monocytes, respectively. In two other functional assays, namely the release of free intracellular calcium and actin polymerization, I-TAC reduced CCR5 activities to minimal levels. Sequence and structure analyses indicate a potential role for K17, K49, and Q51 of I-TAC in CCR5 binding. Our results expand on the potential role of I-TAC as a negative modulator in leukocyte migration and activation, as I-TAC would specifically counteract the responses mediated by many "classical," inflammatory chemokines that act not only via CCR3 but via CCR5 as well.

Eotaxin-3 (CCL26), like eotaxin (CCL11) and eotaxin-2 (CCL24), has long been considered a specific agonist for CC chemokine receptor 3 (CCR3), attracting and activating eosinophils, basophils, and Th2 type T lymphocytes. Although not characterized extensively yet, its expression profile coincides with a potential role in allergic inflammation. We recently reported that eotaxin-3 is an antagonist for CCR2 (Ogilvie, P., Paoletti, S., Clark-Lewis, I., and Uguccioni, M. (2003) Blood 102, 789-784). In the present report, we provide evidence that eotaxin-3 acts as a natural antagonist on CCR1 and -5 as well. Eotaxin-3 bound to cells transfected with either CCR1 or -5 as well as to monocytes expressing both receptors. Further, it inhibited chemotaxis, the release of free intracellular calcium, and actin polymerization when cells were stimulated with known agonists of CCR1 and -5. An analysis of its three-dimensional structure indicated the presence of two distinct epitopes that may be involved in specific binding to CCR1, -2, -3, and -5. Taken together, our data thus indicate eotaxin-3 to be the first human chemokine that features broadband antagonistic activities, suggesting that it may have a modulatory rather than an inflammatory function. Further, eotaxin-3 may play an unrecognized role in the polarization of cellular recruitment by attracting Th2 lymphocytes as well as eosinophils and basophils via CCR3, while concomitantly blocking the recruitment of Th1 lymphocytes and monocytes via CCR1, -2, and -5.