Gerhard Müller-Newen

Principles of interleukin (IL)-6-type cytokine signalling and its regulation.

The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important family of mediators involved in the regulation of the acute-phase response to injury and infection. Besides their functions in inflammation and the immune response, these cytokines play also a crucial role in haematopoiesis, liver and neuronal regeneration, embryonal development and fertility. Dysregulation of IL-6-type cytokine signalling contributes to the onset and maintenance of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, multiple sclerosis and various types of cancer (e.g. multiple myeloma and prostate cancer). IL-6-type cytokines exert their action via the signal transducers gp (glycoprotein) 130, LIF receptor and OSM receptor leading to the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) cascades. This review focuses on recent progress in the understanding of the molecular mechanisms of IL-6-type cytokine signal transduction. Emphasis is put on the termination and modulation of the JAK/STAT signalling pathway mediated by tyrosine phosphatases, the SOCS (suppressor of cytokine signalling) feedback inhibitors and PIAS (protein inhibitor of activated STAT) proteins. Also the cross-talk between the JAK/STAT pathway with other signalling cascades is discussed.

Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies.

Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 0.4% of the worlds population. Mutations in ten genes causing distinct forms of idiopathic epilepsy have been identified so far1,2,3,4,5,6,7, but the genetic basis of many IGE subtypes is still unknown. Here we report a gene associated with the four most common IGE subtypes: childhood and juvenile absence epilepsy (CAE and JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA; ref. 8). We identified three different heterozygous mutations in the chloride-channel gene CLCN2 in three unrelated families with IGE. These mutations result in (i) a premature stop codon (M200fsX231), (ii) an atypical splicing (del74–117) and (iii) a single amino-acid substitution (G715E). All mutations produce functional alterations that provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74–117 cause a loss of function of ClC-2 channels and are expected to lower the transmembrane chloride gradient essential for GABAergic inhibition. G715E alters voltage-dependent gating, which may cause membrane depolarization and hyperexcitability.

Activation of STAT3 by IL-6 and IL-10 in Primary Human Macrophages Is Differentially Modulated by Suppressor of Cytokine Signaling 3

On human macrophages IL-10 acts as a more potent anti-inflammatory cytokine than IL-6, although both cytokines signal mainly via activation of the transcription factor STAT3. In this study we compare IL-10 and IL-6 signaling in primary human macrophages derived from blood monocytes. Pretreatment of macrophages with PMA or the proinflammatory mediators LPS and TNF-α blocks IL-6-induced STAT3 activation, whereas IL-10-induced activation of STAT3 remains largely unaffected. Although LPS induces the feedback inhibitor suppressor of cytokine signaling 3 (SOCS3) in macrophages, inhibition of IL-6 signal transduction by LPS occurs rapidly and does not depend on gene transcription. We also found that pretreatment of macrophages with IL-10 inhibits subsequent STAT3 activation by IL-6, whereas IL-10-induced STAT3 activation is not affected by preincubation with IL-6. This cross-inhibition is dependent on active transcription and might therefore be explained by different sensitivities of IL-10 and IL-6 signaling toward the feedback inhibitor SOCS3, which is induced by both cytokines. In contrast to the IL-6 signal transducer gp130, which has been previously shown to recruit SOCS3 to one of its phosphotyrosine residues (Y759), peptide precipitation experiments suggest that SOCS3 does not interact with phosphorylated tyrosine motifs of the IL-10R. Taken together, different sensitivities of IL-10 and IL-6 signaling toward mechanisms that inhibit the Janus kinase/STAT pathway define an important mechanism that contributes to the different anti-inflammatory potencies of these two cytokines.

Mesenchymal Stem Cells Prevent Progressive Experimental Renal Failure but Maldifferentiate into Glomerular Adipocytes

Glomerulonephritis (GN) is a major cause of renal failure. This study sought to determine whether intrarenal injection of rat mesenchymal stem cells (MSC) can preserve renal function in a progressive rat model of GN. Early in GN (day 10), fluorescently labeled rat MSC localized to more than 70% of glomeruli, ameliorated acute renal failure, and reduced glomerular adhesions. Fifty days later, proteinuria had progressed in controls to 40 +/- 25 mg/d but stayed low in MSC-treated rats (13 +/- 4 mg/d; P < 0.01). Renal function on day 60 in the MSC group was better than in medium controls. Kidneys of the MSC group as compared with controls on day 60 contained 11% more glomeruli per 1-mm(2) section of cortex but also significantly more collagen types I, III, and IV and alpha-smooth muscle actin. Approximately 20% of the glomeruli of MSC-treated rats contained single or clusters of large adipocytes with pronounced surrounding fibrosis. Adipocytes exhibited fluorescence in their cytoplasm and/or intracellular lipid droplets. Lipid composition in these adipocytes in vivo mirrored that of MSC that underwent adipogenic differentiation in vitro. Thus, in this GN model, the early beneficial effect of MSC of preserving damaged glomeruli and maintaining renal function was offset by a long-term partial maldifferentiation of intraglomerular MSC into adipocytes accompanied by glomerular sclerosis. These data suggest that MSC treatment can be a valuable therapeutic approach only if adipogenic maldifferentiation is prevented.

Identification and Functional Characterization of Dendritic Cells in the Healthy Murine Kidney and in Experimental Glomerulonephritis

The kidney tubulointerstitium contains numerous bone marrow-derived antigen-presenting cells, which are often referred to as resident tissue macrophages, although several previous studies had demonstrated characteristics of dendritic cells (DC). In this study, we describe a subset of tubulointerstitial cells expressing the DC marker CD11c. A protocol was established to isolate these cells for in vitro analysis. Renal CD11c(+) cells resembled splenic DC, but not peritoneal macrophages, in morphology, lysosomal content, phagocytic activity, microbicidal effector functions, expression of T cell costimulatory molecules, and ability to activate T cells. Nevertheless, many CD11c(+) renal cells expressed low or intermediate levels of F4/80 and CD11b, indicating that both markers are not absolutely specific for macrophages in the kidney. Subpopulations of renal DC could be distinguished based on their expression of MHC class II and costimulatory molecules and may represent different maturation stages. In nephrotoxic glomerulonephritis, increased numbers of CD11c(+) cells showing DC functionality were found in the tubulointerstitium. Focal accumulation was seen within tubulointerstitial mononuclear infiltrates and adjacent to, but not within, inflamed glomeruli. These results are the first to identify and characterize renal CD11c(+) cells as DC and to demonstrate marked changes in experimental glomerulonephritis.

Plasticity and cross-talk of Interleukin 6-type cytokines

Interleukin (IL)-6-type cytokines are critically involved in health and disease. The duration and strength of IL-6-type cytokine-mediated signaling is tightly regulated to avoid overshooting activities. Here, molecular mechanisms of inter-familiar cytokine cross-talk are reviewed which regulate dynamics and strength of IL-6 signal transduction. Both plasticity and cytokine cross-talk are significantly involved in pro- and anti-inflammatory/regenerative properties of IL-6-type cytokines. Furthermore, we focus on IL-6-type cytokine/cytokine receptor plasticity and cross-talk exemplified by the recently identified composite cytokines IL-30/IL-6R and IL-35, the first inter-familiar IL-6/IL-12 family member. The complete understanding of the intra- and extracellular cytokine networks will aid to develop novel tailor-made therapeutic strategies with reduced side effects.

Platelet Microparticles Enhance the Vasoregenerative Potential of Angiogenic Early Outgrowth Cells After Vascular Injury

Background— Angiogenic early outgrowth cells (EOCs) have been reported to contribute to endothelial regeneration and to limit neointima formation after vascular injury. Vascular pathologies comprise platelet activation and concomitant generation of platelet microparticles (PMPs). We hypothesized that PMPs may interact with EOCs in the context of vascular injury and modulate their regenerative potential. Methods and Results— Using flow cytometry, confocal microscopy, and scanning electron microscopy, we demonstrated the binding of thrombin/collagen-induced PMPs to EOCs with subsequent membrane assimilation and incorporation. This interaction promoted phenotypic alterations of EOCs with increased expression of endothelial cell markers and transfer of the chemokine receptor CXCR4 to EOCs with enhanced responsiveness to its ligand CXCL12/SDF-1&agr;. In addition, PMPs augmented the adhesion of EOCs to extracellular matrix components and to the injured vessel wall and accelerated cytoskeletal reorganization and migration of EOCs. PMPs induced changes in the EOC secretome toward a more proangiogenic profile and amplified the EOC-mediated induction of proliferation, migration, and capillary tube formation by mature endothelial cells. Compared with untreated EOCs, the injection of PMP-treated EOCs resulted in accelerated reendothelialization after arterial denudation injury in athymic nude mice, whereas the EOC-mediated reduction of neointima formation remained unchanged. Conclusions— Our data provide evidence that PMPs can boost the potential of EOCs to restore endothelial integrity after vascular injury. Major mechanisms involve the enhancement of EOC recruitment, migration, differentiation, and release of proangiogenic factors.

Two Distinct Types of Langerhans Cells Populate the Skin during Steady State and Inflammation

Langerhans cells (LCs), the dendritic cells (DCs) in skin epidermis, possess an exceptional life cycle and developmental origin. Here we identified two types of LCs, short-term and long-term LCs, which transiently or stably reconstitute the LC compartment, respectively. Short-term LCs developed from Gr-1(hi) monocytes under inflammatory conditions and occurred independently of the transcription factor Id2. Long-term LCs arose from bone marrow in steady state and were critically dependent on Id2. Surface marker and gene expression analysis positioned short-term LCs close to Gr-1(hi) monocytes, which is indicative of their monocytic origin. We also show that LC reconstitution after UV light exposure occurs in two waves: an initial fast and transient wave of Gr-1(hi) monocyte-derived short-term LCs is followed by a second wave of steady-state precursor-derived long-term LCs. Our data demonstrate the presence of two types of LCs that develop through different pathways in inflammation and steady state.

The role of the carboxyl terminus in ClC chloride channel function

The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-β-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxyl-terminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.

Janus Kinase (Jak) Subcellular Localization Revisited THE EXCLUSIVE MEMBRANE LOCALIZATION OF ENDOGENOUS JANUS KINASE 1 BY CYTOKINE RECEPTOR INTERACTION UNCOVERS THE Jak·RECEPTOR COMPLEX TO BE EQUIVALENT TO A RECEPTOR TYROSINE KINASE

The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor·Janus kinase complexes can be regarded as receptor tyrosine kinases.