Interleukin-4 Receptor Signal Transduction: Involvement of P62

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1 Interleukin-4 Receptor Signal Transduction: Involvement of P62 Den Naturwissenschaftlichen Fakultäten der Friedrich Alexander Universität Erlangen Nürnberg zur Erlangung des Doktorgrades vorgelegt von Susanne Bürgis aus Nürnberg

2 Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen Nürnberg Tag der mündlichen Prüfung: 6. Oktober 2006 Vorsitzender der Prüfungskommission: Erstberichterstatter: Zweitberichterstatter: Prof. Dr. D. P. Häder Prof. Dr. Dr. A. Gessner Prof. Dr. T. Winkler

3 Contents 1 Introduction The biology of interleukin-4 and its receptor Interleukin-4 and its biological functions Interleukin-4-receptor complexes Interleukin-4-receptor signaling The adaptor protein P Link between IL-4R and P Aims of this study Materials and Methods Materials Cytokines Commercial systems Enzymes Antibodies Plasmids Oligonucleotides Cell lines and bacteria Media Animals Methods Genotyping of mice Polymerase chain reaction Sequencing of plasmids and PCR products Isolation of genomic DNA Southern blot Isolation of total RNA Reverse transcription RT PCR Quantitative RT PCR Enzyme linked immuno sorbent assay Generation of a P62 antiserum Transfection of cells with calcium phosphate Western blot Immunoprecipitation Proliferation of cells Stimulation of macrophages iii

4 iv CONTENTS Manipulation of mouse embryonic stem cells Preparation of mouse embryo fibroblasts Class switching of splenic B cells TH2 differentiation of naïve T cells Infection with Leishmania major Oral glucose tolerance of mice OVA induced allergic lung disease Results Studies in cell culture Phosphorylation of signaling intermediates Effect of kinase inhibitors Involvement of P62 in IL-4R signaling Binding of P62 to the IL-4R Colocalization of P62-EGFP and IL-4R-RFP Co-Immunoprecipitation of P62 with the IL-4R Trimolecular Co-Immunoprecipitation Description of the P62 gene deficient mouse model P62 deficiency on genomic level P62 deficiency on transcriptome level P62 deficiency on protein level Steady state phenotype of P62 gene-targeted mice Immunological phenotypes of P62 gene-targeted mice Oral glucose tolerance Functionality of macrophages Class switching of splenic B cells in vitro Differentiation of naïve T cells in vitro Infection with Leishmania major Fibroblast functionality OVA induced allergic asthma Generation of a N388 IL-4R mouse model Targeting strategy Homologous recombination Blastocyst injection and breeding strategy Discussion Mechanism of IL-4 induced proliferation in TF1 cells Binding of P62 to IL-4R and PKCζ Involvement of P62 in IL-4R signal transduction Generation of a N388 IL-4R knock in mouse model Summary 55 A Abbreviations 59 Bibliography 61

5 Chapter 1 Introduction 1.1 The biology of interleukin-4 and its receptor Interleukin-4 and its biological functions Murine interleukin-4 (IL-4) is a pleiotropic cytokine of 140 amino acids that is secreted after removal of a signal peptide as a mature protein of 120 amino acids, and has first been described as a growth factor for B cells by the group of William Paul [58]. Similar to other cytokines, it has a compact, globular fold, which is stabilized by three disulphide bonds [18]. The crystal structure has been solved for the human protein, and based on the amino acid sequence identity, the murine protein is expected to have a similar tertiary structure dominated by a four alpha-helix bundle with a left handed twist [153]. IL-4 is mainly sectreted by CD4 + T cells that also produce IL-5 and IL-13 (TH2 cells) [128]. In mice, basophils have been shown to secrete IL-4 after infection with the nematode Nippostrongylus brasiliensis [93], in humans upon challenge with allergens [127]; likewise, mast cells have been reported to secrete IL-4 [13, 15]. Additional sources of IL-4 are NK1.1 positive CD4 + T cells [20], γ/δ T cells [35] and eosinophils [31]. Recently, also dendritic cells and B cells have been suggested to produce IL-4 [90, 52]. IL-4 is an important cytokine in shaping immune responses and exerts different effects on various hematopoietic cells including lymphocytes (reviewed in [43] and [101]). On B cells, it acts as a growth factor [58] and is responsible for the upregulation of surface molecules such as MHC II [103], the low affinity IgE receptor CD23 [25] and the IL-4R itself [104]. Likewise, IL-4 induces Ig heavy chain class switching of human B cells to IgE and IgG 4 [39] and is indispensable for class switching of murine B cells to IgE and IgG 1 [23, 150]. On T cells, IL-4 acts as a growth factor that induces either proliferation or antiapoptotic effects [60]. IL-4 is also required for the development of TH2 cells from naïve T cells after antigen stimulation. These cells secrete large amounts of IL-4 and other cytokines such as IL-5 or IL-13, thereby initiating a positive feedback loop. Additionally, IL-4 blocks the development of IFN-γ secreting TH1 cells, thus stabilizing a TH2 dominated immune response [59, 128]. In some infectious disease models it is decisive for the outcome of the infection, whether the immune system mounts a TH1- or TH2-dominated immune response. The importance of IL-4 for this decision has been clearly shown in various mouse 1

6 2 CHAPTER 1. INTRODUCTION models using either neutralizing anti-il-4 antibodies [117, 121], soluble IL-4R [113, 44] or IL-4 gene-deficient mice [75]. Apart from lymphocytes, IL-4 also affects other cells of the hematopoietic lineage. It upregulates MHC II expression on monocytes and leads to enhanced antigen presentation by macrophages, while at the same time the production of proinflammatory cytokines is downregulated [10]. IL-4 is also involved in inflammatory processes. It influences the expression of endothelial adhesion molecules like vascular cell adhesion molecule 1 (VCAM-1) [145] or E-selectin [6] and promotes chemokine production, thereby favouring the recruitment of eosinophils to the site of infection. Dysregulation of IL-4 expression results in uncontrolled allergic inflammation in mice and humans [115, 143], so a thorough understanding of IL-4R signal transduction processes is essential for therapeutic applications Interleukin-4-receptor complexes IL-4 binds to a high affinity receptor IL-4Rα (K d 20 to 80 pm [87]), that is a member of the type I cytokine receptor family as it contains conserved cysteine residues and an extracellular WSXWS motif, which is required for ligand binding [85]. The receptor is expressed on a variety of hematopoietic (monocytes, macrophages, mast cells, T cells and B cells) and nonhematopoietic cells (fibroblasts, neuroblasts, keratinocytes and hepatocytes) at relatively low frequencies (100 to 5000 copies per cell). Apart from the glycosylated 140 kd transmembrane protein, a 40 kd soluble IL-4R has been identified, that results from an alternatively spliced mrna [99]. Compared to the transmembrane receptor, the soluble IL-4R binds IL-4 with similar affinity [34] and has been employed in the treatment of human asthma [11] and murine leishmaniasis [44]. Signal tranduction can be initiated through artificial cross-linkage of IL-4Rα chains [80], whereas physiological activity is obtained through heterodimerization of the IL-4Rα chain with either the common gamma chain (γ c chain, CD132) to yield the type I receptor complex, or the IL-13Rα1 chain to yield the type II receptor complex, respectively. The majority of cells, including hematopoietic cells, express the type I IL-4R complex in which γ c chain participates. This protein is also involved in the signal transduction of receptor complexes for IL-2, IL-7, IL-9, IL-15 [138] and IL-21 [108]. First, a complex of IL-4 and IL-4Rα is formed and subsequently γ c chain interacts with this complex [82]; this binding does not increase the affinity for IL-4, but is required for the initiation of signal transduction [119]. Cells lacking the γ c chain (mostly nonhematopoietic cells) can signal via the type II IL-4R complex [16, 56]. This receptor can also transduce signals in response to the cytokine IL- 13 [92]. Additionally to the IL-13Rα1 chain, an IL-13Rα2 chain exists, that binds IL-13 with even higher affinity but does not contribute to signal transduction; it acts as a decoy receptor for IL-13 [158]. However, most recently the IL-13Rα2 chain was described to transduce IL-13 signals in macrophages, thereby fostering TGF-β-mediated fibrosis [36]. Recently the IL-4Rα chain has been proposed to contribute to the signal transduction of progesterone-induced blocking factor (PIBF), which induces a TH2 dominated cytokine

7 1.1. THE BIOLOGY OF INTERLEUKIN-4 AND ITS RECEPTOR 3 production through signaling via STAT6. This novel receptor complex consists of the IL-4Rα chain and the GPI-anchored PIBF receptor [77] Interleukin-4-receptor signaling As a typical member of the hematopoietin receptor family, the IL-4Rα and the associated receptor molecules γ c chain and IL-13Rα1 lack endogenous kinase activity. Thus, for the initiation of signaling cascades, intracellular kinases and adaptor proteins have to be recruited. Janus tyrosine kinases (Jaks) are known to contribute to cytokine signaling [62]: Jak-1, Jak-2 and Jak-3 have been shown to interact with the IL-4R. The IL-4Rα chain itself recruits Jak-1 and in some cell lines also Jak-2 [101]; in doing so, binding is mediated by a conserved motif of the IL-4Rα chain, located in close proximity to the transmembrane region and containing several acidic amino acids (box-1 motif) [27, 95]. The γ c chain also contains a box-1 motif and has been shown to bind Jak-3, whereas the IL-13Rα1 chain is associated with Jak-2 or the tyrosine kinase 2 (Tyk2) [106]. Activation of Jaks or other IL-4R associated kinases leads to tyrosine phosphorylation of the IL-4Rα chain [130]. Five conserved tyrosine residues of the intracellular receptor portion are phosphorylated, namely tyrosines Y475, Y550, Y578, Y606 and Y684 (numbering according to Mosley et al. [99]). Subsequently, adaptor proteins with Src homology 2 domains (SH2) or phosphotyrosine-binding domains (PTB) can bind to the receptor. Two major pathways for IL-4R signal transduction have been described [101]: one employs insuline receptor substrates (IRS) and transduces mainly IL-4 induced proliferation signals (IRS-pathway), the other uses signal transducers and activators of transcription (STATs) and mediates gene activation in response to IL-4 (Jak/STAT-pathway). Figure 1.1: IL-4R type I and type II. Schematic representation with receptor chains involved and conserved interaction domains for adaptor proteins.

8 4 CHAPTER 1. INTRODUCTION Signaling via the IRS-pathway is initiated by the recruitment of IRS molecules, that possess a PTB domain, to the phosphorylated consensus motif 466 PIxxxxNPxYxSxSD 480 within the IL-4Rα chain [71] (see figure 1.1). This motif is also known as insulin-/il-4rmotif (I4R) due to the homology in receptor sequences [155]. Subsequently, IRS molecules are multiply phosphorylated at up to 20 different amino acids by Jaks [140, 160], after which they act as adaptor proteins linking other signal transduction molecules to the receptor [139, 141]. Phosphorylated IRS molecules have been described to bind to the regulatory subunit of the phosphoinositide-3 kinase (PI3-kinase); the activated PI3-kinase phosphorylates membrane lipids that act as second messengers and are crucial for the survival of the cell [37]. Another binding partner of phosphorylated IRS is the adaptor molecule Grb-2, that establishes a link to the Ras/Raf-pathway [26]. Similarly, signaling via the STAT6-pathway requires the recruitment of cytoplasmatic STAT6 molecules to the IL-4Rα chain. The three conserved tyrosine residues Y550, Y578 and Y606 of the IL-4Rα act as binding sites for the SH2 domain of STAT6 (see figure 1.1), and bound STAT6 is phosphorylated by Jaks. After tyrosine phosphorylation, STAT6 dissociates from the receptor and translocates as a homodimer to the nucleus, where it acts as a transcription factor binding to promotor consensus motifs (GAS-elements) of IL-4 inducible genes [63, 91]. STAT6 regulates genes responsible for allergic reactions, a TH2- dominated immune response and IgE production [110]. Figure 1.2: IL-4R deletion mutants. Schematic representation of C-terminal IL-4Rα deletion mutants and their ability to proliferate upon IL-4 stimulation and to activate signaling intermediates.

9 1.2. THE ADAPTOR PROTEIN P62 5 However, identification of functional regions within IL-4Rα being strictly responsible for either proliferation or gene activation is difficult. On the one hand, the IRS-pathway also phosphorylates high mobility group protein-i (HMG-I), a DNA-binding protein involved in the regulation of Iǫ expression in response to IL-4 [154], hence influencing gene expression (IgE class switching). Also, cell lines transfected with truncated human IL-4Rα constructs, that lack all STAT6 binding sites, were still able to signal weak IL-4 induced gene activation as measured by surface expression of CD23 [120]. More recently, an acidic serine rich motif of the IL-4Rα termed ID-1 ( 347 VQSVEEEEDEMVKEDLSMSPENSG 370 ) has been connected to IL-4 induced activation of STAT6 and also STAT5 [97, 136], which resulted in the induction of TH2 differentiation in activated T cells. On the other hand, activated STAT6 also leads to the expression of genes important for survival and proliferation of lymphocytes like growth factor-independent gene-1 (GFI-1) or E4 binding protein 4 (E4BP4) [162]. Data from our own laboratory also challenge the model of IL-4R signal transduction described above: using a cell culture system of human TF-1 cells, that were stably transfected with truncated murine IL-4Rα constructs, it was shown that IL-4 induced proliferation required neither phosphorylation of IRS-2 nor STAT6 (see figure 1.2), whereas phosphorylation of Jak1 was a strict prerequisite for functionality [144]. As functional receptors lacked binding sites for both IRS molecules and STAT6, and alternative activation of these adaptor proteins was not observed, the existence of an alternative signal transduction pathway for IL-4 signals, employing additional adaptor proteins, was postulated. 1.2 The adaptor protein P62 In the literature P62 protein has been described in a variety of different contexts and under various synonyms: in humans, the protein has been identified as a phosphotyrosineindependent ligand of the Src homology 2 (SH2) domain of p56 lck [68, 107]; in rat, the protein was shown to interact with the regulatory domain of the atypical protein kinase C- ζ [114], and was therefore termed PKCζ interacting protein (ZIP). In the murine system, P62 has been described as an oxidative stress protein induced in peritoneal macrophages (A170) [64], and also as signal transduction and adaptor protein (STAP) expressed in osteoblasts [105]. Amino acid sequences are highly conserved between species, the murine protein shares 97% and 90% homology with rat and human P62, respectively. Proteins share several structural motifs, including an SH2 binding domain, an acidic interaction domain (AID) that binds the atypical PKCζ, a ZZ zink finger, a binding site for TNF receptor associated factor 6 (TRAF6), two PEST sequences that are associated with the rapid degradation of proteins, and a ubiquitin associated (UBA) domain (see figure 1.3) [40]. Two splice variants of rat P62, termed ZIP2 and ZIP3 have been identified, that lack the TRAF6 binding domain or the PEST and UBA domains, respectively [24, 46]. Endogenous P62 is located either in the cytoplasm or as a membrane associated protein [114], and is sometimes detected in lysosome targeted endosomes colocalizing with Rab7 and partially with lamp-1 [123]. On the contrary, in HeLa cells stimulated with sorbitol, P62 has been detected in the nucleus consistent with its suggested role as a transcription factor [137]. For example, P62 has been shown to potentiate transactivation by chicken

10 6 CHAPTER 1. INTRODUCTION Figure 1.3: Schematic domain organization of murine P62 ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily of ligand-activated transcription factors [89]. As P62 lacks any classical nuclear localization signals itself, it is supposed to shuttle into the nucleus bound to adaptor proteins [40, 109]. In earlier publications, P62 has been assumed to possess either serine/threonine kinase activity [107], or, due to a number of putative target sites for several protein kinases, to be a substrate for phosphorylation itself [114]. Today, the general notion prevails, that P62 is neither a kinase nor the substrate of a kinase, but acts as a scaffold protein or a molecular adaptor, linking the functionality of otherwise promiscous cellular enzymes to discrete signal transduction events. The binding of P62 to atypical protein kinases (apkcs), namely PKCζ and PKCλ/ι, seems to be of outstanding functional relevance. The apkcs share a conserved catalytic domain with the classical and novel PKCs, but possess a clearly distinct regulatory domain [65, 98]. They are insensitive to Ca 2+ as well as diacylglycerol and phorbol esters, and in contrast to other family members, they contain only one zinc finger domain and no C2 domain. On a functional level, apkcs are essential components of signal transduction cascades regulating cell growth and survival [7, 29], as well as cytoskeletal rearrangements [149] and cell polarity [83]. The interaction of P62 with apkcs, especially PKCζ, has been reported by independent groups [17, 114, 123], and the interacting domains have been mapped using deletional mutants: P62 utilizes a novel acidic motif comprising amino acids 69 to 81 (AID domain) for the interaction [124], whereas the regulatory domain of PKCζ (PB1 domain) is involved. More recently, the solution structure of apkc s PB1 domain and its mode of interaction with P62 was determined [57]. Furthermore, the crystal structure of the interacting motifs was solved [157], confirming earlier results regarding this interaction. The interaction of P62 and PKCζ has been shown to target PKC activity to several pathways and cellular functions. For example, P62 links the Kvβ2 subunit of the voltage-gated potassium channel as well as the ρ subunit of the ligand-gated ion channel GABA C R to PKCζ [24, 46]. In 2002 Burnol et al. showed that growth factor receptor-bound protein 14 (Grb14), a negative regulator of insulin signaling, is phosphorylated by PKCζ as a consequence of the interaction of Grb14 and the heterodimeric P62/PKCζ complex. As phosphorylated Grb14 is an even stronger inhibitor of insulin signaling, the cellular metabolism is influenced by the scaffold protein P62 [17].

11 1.3. LINK BETWEEN IL-4R AND P62 7 Selective activation of the transcription factor nuclear factor κb (NF-κB) in response to different extracellular stimuli is another funtion of P62, which has been extensively studied. For example, signals induced by nerve growth factor (NGF) are transduced via the neurotrophin receptors p75 and TrkA and result in P62 mediated NF-κB activation. P62 directly binds to TrkA, and to p75 using TRAF6 as a molecular bridge. PKCζ, that is recruited to this macromolecular complex, phosphorylates IκB kinase, leading to phosphorylation and degradation of inhibitor of κb (IκB) and subsequent activation of NF-κB, which results in cell survival. P62 has also been shown to participate in TrkA internalization, connecting receptor signals with the endosomal signaling network required for neuronal differentiation [41, 67, 122, 159]. In a similar manner, signal transduction processes in response to the cytokines tumor necrosis factor-α (TNF-α) and IL-1 are influenced by P62: P62 binds to RIP, a death domain kinase that associates with the TNF receptor 1 (TNF-R1) through interaction with the adaptor molecule TRAAD, thereby recruiting PKCζ. As a result, IKKβ-kinase functionality (mediated by PKCζ) is targeted to the TNF receptor [81, 125]. Likewise, the IL-1 receptor is connected via myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor associated kinase (IRAK) and TRAF6 to the scaffold protein P62, which recruits PKCζ and consequently causes NF-κB activation upon stimulation with IL-1 [124]. The involvement of P62 in these signal transduction pathways has been investigated mainly in cell culture systems employing sirna approaches or using dominant negative variants of participating proteins. To verify these oberservations, a P62 gene-deficient mouse model was established. The first study on this mouse model reported an impaired induced osteoclastogenesis in P62 gene-deficient mice, whereas the basal physiology of bones was not affected [33]. Stimulation with the calciotropic hormone parathyroid hormone-related protein (PTHrP) induces osteoclastogenesis via the receptor activator of NF-κB ligand (RANK-L), which is a member of the TNF-α family of cytokines. Engagement of RANK leads to the formation of a ternary complex of TRAF6, P62 and apkc. Most likely, PKCλ/ι is involved in this complex formation, since PKCζ gene-deficient mice displayed no osteopetrotic phenotype. Recently, Rodriguez et al. reported that the loss of P62 leads to mature-onset obesity [116]. Mice older than five months displayed increased body fat, a reduced metabolic rate and insulin- as well as leptin-resistance. Most likely this phenotype is due to an enhanced basal activity of the extracellular signal-regulated protein kinase (ERK) in adipose tissue, which has been shown to be important for adipogenesis and obesity [12]. P62 can directly interact with ERK, and in a WT situation this interaction seems to block ERK activity, resulting in reduced adipocyte differentiation. 1.3 Link between IL-4R and P62 Both IRS- and STAT6-independent IL-4 induced proliferation in a TF1 cell culture system was observed in the laboratory of Prof. Gessner. As a logical consequence, we postulated that a third signal transduction pathway for IL-4R signals, independent of the signal transduction proteins described until now, must exist. To identify proteins involved in this hypothetical signal pathway, a yeast two hybrid (Y2H) screen was performed. In

12 8 CHAPTER 1. INTRODUCTION this experiment, P62 was detected as an interaction partner of the cytoplasmic portion of the shortest functional IL-4R mutant. Additionally, a short peptide stretch of the IL-4R ( 297 KTDFPKAAPTKSPQSPGKA 315 ) was mapped as the region interacting with P62 in a pepscan experiment. Mutation of serine 308, completely abolished IL-4 induced proliferation in cells carrying the truncated N388 IL-4R, whereas cells transfected with a mutated WT-IL-4R were unaffected [144]. This observation indicated a functional relevance of the putative binding motif for P62. In 2004 Durán et al. linked IL-4 signal transduction to PKCζ, which is a functionally important binding partner of P62 [33]. In this report, PKCζ gene-deficient mice displayed reduced liver damage in a ConA-induced hepatitis model, which was due to impaired IL- 4R signaling, resulting in reduced activation of JAK1 and STAT6. Based on our previous data, the established link between P62 and PKCζ, and the recently reported connection between PKCζ and IL-4R signaling, we propose that P62 is invoved in IL-4R signaling, possibly via an IRS- and STAT6-independent pathway. To test this hypothesis, a mouse model with a mutated P62 gene, in which exons two to four have been removed, was generated in the group of Prof. Gessner.

13 1.4. AIMS OF THIS STUDY Aims of this study IL-4, together with IL-13, are key cytokines in atopic diseases and allergic asthma. Polymorphisms in the IL-4R gene have been shown to associate with susceptibility to and severity of atopic asthma [61]. With approximately 300 million people worldwide suffering from asthma (Global Initiative for Asthma, GINA), this disease is an important health problem. Therefore, a thorough understanding of IL-4R signaling pathways is important, especially with regard to therapeutic implications. To date, two signaling pathways have been described for IL-4: one functions via Jak1 and STAT6, the other via IRS-molecules. In the laboratory of Prof. Gessner, IL-4 induced proliferation was observed, which was independent of STAT6- and IRS-phosphorylation. Additionally, P62 was detected as a binding partner of the IL-4R in a Y2H screen. Based on these data, the existence of a third signal transduction pathway for IL-4 was postulated, presumably independent of STAT6 and IRS, but depending on the adaptor protein P62. Therefore, this study investigated the following topics: First, the mechanism of STAT6- and IRS-independent proliferation in TF1 cells was investigated. Cells transfected with either WT IL-4R or 388 IL-4R were treated with different kinase inhibitors to discriminate between different proliferation pathways (PI3 Kinase/Akt pathway vs. MAP kinase pathway). Second, the binding of IL-4R to P62, a novel interaction partner of the receptor, was verified. It was tested whether stimulation of cells with IL-4 or the expression levels of P62 exerted any influence on this interaction. Additionally, the functional relevance of the interaction between P62 and IL-4R was investigated both in vitro and in vivo. For this purpose, P62 gene-deficient mice were characterized in steady state and were subsequently tested for their ability to respond to IL-4 stimulation. Finally, the functionality of the truncated 388 IL-4R, which lacks binding sites for IRS and STAT6, and which was able to mediate proliferation in vitro, should be further analyzed. Therefore, a transgenic mouse model carrying this 388 IL-4R was established.

14 10 CHAPTER 1. INTRODUCTION

15 Chapter 2 Materials and Methods 2.1 Materials Cytokines All recombinant cytokines (mil-4, hil-4, mil-13, mil-1α, mil-1β and mil-10) for cell stimulation and proliferation studies were purchased from Strathmann Biotech AG, Hamburg. IL-2, IL-3 and IL-4 for cell culture were isolated from supernatants of X63Ag8-653 cells transfected with the respective cdnas [70] Commercial systems Unless stated otherwise, all systems were used according to manufacturers recommendation. Kit DuoSet Mouse Eotaxin 1/CCL11 ECL plus TM Western Blotting Detection System EndoFree TM Plasmid Maxi Kit Long template PCR Kit PureLink HiPure Plasmid Midiprep Kit pgem-t easy TM Vector Systems QIAprep TM Plasmid Midi Kit QIAprep TM Spin Miniprep Kit QIAquick TM Gel Extraction Kit QIAquick TM PCR Purification Kit QIAshredder TM Ready-to-go TM DNA Labelling Beads (-dctp) RNeasy TM Mini Kit StreptABComplex/HRP Manufacturer R&D Systems Amersham Qiagen Roche Invitrogen Promega Qiagen Qiagen Qiagen Qiagen Qiagen Amersham Qiagen DAKO 11

16 12 CHAPTER 2. MATERIALS AND METHODS Enzymes Restriction enzymes were used in appropriate buffers recommended by the manufacturer. Digestions were carried out in 10-fold the volume compared to the enzyme volume and incubated at 37 C for 4 h up to 12 h. Enzyme Pfu Ultra Hotstart DNA Polymerase Platinum Taq TM DNA Polymerase Restriction enzymes Superscript II TM -reverse transcriptase Shrimp alkaline phosphatase (SAP) T4 DNA ligase Manufacturer Statagene Invitrogen New England Biolabs or Fermentas Invitrogen Pharmacia-Biotech Invitrogen Antibodies Antibodies were stored at 4 C or 20 C and were diluted to the appropriate working concentration shortly prior to use. Antibodies for Western Blotting were reused and stored at 4 C in Western Blot blocking buffer supplemented with sodium azide. Antibody Application Manufacturer mouse anti Flag IgG FITC Western Blot, IP Sigma rabbit anti goat IgG POX Western Blot Dianova goat anti GST IgG (polyclonal) Western Blot Pharmacia-Biotech mouse anti HIS IgG1 Western Blot Qiagen rat anti mouse IL-4R (M1) Western Blot, IP Beckmann [5] rabbit anti mouse IL-4R serum Western Blot AG Gessner goat anti mouse IgG POX Western Blot Dianova goat anti rat Cy5-conjugated FACS Jackson donkey anti rabbit POX Western Blot Dianova rabbit anti P62 polyclonal serum Western Blot AG Gessner rabbit anti PKCζ Western Blot Sigma PathScan Multiplex Western Cocktail I Western Blot Cell Signaling anti CD40L stimulation of cells AG Winkler anti IFNγ ELISA capture R&D Systems anti IFNγ-bio ELISA detection BD Biosciences anti IL-4 (11B11) ELISA capture BD Biosciences continued on next page

17 2.1. MATERIALS 13 continued from previous page Antibody Application Manufacturer anti IL-4-bio ELISA detection BD Biosciences anti IL-13 ( ) ELISA capture R&D Systems anti IL-13-bio ELISA detection R&D Systems anti IgE (R35-72) ELISA capture BD Biosciences anti IgE-bio (R35-118) ELISA detection BD Biosciences anti IL-5 (TRF-K5) ELISA capture cell sciences anti IL-5-bio ELISA detection Pharmingen anti IL-12p40 ELISA capture BD Biosciences anti IL-12p40-bio ELISA detection BD Biosciences anti B220-bio (RA3-6B2) cell depletion, BD Biosciences FACS anti CD3-bio (145-2C11) cell depletion BD Biosciences anti CD4-bio cell depletion Caltag anti CD8-bio (53-6.7) cell depletion BD Biosciences anti CD62L-bio (MEL-14) cell enrichment BD Biosciences anti CD11b-bio (M1/70) cell depletion BD Biosciences Pan NK (DX5)-bio cell depletion Pharmingen Ter119 / Ly76-bio cell depletion BD Biosciences Gr1-bio (RB6-8C5) cell depletion, BD Biosciences FACS c-kit-pe FACS Caltag Lab. CCR3-PE FACS R&D Systems CD11b-FITC FACS BD Bioscience CD80-PE (B7.1) (16-10A1) FACS Pharmingen CD86-FITC (B7.2) FACS BD Biosciences CD11c-bio (HL3) FACS BD Biosciences MHC II-PE FACS Biotechnology F4/80-bio FACS Caltag CD23-FITC (B3B4) FACS Pharmingen CD69-PE (H1.2F3) FACS BD Biosciences CD44-bio (IM7) FACS Pharmingen CD3-PE FACS Caltag B220-PE (CD45R) FACS Caltag CD62L-PE FACS Caltag Gr1-PE (RB6.8C5) FACS BD Biosciences Plasmids All plasmids were isolated with the QIAprep Plasmid Midi Kit or the PureLink HiPure Plasmid Midiprep Kit from cultures of E. coli DH10B. Plasmids were stored at 20 C and were digested with the appropriate restriction enzymes prior to use.

18 14 CHAPTER 2. MATERIALS AND METHODS Plasmid Application Reference pgem-t easy subcloning Promega notes pegfp-n1 EGFP fusion constructs [112] pdsred1-n1 (T4mut) RFP fusion constructs [8] pflag-cmv-2 FLAG expression constructs [2] pci-tpa vector for DNA vaccination [156] pcdna 3.1 myc-his expression constructs Invitrogen prosa26-1 STOP targeting construct for IL 4R knock in T. Buch, Universtity of Cologne Oligonucleotides Oligonucleotides were ordered from Thermo Hybaid and stored at 20 C at a concentration of 80 pmol/µl. Primers were used at a final concentration of 0.2 µm for PCR reactions. Name Sequence 5 to 3 Usage HPRT-P1 GTT GAA TAC AGG CCA GAC TTT GTT G qrt PCR HPRT-P2 GAT TCA ACT TGC GCT CAT CTT AGG C qrt PCR PBGD-P1 ATG TCC GGT AAC GGC GGC qrt PCR PBGD-P2 CAA GGC TTT CAG CAT CGC CAC CA qrt PCR mu-eotaxin 1 ATG CAG AGC TCC ACA GCG CTT CTA qrt PCR LC1 fwd mu-eotaxin 1 TGT AGC TCT TCA GTA GTG TGT TGG qrt PCR LC1 rev mu-eotaxin-p1 TAT CAC CCT GAC TGA CCT GTA ACT CA RT-PCR mu-eotaxin-p2 CAC TTA AAG GCA GAG GCA GGT AA RT PCR Neo-P3 GGG CGC CCG GTT CTT TTT G mouse screen Neo-P4 ACA CCC AGC CGG CCA CAG TCG mouse screen Cre-P1 ACC AGC CAG CTA TCA ACT CG mouse screen Cre-P2 TTA CAT TGG TCC AGC CAC C mouse screen EGFP-P1 CGT CCA GGA GCG CAC CAT CTT CTT mouse screen EGFP-P2 ATC GCG CTT CTC GTT GGG GTC TTT mouse screen Stat6-P1 CTG GAC CTC ACC AAA CGC mouse screen Stat6-P2 CCC GGA TGA CGT GTG C mouse screen IL-4R I6 fwd CCC TTC CTG GCC CTG AAT TT mouse screen IL-4R E10 rev ACC TGT GCA TCC TGA ATG AT mouse screen IL-4R E8 fwd GTA CAG CGC ACA TTG TTT TT mouse screen IL-4R E8 rev CTC GGC GCA CTG ACC CAT CT mouse screen continued on next page

19 2.1. MATERIALS 15 continued from previous page Name Sequence 5 to 3 Usage P62 Ex2-3-4 P1 AAT CCG GGG CTT CCT TCC TG mouse screen P62 Ex2-3-4 P2 GCC CTT CCC CTC GCA CAC G mouse screen P62-3 TCA GGA AAT TGA CAT TGG GAT CTT mouse screen P62-5 AAC AAC TTA GAT GGA GCC TGA ATG mouse screen mp62 Ex1 GAG CCG AGT GGC TGT GCT GTT CCC cloning P62 LCP4 GCC AGG CCT AGG GGA AGC AGA G cloning s IL-4R P1 GCC CCA GTG GTA ATG TGA AGC CCT sequencing ROSA26 AGC ATA GAA GGG GCT TTC CCA GGA G probe Sonde 1 fwd Southern ROSA26 AGT TCC TAT CTC AGA TGG CTG CTG C probe Sonde 1 rev Southern pm5neo- CGG GCG CGC CGC CGC GCC GCG CGT cloning Rosa26-Stop1 CTT GTC TGC TG sequencing mouse screen pm5neo- GCG GCC GGC CCA CAA GCT TAT CGA sequencing Rosa26-Stop-2 TGA TAA GTG TC p-rosa26-sa GGA GGC AGG AAG CAC TTG CTC TCC C sequencing mouse screen 570-Stop- CCC GGC CGG CCT TAG TTC TCA GTG cloning and ROSA26 AGC CGA GCC AT sequencing ROSA26-flank CCT AAA GAA GAG GCT GTG CTT TGG sequencing nidogen-p1 CCA GCC ACA GAA TAC CAT CC PCR nidogen-p2 GGA CAT ACT CTG CTG CCA TC PCR WT IL-4R TTT AGA TCT TTT GTG TCC CCA ATG cloning DSred BglII f. GGG CGG WT IL-4R AAA AGA TCT AGA AAC AAT GCC cloning DSred BglII r. CAG GGC IL-4R P3 TTC AGC AAG CAA GGC AGC AGC sequencing IL-4R P4 CGA GGC ACC TTT TGT GTC CCC sequencing IL-4R P5 TCG GGA AGC TCA GCC TGG GTT sequencing mouse screen IL-4R P6 TCA ACC AAG TAC CCG CAC TGG A sequencing IL-4R P19 GGA ATT CCT AGT TCT CAG TGA GCC sequencing GAG CCA T IL-4R P21 CCG CAG AAT TCA TTG TCT ATA ATG sequencing TGA CCT AC IL-4R P22 CCG GAT CCA TTA AGA AGA TAT GGT sequencing GGG ACC AG continued on next page

20 16 CHAPTER 2. MATERIALS AND METHODS continued from previous page Name Sequence 5 to 3 Usage IL-4R P23 GGG AAT TCC TAC TCC TGG CTT CGG sequencing GTC TGC TTA TC Neo ext P1 ACC TGC GTG CAA TCC ATC TTG TTC sequencing Neo ext P2 CGC CTT CTT GAC GAG TTC TTC TGA sequencing P62 Myc-tag TTT AAG CTT TAG ACC GCG GTT ATG cloning Hind III GCG TCG P62 Myc-tag AAA CTC GAG CAA TGG TGG AGG GTG cloning XhoI CTT CGA P62 Start TTT AAG CTT GCG TCG TTC ACG GTG cloning (pflag) AAG GCC P62 Stop GGG AGA TCT TCA CAA TGG TGG AGG cloning (pflag) GTG CTT mu-p62 P1 CCC GGA TCC GCG TCG TTC ACG GTG cloning AAG GCC TAT C mu-p62 P2 AAA GGA TCC TCA CAA TGG TGG AGG cloning GTG CTT CGA A mu-p62 P0 CCA GCT GTT TCG TCC GTA CCT AGA cloning CCG mu-p62 GGG GGA TCC CGC AAT GGT GGA GGG cloning End BamHI TGC TTC GAA TA WT IL-4R TTT AGA TCT TTT GTG TCC CCA ATG cloning DSred fwd GGG CGG WT IL-4R AAA AGA TCT AGA AAC AGC AAT GCC cloning DSred rev CAG GGC IeF TGGGATCAGATCTTTGAG PCR CeR CCAGGGTCATGGAAGCAGTG PCR ImF CTCTGGCCCTGCTTATTGTTG PCR Cell lines and bacteria Cell Line Origin Reference 3T3 mouse fibroblast (Swiss mouse) [147] HEK293T embryonal kidney cell, human [47] CTLL-2 mouse T-lymphocyte (C57BL/6) [45] TF-1 erythroleukemia, human [74] Balb/c ES cell embryonal stem cell (Balb/c) Prof.Nitschke (University of Erlangen)

21 2.1. MATERIALS 17 Strain Genotype Reference DH10B F mcra (mrr-hsdrms-mcrbc) Invitrogen [48] φ80lacz M15 lacx74 reca1 deor arad139 ara-leu7697 galu galk rpsl (Str R ) enda1 nupg λ BL21(DE3) F ompt hsds B (r B m B ) gal dcm Sigma Aldrich Media Media for propagation of cell lines or primary cells was purchased ready to use and was kept at 4 C. Medium for bacteria was prepared from powder and autoclaved prior to use. Bacteria were cultured in LB-Medium (10 g/l tryptone; 5 g/l yeast-extract; 10 g/l NaCl). For plates 20 g/l agar were added. Suspension cells were kept in RPMI 1640 medium (PAA) supplemented with 1% 100 x Pen/Strep (PAA), 1% 1 M HEPES Buffer Solution (PAA), 10% FCS (fetal bovine serum from Sigma) and 1% 200 mm L-glutamine (Gibco). Adherent cells were cultured in D-MEM (high glucose, plus L-glutamine, plus pyruvate from Gibco). Medium was supplemented with 1% 100 x Pen/Strep (PAA), 1% 1M HEPES Buffer Solution (PAA), 10% FCS (fetal bovine serum from Sigma) and 1% 200 mm L-glutamine (Gibco). MEFs and ES cells were kept in D-MEM (high glucose, plus L-glutamine, plus pyruvate from Gibco). Medium was supplemented with 15% fetal bovine serum (PAN TM Biotech GmbH), 1% MEM Non Essential Amino Acids (Gibco), 1% Pen/Strep (Gibco) and 0.001% β-mercaptoethanol (Gibco). Cells were detached with 1 x Trypsin EDTA from Gibco. To cultures of ES cells, additionally ESGRO leukemia inhibitory factor (LIF) from Chemicon was added at a concentration of 120 U/ml Animals Mice were obtained from the internal animal breeding house of the Institute of Clinical Microbiology, Immunology and Hygiene or purchased from Charles River Laboratories (Sulzfeld, Germany). STAT6 gene-deficient and IL-4R gene-deficient mice were on a Balb/c background (minumum F10), P62 gene-targeted mice for most experiments were also Balb/c background (F6 speed congenics and subsequently F10 conventional backcross) or F6 C57/Bl6. Animals recieved humane care according to the legal requirements in Germany. Mice were kept under controlled conditions (22 C, 12h day/night rhythm) and were fed a standard laboratory chow (Altromin 1434) ad libitum. Mice were used for experiments between six and ten weeks of age weighing 20 to 25 g.

22 18 CHAPTER 2. MATERIALS AND METHODS 2.2 Methods Genotyping of mice Genomic DNA from mice was obtained from tail biopsies lysed in 50mM KCl, 10mM Tris HCl ph 8.3, 2.5 mm MgCl 2, 0.1 mg/ml gelatine, 0.45% TritonX-100, 0.45% Tween-20 in water supplemented with Proteinase K. Samples were incubated at 56 C with gentle agitation over night. After boiling at 95 C for 5 min, tail DNA was subjected to a PCR reaction with the corresponding primers; resulting bands were resolved by agarose gel electrophoresis. The following PCR conditions were used: PCR Cycles Annealing Elongation Product Size P62 KO C, 60 sec 72 C, 60 sec 700 bp P62 WT C, 60 sec 72 C, 60 sec 300 bp STAT6 WT C, 30 sec 72 C, 45 sec 250 bp STAT6 KO (Neo) C, 30 sec 72 C, 45 sec 500 bp GFP /4get C, 30 sec 72 C, 40 sec 400 bp Cre C, 30 sec 72 C, 35 sec 200 bp C, 30 sec 72 C, 35 sec IL-4R WT C, 30 sec 72 C, 30 sec 170 bp IL-4R KO C, 30 sec 72 C, 60 sec 1300 bp 388 IL-4R ROSA KI C, 40 sec 72 C, 50 sec 630 bp (IL-4R P5 pm5neo-rosa26-stop-1) 388 IL-4R ROSA KI C, 40 sec 72 C, 60 sec 870 bp (IL-4R P5 p-rosa26-sa) Polymerase chain reaction PCR reactions for amplification of plasmid-dna, genomic DNA or cdna were carried out in 40µl total volume. Each sample included: ng DNA 8 pmol 5 primer 8 pmol 3 primer 1.25 U Taq-polymerase 2.5 mmol dntps 4 µl 10 x Buffer w/o MgCl 2 2 mmol MgCl 2 An initial denaturing step of 5min at 95 C was performed. Cycling conditions were dependent on several parameters (length of sequence to be amplified and melting temperature

23 2.2. METHODS 19 of primers employed) and were optimized for each pair of primers separately. One last elongation step of 5min at 72 C was added. Products were assessed for purity and specificity by agarose gel electrophoresis Sequencing of plasmids and PCR products PCR products used for cloning and expression plasmids were either sequenced at MWG Biotech (Ebersberg, Germany) or at the internal sequencing facility of the Institute for Clinical Microbiology, Immunology and Hygiene. Shortly, the BigDye Terminator Cycle Seq kit (Applied Biosystems) was used according to manufacturer s protocol with the corresponing primers. The PCR reaction employed: 1x 5 min 95 C 25x 10 sec 95 C 5 sec 50 C 4 min 60 C 1x 4 C PCR products were precipitated with EtOH and solved in water. Analysis was carried out with an ABI PRISM 310 Genetic Analyzer and the DNAStar SeqMan software Isolation of genomic DNA Genomic DNA from Balb/c ES cell clones was isolated for Southern Blot analysis. Shortly, cells were harvested from confluent 6-well cell culture plates or 10cm Petri dishes and were washed twice with PBS to remove residual FCS. Afterwards, cells were resuspended in 500 µl PBS and 500 µl 2 x Bradley Lysis Buffer supplemented with Proteinase K was added. Samples were incubated over night at C with gentle agitation. 2 x Bradley Lysis Buffer: 20 mm Tris/HCl (ph 7.5) 20 mm EDTA 20 mm NaCl 10% SDS Proteinase K 2 mg/ml Cell lysates were extracted twice with 1Vol phenol, once with 1Vol phenol/chloroform and subsequently with 1 Vol chloroform. Genomic DNA was precipitated from the aqueous phase by adding 2 Vol EtOH and 1/10 Vol 3 M sodium acetate (ph 5.2). The precipitate was washed once with 70% EtOH and solved in 100 µl of water for downstream applications.

24 20 CHAPTER 2. MATERIALS AND METHODS Southern blot The method was performed essentially as described previously by Southern [133]. Briefly, 10-20µg of genomic DNA were digested over night with appropriate restriction enzymes. Digests were precipitated with EtOH and loaded on a 0.8% agarose gel in 30 µl total volume. Gels were run for approximately 4 h at 100 V. Photographs of the gels were taken with an Image Master VDS (Pharmacia Biotech) to document the position of the marker (gene ruler 1 kb). Afterwards gels were treated 20 min with 0.25M HCl followed by 30min neutralizing buffer (0.5M NaOH; 1.5M NaCl) to facilitate blotting. DNA was transfered to a nylon membrane (Biodyne by PALL) by upward capillary transfer with a high salt buffer (20 x SSC: 3 M NaCl; 0.3 M Na-Citrate ph 7.0) over night. Blots were dried for 20min at 37 C and stored at room temperature min prior to hybridisation, blots were incubated at 55 C in ULTRAhyb Hybridization Buffer (Ambion) with gentle rotation. Radioactive probes were generated with the Ready-To-Go kit (Amersham) according to the manufacturer s protocol. Excessive [α 32 P] dctp Nucleotides were removed with the QIAquick Nucleotide Removal kit. The denatured probe was added to the Hybridisation solution at a concentration of 10 6 cpm/ml and incubated at 42 C over night. The following day, blots were washed twice for 10min with wash bufferi (2 x SSC; 0.1% SDS; RT) and twice for 15 min with wash bufferii (0.1 x SSC; 0.1% SDS; 42 C); afterwards an X-ray film was exposed to the blot for 1-3 days Isolation of total RNA Isolation of total RNA from mammalian cells was carried out with the RNeasy kit from Qiagen. For one preparation 10 6 to 2 x 10 7 cells were used. Qualtity and quantity of isolated RNA was assessed by photometry (ratio 260 nm/280 nm) and gel electrophoresis Reverse transcription RT PCR Total RNA isolated from mammalian cells was used as template for RT PCR to generate cdna. 1ng to 5µg of RNA were incubated with 1µl oligo(dt) primer in the presence of 0.4 µl 25 mm dntps for 5 min at 65 C in 13 µl total volume. Samples were kept on ice and 4 µl 10 x Reaction Buffer and 2 µl DTT (0.1 M) were added. After preincubation at 42 C for 2 min, 1 µl SuperScript II reverse transcriptase was added to each tube and the reaction was carried out at 42 C for 50min, followed by 70 C for 15min. Obtained cdna was diluted 1:5 for downstream applications Quantitative RT PCR Levels of mrna were determined by quantitative RT-PCR with the Roche Light Cycler PCR System employing the SYBR Green method. The reaction was carried out in 20 µl total volume and contained per sample 1U Platinum Taq polymerase, 2µl 10 x Reaction Buffer, 4mM MgCl 2, 0.4µM of each primer, 0.25mM dntps, 0.5xSYBR Green, 0.5 mg/ml BSA, 5% DMSO and 2µl cdna or standard-plasmid.

25 2.2. METHODS 21 Cycling conditions were as follows: 1x 10 min 95 C 50x 15 sec 95 C 10 sec annealing 15 sec 72 C The annealing temperature was specific for each pair of primers (Eotaxin 1 60 C; HPRT 63 C; PBGD 59 C). Fluorescence was measured after each elongation step. Specificity of the product was verified by a melting curve and by gel electrophoresis. A six point standard curve using 10 fold serial dilutions and a high standard of 10 4 fg was generated with every run. Data was analyzed with the LightCycler 5.32 software and expression levels were normalized to the housekeeping genes HPRT and PBGD Enzyme linked immuno sorbent assay Enzyme linked immuno sorbent assays (ELISAs) were carried out to determine the amount of protein released by cultured cells. ELISAs were used to measure Cytokines (IL-4, IFNγ, IL-13, IL-5, IL-12p40), Chemokines (Eotaxin 1) or antibodies (IgE). Briefly, 96 well round bottom ELISA plates were coated with the corresponing capture antibody in an appropriate concentration over night at 4 C. Plates were washed (PBS; 0.05% Tween) and unspecific binding sites were blocked with 10% FCS or 1% BSA for several hours at RT. Samples and standards were incubated over night at 4 C. After washing, the biotinylated detection antibody was added at an appropriate concentration for 2 h at RT. The detection antibody was coupled to strepavidin HRP (20 min, RT) and after another wash step, substrate (TMB) was added. The reaction was stopped by adding 0.5 Vol of 1 N HCl and plates were read at 450 nm on a Dynatech MR5000. Samples were analysed as duplicates and a 9 point standard curve was generated for each measurement Generation of a P62 antiserum A New Zealand White (NZW) rabbit (Charles River) was used for gene gun vaccination. Gene gun ammunition was prepared by cloning the complete ORF of the P62 protein into the BamHI cloning site of the pci TPA vector. Endotoxin free plasmid was obtained with the EndoFree TM Plasmid Maxi Kit (Qiagen). Gold particles carrying the vaccination plasmid were coated on the inside of a polypropylene tube according to manufacturer s recommendation (Helios gene gun). Briefly, 50 mg gold particles were incubated with 100 µl 50 mm spermidine solution and 100µl of plasmid DNA (1 µg/µl). With gentle agitation 200µl 1 M CaCl 2 were slowly added, and after 10 min incubation at RT gold particles were washed three times with EtOH. Particles were resuspended in 6 ml EtOH supplemented with 50 µg/µl polyvinylpyrrolidone (PVP) and applied to the polypropylene tube. The tube was cut into 1 cm pieces that were used

26 22 CHAPTER 2. MATERIALS AND METHODS as gene gun ammunition. Gold particles were applied intradermally with a pressure of psi. Prior to the first vaccination, preimmune serum was collected, and the rabbit was then immunized with 6 x 1 µg DNA at a time. The rabbit was bled 8 days after the 3 rd and 5 th vaccination Transfection of cells with calcium phosphate Adherent cells were transfected by Ca 3 (PO 4 ) 2 precipitation. Briefly, 5 x 10 6 cells were seeded in a 10 cm Petri dish and after 8 h medium was replaced with 9 ml of fresh, prewarmed medium. 20 µg plasmid DNA were mixed with 48 µl 2.5 M CaCl 2 in a total volume of 480µl. This mixture was slowly added to 480µl of 2 x HBS-Buffer to precipitate the DNA. After 20 min at RT, 5 µl Chloroquin (100 mm) were added and the solution was applied to the cell culture. Medium was replaced after 8 h and cells were used for downstream applications 48 h after transfection. 2 x HBS-Buffer: 50 mm HEPES 280 mm NaCl 1.5 mm Na 2 HPO Western blot Western Blot was used to specifically identify proteins in complex mixtures. The sample mixture was resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) [79] and subsequently transfered to a nitrocellulose membrane by electroblotting (2 h, 1 ma). Unspecific binding sites were blocked with blocking buffer (5% powdered milk in TBST) and membranes were incubated with specific antibodies in blocking buffer over night at 4 C. The next day membranes were washed with TBST (3 x 15 min; RT) and incubated with peroxidase-coupled (POX) secondary antibody in blocking buffer (1 h, RT). Again membranes were washed with TBST (3x15min) and blots were developed with the ECL-Plus Detection Kit (Amersham). For reprobes, bound antibodies were removed with the Antibody Stripping Solution from Chemicon. Transfer-Buffer: TBST-Buffer: 200 mm Glycin 100 mm NaCl 20 mm Tris HCl ph mm Tris HCl ph % Methanol 0.005% Tween Immunoprecipitation Proteins and their interaction partners can be concentrated employing the interaction between a specific antibody and its antigen. The resulting immune-complexes are precipi-

27 2.2. METHODS 23 tated through interaction with Protein A/G-agarose beads as described by Ausubel and colleagues [4]. Briefly, 5 x x 10 7 cells were harvested and washed twice with PBS. Cells were lysed in 1 ml of RIPA Lysis Buffer supplemented with protease and phosphatase inhibitors for 1 h on ice. Nuclei were precipitated by centrifugation (20 min; rpm; 4 C) and the appropriate antibody was added to the cell lysate at a concentration of 5 µg/ml. Samples were incubated over night at 4 C with gentle rotation. The next day, 30µl Protein A/Gagarose beads equilibrated in RIPA Wash Buffer were added to each sample and the mixture was incubated for 1 h at 4 C with gentle rotation. Beads were precipitated by centrifugation and washed four times with 1 ml RIPA Wash Buffer. Samples were boiled for 5 min with 40 µl Laemmli Loading Buffer and resolved on a SDS Page gel. After Western blotting samples were analysed for the presence of the precipitated protein and its interaction partners, respectively. RIPA Lysis-Buffer: RIPA Wash-Buffer: 50 mm Tris HCl ph mm Tris HCl ph mm NaCl 150 mm NaCl 2 mm EDTA 1 mm EDTA 1% Igepal CA % Igepal CA % Sodium desoxycholate 0.25% Sodium desoxycholate Protease Inhibitors: Phosphatase Inhibitors: 1 µl/ml Aprotinin 1 mm PMSF 1 µl/ml Leupeptin 1 mm Na 3 VO 4 1 µl/ml Pepstatin A 1 mm NaF 1 mm sodium-pyrophosphate Proliferation of cells Proliferation of cells was measured via incorporation of [ 3 H]-thymidine into de novo synthesized DNA. Cell lines, that were cultured with growth factors, like TF1 or CTLL 2 were starved without growth factors for 16 h or 8 h, respectively. Cells were seeded on flat bottom 96 well plates at a density ranging from 5x10 3 to 5x10 5 cells per well. IL-4 (in serial dilutions starting from 20 ng/ml) and other growth factors or inhibitors were added in the appropriate concentrations in a total volume of 200µl. After 48h, cells were pulsed with 0.5 µci/well [ 3 H]-thymidine in 50 µl volume and cells were cultured for another 24 h. Subsequently, cells were transferred to a filter with a cell-harvester (Berthold) and incorporated radioactivity was measured with a betacounter (Berthold). Samples were analyzed as triplets and data was normalized to maximum stimulation Stimulation of macrophages Mice were injected with 2 ml 4% thioglycolate intraperitoneally, and peritoneal exsudate cells (PECs) were harvested on day 4 post injection by rinsing the peritoneum with PBS.

28 24 CHAPTER 2. MATERIALS AND METHODS Cells were seeded on 24 well plates at a concentration of 1 x 10 6 /ml and were allowed to adhere for a minumum of 6 h. Cells were washed to remove non adherent cells; the cells bound to the tissue culture plate were largely macrophages. Macrophages were stimulated with lysate of Borellia burgdorferi (10 µl of a lysate of MOI 10 per well), with IL-1α, IL-1β or with LPS (100 ng/ml). 30min prior to stimulation, cytokines (IL-4 or IL-13) were added in the appropriate concentrations. Supernatants of the stimulated cells were taken after 48 h and analyzed for the production of the proinflammatory cytokines IL-12 (IL-12p40), MCP-1, IL-6 and PGE2 by ELISA Manipulation of mouse embryonic stem cells Balb/c embryonic stem cells (ES cells) were obtained from Prof. Nitschke (Department of Genetics; University of Erlangen) at passage 14. Cells were kept in 10 cm Petri dishes covered with mitotically inactive primary mouse embryo fibroblasts (MEFs) in a tissue culture incubator at 37 C with 10% CO 2. D MEM medium was supplemented with 15% FCS tested for ES culture (PAN), and 120 U/ml ESGRO LIF (Chemicon). For propagation, cells were detached with Trypsin (7 min, 37 C) and seeded 1:3 on new Petri dishes. On day one, 2x10 7 ES cells were electroporated with 20µg targeting vector DNA (500µF, 250V). Cells were harvested, resuspended in 800µl of PBS and linearized vector DNA was added 10 min prior to electroporation. After electroporation cells were kept 10 min at RT, and were then transferred to 50 ml fresh medium and seeded on five 10 cm Petri dishes with irradiated feeder cells. After 24h medium was replaced by fresh medium containing 350 µg/ml Neomycin for positive selection of clones. Medium was changed every day and after 9 to 11 days, clones were picked: Briefly, plates were washed with PBS and clones were mechanically isolated with a P20 pipet set to 10 µl. Cells were transfered to individual wells of a 96 well plate containing trypsin and incubated for 5 min at 37 C. The reaction was stopped by adding 200 µl fresh medium. Colonies were expanded to 24 well plates and finally to 6 well plates and samples were frozen and used for isolation of genomic DNA to screen clones for homologous recombination. Positive clones were thawn on 6 well plates and propagated to four 10 cm plates. Aliquots were frozen for subsequent injection and again DNA was isolated and analyzed by Southern Blotting to confirm successful targeting. ES cells were injected into blastocysts at the Department of Genetics, University Erlangen (Prof. Winkler) Preparation of mouse embryo fibroblasts Mouse embryo fibroblasts (MEFs) were derived from a strain transgenic for the neomycinresistance gen (STAT6 KO) or from Balb/c WT and P62 gene-targeted mice, respectively. Female mice were sacrificed on d12 14 postcoitus and the uterine horns were dissected out. Embryos were isolated, decapitated and eviscerated. The material was placed in a fresh Petri dish containing 1 ml trypsin per embryo and was cut into pieces and subesquently incubated for 5 min at 37 C. 20 ml medium was added and the suspension was passed through a sterile needle with a diameter of 0,9 mm. Volume was adjusted to 10 ml per

29 2.2. METHODS 25 dissected embryo and aliquots of 10 ml were seeded per tissue culture dish. The next day, medium was removed and plates were washed several times with PBS. Cells were cultivated to 80% confluency and subsequently split 1:3 to expand the culture. MEFs were frozen as backups (2 vials/plate) or irradiated (2000 Rad) for immediate use Class switching of splenic B cells A single cell suspension of splenic cells was obtained by passing spleens through a cell strainer (BD Falcon TM ). Lysis of erythrocytes was achieved by incubation with NH 4 Cl (5 min, 37 C). Afterwards cells were enriched for B cells by depletion of cells positive for CD3, CD11b, Gr1, NK DX-5 and TER-119 with the magnetic labeling and separation protocol (BD Biosciences) according to manufacturer s recommendation. Briefly, cells were treated with Mouse Fc Block TM on ice for 15 min and subsequently labeled with biotinylated antibodies specific for the surface markers mentioned above (15 min, 4 C; PBS, 0.5% BSA, 2mM EDTA, 0.1% sodium azide). Cells were washed and incubated with 150 µl of BD TM IMag Streptavidin Particles Plus per spleen on ice for 30 min. Samples were adjusted to a volume of to 2 ml with PBS, 0.5% BSA, 2 mm EDTA, 0.1% sodium azide and placed on the BD IMagnet for 6 min. The procedure was repeated twice and unbound cells were collected as the enriched fraction. Purity of enriched cells was assessed by FACS staining plotting CD3 + versus B220 + cells. Generally, a purity greater than 90% B220 + cells was obtained. For class switching of splenic B cells towards IgE production, 5x10 5 cells per well (24 well plate, culture volume 1 ml) were cultured either with medium alone, or in the presence of 5% α CD40 antibody (hybridoma supernatant; generous gift from Prof. Winkler, Department of Genetics, University Erlangen) in the absence or presence of 20 ng/ml recombinant IL-4. Supernatants were collected on days 3, 5, 7 and 9 for ELISAs. On the same days cells were analyzed for viability by trypan blue exclusion and stained for the activation markers CD23 and MHCII. Additionally, total RNA was isolated on days 2 and 4 for detection of switch transcripts after RT PCR. Germ line transcripts were amplified with primers IeF and CeR; post switch transcripts were amplified with primers ImF and CeR TH2 differentiation of naïve T cells Single cell suspensions were obtained from spleens, erythrocytes were removed and cells were enriched for naïve T cells (CD4, CD62L double positive) either by magnetic labeling (BD Biosciences) or by cell sorting with the MoFlo cell sorter (Nikolaus Fiebiger Center, Erlangen). Purity of cells ranged from 90% (magnetic sorting) to 98% (MoFlo). For differentiation experiments 10 6 cells per ml of culture medium were used per well of a 24 well cell culture plate coated with 5 µg/ml α CD3 antibody and 5 µg/ml α CD28 antibody in coating buffer (1 h; 37 C; 50 mm Tris HCl ph 9.5). Cells were stimulated to drive development either towards TH1 (2 µg/ml α-il-4 (11B11); 1 ng/ml IL-12) or towards TH2 (10 µg/ml α-ifn-γ (XMG-1); 100 ng/ml IL-4). After 4 days cells were washed to remove stimuli and expanded for 2 days with 5 ng/ml IL-2. Subsequently, cells were seeded at a concentration of 10 6 per ml and restimulated for 24h either with medium, α CD3 (5 µg/ml platebound) or PMA/IONO (50 ng/ml and 750 ng/ml). Supernatants

30 26 CHAPTER 2. MATERIALS AND METHODS were analyzed by ELISA for the production of IFN-γ as key cytokine of TH1 differentiated cells and for the production of IL-4, IL-13 and IL-5 as typical TH2 cytokines Infection with Leishmania major Mice were infected with the Leishmania major strain MHOM/IL/81/FEBNI. The parasite was maintained in culture by passages of promastigotes cultured in vitro on rabbitblood-agar plates, followed by in vivo culture in infected Balb/c mice as described [131]. For infection experiments, Leishmania were harvested and washed several times with PBS. 2 x 10 6 promastigotes were injected in the right hind foot pad of mice. Foot pad swelling was measured over a period of three weeks and the ratio of foot pad thickness of infected vs. uninfected pad was plotted against time Oral glucose tolerance of mice Glucose tolerance tests were performed with age-matched female mice fasted for 12 h. Mice were injected with 2 mg/g glucose intraperitoneally at time point 0. Tail blood was collected at time points -30, 30, 60, 120 and 180 min, and blood glucose concentration was determined with a FreeStyle TM bloodsugar measuring system (Disetronic Medical Systems, Sulzbach) OVA induced allergic lung disease Mice were immunized intraperitoneally with 200 µg chicken egg ovalbumin (OVA) on days 1, 14 and 20. Starting six days after the last injection, lightly anesthetized mice recieved two intranasal administrations of 100 µg OVA twice a day for four subsequent days. Mice were sacrificed 18h after the last challenge, and lungs were lavaged three times with 1ml of HBSS containing 10 mm Na EDTA and 10 mm HEPES. Bronchoalveolar lavage fluid (BALF) was collected for analysis of cytokines, cell numbers were determined and cells were differentially counted after cytospin and hematoxylin/eosin staining.

31 Chapter 3 Results 3.1 Studies in cell culture For proliferation studies the human myeloid progenitor cell line TF1 was used, that was stably transfected with either the murine WT IL-4R or the murine N388 IL-4R. These cells proliferate upon mu-il-4 stimulation even if both STAT6 and IRS2 remain unphosphorylated, which is the case in cells that carry the truncated receptor [144]. To further elucidate the mechanism responsible for this STAT6 and IRS2 independent proliferation, the respective contribution of the PI3 Kinase/Akt pathway and the MAP kinase pathway was investigated Phosphorylation of signaling intermediates Proliferation in response to cytokines can be mediated by two distinct pathways: either the PI3 Kinase/Akt pathway or the MAP kinase pathway (see figure 3.1). Transfected TF1 cells were stimulated with IL-4 for 0, 10 or 20 min and cell lysates were assessed for phosphorylation of signaling intermediates involved in these two pathways. No phosphorylation of the Akt kinase was detectable, as might have been expected from the lack of IRS2 phosphorylation, because IRS2 acts upstream of the PI3 Kinase in IL- 4R signaling. On the other hand, both ERK1/2 and p90rsk, two signaling molecules of the MAP kinase pathway, were readily phosphorylated after IL-4 stimulation. This observation holds true for both cell lines carrying either the WT or the N388 IL-4R, indicating that proliferation in TF1 cells is mediated preferentially via the MAP Kinase pathway Effect of kinase inhibitors To further clarify the contribution of the different pathways, TF1 cells carrying either the WT or the N388 IL-4R were stimulated with IL-4 in the presence of various kinase inhibitors. 3T3 cells were used as a toxicicity control as these cells proliferate in the absence of growth factor signaling [147] (data not shown). Both U0126 and PD98059, two inhibitors of the MAP kinase kinase MEK, abrogated IL-4 induced proliferation in TF1 cells at low concentrations (see figure 3.2). At inhibitor 27

32 28 CHAPTER 3. RESULTS Figure 3.1: Proliferation signals initiated by cytokines are mediated via two distinct pathways. (A) Schematic representation of the PI3 kinase/akt pathway and the MAP kinase pathway addressed by various growth factors (adapted from Cell Signaling Technology). Inibitors and their targets are indicated in red. (B) Phosphorylation pattern of signaling intermediates. TF1 N388 cells were stimulated with 20 ng/ml mu-il-4 for 0, 10, or 20 min after which whole cell lysates were prepared and probed for phosphorylation of Akt, S6 ribosomal protein, ERK1/2 and p90 RSK. concentrations of 2 µm and 1.5 µm, respectively, only 50% of the maximun proliferation was observed for both cell lines, thereby indicating that the MAP kinase pathway is involved in mediating proliferation signals. Rapamycin, an inhibitor of the P70S6 kinase by means of binding to mtor, exerted no influence on TF1 proliferation at concentrations of up to 400 nm, which was markedly higher than reported IC50 values of 50 pm [22]. Two other inhibitors of the PI3 Kinase/Akt pathway, namely PI3 Kinase inhibitors LY and Wortmannin were tested in the TF1 system. 62nM Wortmannin reduced TF1 proliferation by only 12% although the IC50 for this substance has been reported to range between 2 and 4nM [3]. LY led to impaired TF1 proliferation with an IC50 of about 2.5µM and almost no proliferation at 20 µm, which is in good agreement with data reported in the literature (IC50 = 1.4µMol [151]).

33 3.1. STUDIES IN CELL CULTURE 29 Figure 3.2: Proliferation of TF1 N388 IL-4R cells in the presence of kinase inhibitors. (A) U0126, (B) PD98059, (C) Rapamycin, (D) Wortmannin, (E) LY and (F) PKCζ pseudosubstrate. Cells were starved without growth factors for 24 h and were subsequently stimulated with 20 ng/ml recombinant mu-il-4. Kinase inhibitors were added 30 min prior to stimulation. After 48 h of stimulation, cells were pulsed with [ 3 H]-thymidine for 20 h and incorporation of radioactivity was measured in a beta counter. Data are expressed as % maximum stimulation (in the absence of inhibitors) and are represenative for at least three independent experiments. As we observed proliferation to be mediated predominantly by the MAP-kinase pathway, we hypothesized that P62 and PKCζ might be involved in the signal transduction (see chapter 1). Therefore, we inhibited PKCζ functionality with a specific pseudosubstrate in TF1 proliferation studies. A concentration of 20 µm PKCζ inhibitor reduced IL-4 induced proliferation by approximately 50%, which indicated the involvement of PKCζ in TF1 proliferation. With all inhibitors tested, no difference was observed between cells carrying the WT IL- 4R or the N388 IL-4R, indicating that proliferation in TF1 cells is only to a small extent dependent on the IRS2-triggered PI3 Kinase pathway.

34 30 CHAPTER 3. RESULTS 3.2 Involvement of P62 in IL-4R signaling One aim of this thesis was to confirm the involvement of the P62 protein in an alternative IL-4R signal transduction pathway (see chapter 1.3), and to evaluate its relative importance both in vitro and in vivo. To achieve this goal, the interaction of the two proteins had to be confirmed by independent techniques. Subsequently, a P62 gene deficient mouse, generated in the laboratory of Prof. Gessner, was analysed on genomic level and was characterized in steady state as well as in immunological settings Binding of P62 to the IL-4R It has been mentioned in section 1.1 that P62 has been identified as a novel interaction partner of the IL-4R in a Yeast-Two-Hybrid screen. To verify this result, several independent techniques were applied Colocalization of P62-EGFP and IL-4R-RFP As a first approach, the two putative binding partners were overexpressed as fusion proteins with green fluorescent protein (GFP) or red fluorescent protein (RFP), respectively. For this purpose, the complete open reading frame (ORF) of P62 was cloned into the BamHI site of the expression vector pegfp-n1, whereas the cdna coding for the 388 IL-4R was inserted into the BglII site of the pdsred1-n1 plasmid. 20 ng of each plasmid were transiently transfected into HEK293T cells by calcium phosphate precipitation and cells were analyzed after 48 h by confocal laser microscopy. Both overexpressed proteins resided in the cytoplasm near the cell membrane and colocalization of the two proteins was detected as yellow fluorescence in the overlay (see figure 3.3 A) Co-Immunoprecipitation of P62 with the IL-4R As a second approach the interaction of the binding partners was shown by immunoprecipitation. Again, both proteins were overexpressed in HEK293T cells, cells were gently lysed 48 h after transfection and the IL-4R was precipitated with a specific antibody coupled to protein A/G-agarose. The isolated protein complex was loaded on a polyacrylamide-gel and was analyzed for the presence of P62 by Western blotting. In initial experiments P62 was expressed as a tagged protein with either an N-terminal Flag-tag (pflag-cmv-2) or a C-terminal His-tag (pcdna3.1/myc-his). The P62 protein bound to the WT IL-4R as well as to the 388 IL-4R, thereby confirming the previous observation of the Yeast-Two-Hybrid experiment (see figure 3.3 B). Furthermore, interaction of the binding partners was shown in a more physiological setting without overexpression of P62: TF1 cells, that were stably transfected with IL-4R constructs, were utilized in a coimmunoprecipitation experiment as described above. Endogenous P62 was proven to bind to the receptor even in the absence of IL-4 stimulation (see figure 3.3 C).

35 3.2. INVOLVEMENT OF P62 IN IL-4R SIGNALING 31 Figure 3.3: Binding of P62 to the IL-4R. (A) HEK293T cells transfected with plasmids encoding the fusion proteins P62-GFP and 388 IL-4R-RFP. Confocal imaging 48 h after transfection. (B) and (D) Immunoprecipitation of P62 with an α-il-4r specific antibody. HEK293T cells were transfected with plasmids coding for IL-4R variants and tagged P62. Whole cell lysates were prepared after 48 h and incubated with an IL-4R specific antibody coupled to agarose beads. (B) Transfection with IL-4R variants and P62-Flag. Precipitates were analyzed for the presence of P62 with an α-flag antibody after Western Blotting. (D) Trimolecular Immunoprecipitation. Cells were transfected with plasmids encoding IL-4R, P62-His and PKCζ-Flag. Precipitates were probed with antibodies specific for IL-4R, His-Tag and Flag-tag. (C) Cell lysates of TF1 cells stably transfected with either WT IL-4R or 388 IL-4R were incubated with an IL-4R specific antiboby. Precipitates were probed for the presence of the endogenous P Trimolecular Co-Immunoprecipitation Finally, we conducted a trimolecular immunoprecipitation to identify possible downstream binding partners of P62. The atypical protein kinase C isoform PKCζ is described as a binding partner of P62, which is also known as ZIP (PKCZeta Interacting Protein) [57, 123, 157]. In several signaling pathways, e.g. the NGF or TNFα pathway, P62 acts as a scaffold protein linking receptor signals to NFκB via the atypical PKCs [40]. In addition to this, a recent publication by Moscat et al. connected IL-4R function to PKCζ for the first time [33]. Based on these data we hypothesized that P62 might function

36 32 CHAPTER 3. RESULTS as an adaptor protein directly linking the IL-4R to downstream events mediated by this atypical PKC. To test this hypothesis, HEK293T cells were transiently transfected with expression constructs coding for the IL-4R, His-tagged P62 and FLAG-tagged PKCζ. Cells were lysed 48 h after transfection and the immunoprecipitation was carried out. The precipitates were analysed by Western Blotting with specific antibodies (see figure 3.3 D). In independent experiments binding of P62 to the WT IL-4R even in the absence of IL-4 stimulation was confirmed. PKCζ was also detected in this complex, thereby supporting our hypothesis. However, PKCζ was also found in association with the IL-4R in samples that were not transfected with the P62 construct, arguing for a possible role of endogenous P Description of the P62 gene deficient mouse model A P62 gene-deficient mouse model was established in the laboratory of Prof. Gessner. For this purpose, the coding exons two, three and four of the P62 gene located on chromosome 11 were replaced by a neomycin resistance cassette in 129/SV ES cells (unpublished data of Gessner). Clones growing under positive/negative selection were subjected to Southern blotting and clones that underwent homologous recombination were injected into blastocysts. The transgene showed successful germline transmission; resulting heterozygous mice were mated to yield homozygous offspring. As the genetic alteration affected only a part of the P62 gene and did not knock out the entire locus, it was necessary to assess the degree of alteration both on genomic and on protein level P62 deficiency on genomic level Although the genetic alteration introduced into ES cells has been analyzed by Southern blotting prior to blastocyst injection, the genotypes of the resulting P62 gene-deficient mice and their littermates were confirmed in a Southern blot experiment. Genomic DNA was isolated from livers of the respective mice by phenol/chloroform extraction and 20 µg of each sample were digested with HindIII. Samples were resolved by agarose gel electrophoresis and blots were incubated with a radioactive probe located in exon 1 beyond the short arm of homology (see figure 3.4). Samples showed the expected bands of 6.8 kb for the WT allele and 5.2 kb for the gene-targeted allele, thereby confirming the successful genetic alteration. From this experiment it cannot be concluded whether the neomycin cassette is still present in the gene-targeted configuration, as the newly introduced HindIII site lies upstream of the loxp site that acts as recognition sequence of the Cre recombinase. Deletion of the neomycin resistance cassette was verified by an additional Southern Blot experiment that employed a probe located in the neomycin cassette (data not shown) P62 deficiency on transcriptome level As the first exon of the P62 gene is still present in the gene-targeted mice, it was investigated if any transcript from this locus could be detected. For this purpose splenocytes

37 3.2. INVOLVEMENT OF P62 IN IL-4R SIGNALING 33 Figure 3.4: Southern blot of P62 gene-targeted mice. (A) Genomic organization of the P62 locus. The targeting vector replaces exons two to four with a neomycin resistance cassette. (B) Southern blot with genomic DNA isolated from ES cells or livers of mice. The 5 probe is located in exon 1 (closed bar in (A) and recognizes a 6.8 kb fragment in the WT or a 5.2 kb fragment in the gene-targeted configuration. isolated from P62 gene-targeted mice were stimulated for 24 h with ConA, after which total RNA was isolated and transcribed into cdna using oligo(dt) primers. The obtained cdna was used as template in a PCR reaction amplifying the P62 core sequence with primers flanking the knocked out exons. Both from WT and from knock out cdnas a product was amplified that was subsequently sequenced. Sequencing provided additional evidence for the genomic alteration and showed that an internal deletion of bases 205 to 753 occured in mrna. Since the deleted region covered a multitude of three bases, a truncated mrna resulted without changing the reading frame, thereby making the existence of a residual protein theoretically possible.

38 34 CHAPTER 3. RESULTS Figure 3.5: Shematic view of P62 domain organisation (adapted from Wooten et al. [40]). The amino acids missing in the hypothetical gene-targeted protein (69 to 251) are indicated by a closed box P62 deficiency on protein level For the reasons stated above, it was necessary to analyze the P62 gene-targeted mice on protein level for the existence of a truncated protein generated from the manipulated locus. Any residual truncated protein was expected to be non-functional based on literature data. If the P62 protein mediates its effects via binding to PKCζ, an intact binding site for this downstream partner will be a prerequisite for its functionality. This binding site has been mapped by several groups to the region comprising the acidic interaction domain (AID-domain; see figure 3.5) [114, 157, 159]. The truncated protein derived from the gene-targeted mrna lacks this binding site (amino acids 69 to 251), and subsequently the protein will no longer be able to recruit the atypical protein kinase Cζ. Moreover, nonphysiological mrna might have a short half-life and might be rapidly degraded by Figure 3.6: Immunoprecipitation of PKCζ. HEK293T cells were transfected with plasmids coding for PKCζ-Flag and P62-His, either WT or the truncated protein hypothetically generated from the gene-targeted configuration. Whole cell lysates were prepared after 48 h and incubated with a Flag-specific antiboby coupled to agarose beads. Precipitates were analyzed for the presence of P62 with an antibody specific for the His-tag and were reprobed for the presence of PKCζ.

39 3.2. INVOLVEMENT OF P62 IN IL-4R SIGNALING 35 the cell machinery, or the deletion in the protein could interfere with its folding. Still, we performed immunoprecipitation studies in HEK293T cells, which overexpressed the truncated P62 and PKCζ in order to confirm the theoretical inability of the truncated P62 protein to recruit PKCζ. After immunoprecipitation of PKCζ, WT P62 was readily detectable in the precipitates, whereas the truncated P62 protein was not able to bind to the kinase (see figure 3.6), as would have been expected from literature data. Additionally, we investigated protein expression in a Western blot experiment. As no specific antibody against P62 existed, we immunized a rabbit with the complete P62 ORF cloned into the BamHI site of the pci TPA DNA vaccination vector. After five rounds of DNA vaccination the antiserum recognized overexpressed P62 (both WT and the theoretically existing deleted protein variant) in lysates from transfected cells, but in organ lysates of both WT or P62 gene-targeted animals no specific band was detectable (data not shown). Due to these constrictions, we cannot rule out the possibility of a residual P62 protein being present in the gene-targeted mice, but if such a protein existed, it would not be able to recruit PKCζ Steady state phenotype of P62 gene-targeted mice The newly established P62 gene-targeted mouse line was first characterized in steady state. Mice appeared and behaved normal, were fully fertile and viable and were born in expected Mendeleian ratios. As these mice were to be further used in immunological experiments, we carefully investigated their hematopoietic compartments with regard to their specific relative compositions as well as absolute numbers of cells. Dendritic cells were analyzed for the specific marker CD11c and the activation markers MHC II, CD80 and CD86. Mast cells were characterized by expression of c-kit, CCR3 and GR-1. Additionally, macrophages present in peritoneal exsudat cells (PECs) were stained with CD11b, Gr-1 and CD19 and after activation also with MHC II, CD80 and CD86. In all these compartments, there were no differences detectable between littermates of WT, P62 gene-targeted or heterozygous genotype (data not shown). IL-4 is an important cytokine for the function of lymphocytes, exerting non-redundant effects on both T and B cells. Also, P62 has been described as an apaptor protein involved in signaling of the T cell receptor [107]. Therefore, we also examined cells isolated from thymus, spleen or bone marrow for their lymphocyte composition. Again, littermates of WT, P62 gene-deficient and heterozygous genotype were used and isolated cells were stained for B and T cell markers (see figure 3.7). In the thymus all mice displayed mostly double positive T cells with little amounts of single positives, mostly CD4 positives. As anticipated, bone marrow contained approximately 80% B cells staining for B220 and 10% Ter119 positive erythroid cells. In spleens, the ratio of B vs. T cells and CD4 vs. CD8 positive cells was normal. Taken together, P62 knock out mice showed no hematopoietic abnormalities in all compartments analyzed.

40 36 CHAPTER 3. RESULTS Figure 3.7: FACS analysis of lymphoid compartments. Single cell suspensions of cells isolated from lymphoid compartments of P62 gene-deficient or WT mice were analyzed for their surface expression of hematopoietic markers. (A) Thymocytes were analyzed for expression of CD4 and CD8. (B) Cells isolated from bone marrow were analyzed for B220 and Ter119. (C) Spleen cells were stained for CD4, CD8 and B220. All plots represent one individual sample and are representative for at least five animals analyzed Immunological phenotypes of P62 gene-targeted mice For all following experiments, in which P62 gene-targeted mice were analyzed in immunological settings, a defined genetic background was a strict prerequisite. To shorten the necessary time for backcrossing, a speed congenic approach was followed as previously described by Longmate et al. [152]. In each generation of backcrossing, males were analyzed for their content of target strain and donor strain DNA by PCR reactios with alltogether 62 primer pairs distributed over the whole genome. After six generations all markers displayed Balb/c configuration, so backcrossing was continued in the classical way. Mice used for experiments were minumum F6 speed congenic and in later experiments F10. After backcrossing, the P62 gene-targeted mice were analyzed for IL-4 mediated phenotypes Oral glucose tolerance The IL-4R and the insuline receptor share a motif of striking sequence homology, namely the I4R (insuline/il-4r) motif comprising amino acids P466 to D480, that is also present in insuline like growth factor-1 receptor (IGF-1R) [49, 71]. This sequence homology alone already suggests similarities in signal transduction pathways. Additionally, a publication by Burnol et al. [17] directly linked the P62 protein to insulin signaling by showing that

41 3.2. INVOLVEMENT OF P62 IN IL-4R SIGNALING 37 Figure 3.8: Glucose tolerance of P62 gene-targeted mice. Female mice, either WT, genedeficient or HZ for P62 were fasted overnight and challenged with 2 mg/g glucose solved in PBS by i.p. injection. Blood glucose levels were monitored over a period of 2 h. Data are expressed as mean ± SEM (n=3). Grb14, a negative regulator of insulin signaling, forms a ternary complex with P62 and PKCζ. This complex formation results in phosphorylation of Grb14 by PKCζ, which is increased upon insulin stimulation; the phosphorylated Grb14 displays elevated inhibition of insuline receptor tyrosine kinase activity. Taken together, a negative feedback loop in insuline receptor signaling was shown to signal via P62. Based on these observations, P62 gene-targeted mice and their littermates were analyzed in a glucose tolerance test. Mice were fasted overnight and blood glucose levels were determined prior to injection with 2 mg/g glucose i.p. Blood glucose levels were determined at time points 30, 60, 120 and 180 min (see figure 3.8). All goups of mice showed a similar trend of glucose levels over time, starting with 100 to 150 mg/dl glucose prior to challenge, reaching peak levels approximately after 30 min, and declining to starting levels by 180 min Functionality of macrophages Macrophages are important mediators of inflammatory processes through release of proinflammatory cytokines TNFα, IL-1, PGE2 or IL-12 [66]. Upon stimulation with LPS or Borrelia burgdorferi, peritoneal exsudate cells (PECs), that mainly consist of macrophages, release these cytokines that can be measured in cell culture supernatants by ELISA. IL-4, and to a lesser extent IL-13, are able to suppress this proinflammatory cytokine milieu when added 30 min prior to stimulation [50, 53]. Based on these data the question arose, if macrophages from P62 gene-targeted mice are still able to transduce these antiinflammatory IL-4 signals. To address this, PECs were isolated from P62 gene-deficient and WT mice as well as IL-4R KO mice and were stimulated in vitro with LPS or B. burgdorferi-lysate. Supernatants were taken after 48 h

42 38 CHAPTER 3. RESULTS Figure 3.9: Cytokine release by stimulated macrophages. PECs were isolated from P62 genetargeted, Balb/c WT and IL-4R KO mice on d4 after thioglycolate injection and were enriched for macrophages. Cells were stimulated with 100 ng/ml LPS or 10 µl B. burgdorferi-lysate (MOI=10, Bb). 30 min prior to stimulation 20 ng/ml recombinant IL-4 was added to test its antiinflammatory potential on IL-12p40 release. Supernatants were taken after 48h and analyzed by ELISA. Data are expressed as percent of maximum stimulation and are representative of three independent experiments. and were analyzed for the presence of the proinflammatory cytokine IL-12 (see figure 3.9). All groups of mice showed a strong increase in IL-12 release after stimulation with both LPS and B. burgdorferi-lysate with absolute levels of IL-12 of about 1500 pg/ml. Addition of 20 ng/ml recombinant IL-4 prior to stimulation reduced the proinflammatory response by approximately 75% in both WT and P62 gene-targeted samples, whereas IL-4R KO cells exhibited no change in secreted IL-12 levels. Similar results were obtained for the measurement of monocyte chemoattractant protein-1 (MCP-1), another proinflammatory cytokine produced by macrophages (data not shown) Class switching of splenic B cells in vitro IgE class switching of murine B cells is known to be mediated exclusively by IL-4 stimulation [23] and requires transcriptional activation of the Cǫ locus which manifests itself in sterile heavy chain transcripts [42]. Additionally, B cells of IL-4 knock out mice [78] and IL-4R knock out mice [102] show severely reduced IgE secretion as compared to WT, thereby confirming the necessity of IL-4 signals for this important lymphocyte function. Based upon these data, we examined P62 gene-targeted mice for their ability to undergo IgE class switching. Firstly, upregulation of the activation markers MHC II and CD23 on B cells was analyzed by FACS, secondly the generation of sterile germline Ig transcripts [73] was verified, and thirdly the generation of IgE antibodies was directly measured by ELISA at several timepoints after stimulation. As anticipated, IL-4R knock out mice were not able to undergo IgE class switching upon stimulation with αcd40 and IL-4 or LPS and IL-4, whereas WT mice showed the published phenotype (see figure 3.10). P62 gene-targeted mice behaved like WT, being activated upon IL-4 stimulation and secreting comparable amounts of IgE with the same kinetics as WT mice.

43 3.2. INVOLVEMENT OF P62 IN IL-4R SIGNALING 39 Figure 3.10: Class switching of splenic B cells towards IgE in vitro. Spleenocytes were isolated from P62 gene-targeted and WT mice and were enriched for B cells. Cells were stimulated with αcd40 and 20 ng/ml IL-4 to induce class switching towards IgE. (A) On day 4 cells were surface-stained for activation markers MHC II and CD23 in FACS analysis. Data are depicted as overlay of the fluorescence of αcd40 stimulated cells (grey areas) vs. αcd40 plus IL-4 stimulated cells (white areas) and are representative for three individual mice. (B) PCR for germ line transcripts (GLTs) and post switch transcripts (PST). RNA was isolated from αcd40 + IL-4 stimulated cells on d 2 and d 4 and was transcribed into cdna with oligo-(dt) primer. cdna from WT and P62 gene-targeted mice was analyzed for the presence of GLTs at d 2 and CTs at d 4, respectively. (C) Supernatants from stimulated cultures were collected on days 3, 5, 7 and 9 and were analysed by ELISA for production of IgE antibodies. Data are expressed as mean ± SEM (n 3). We also detected no differences in the capacity of B cells to proliferate upon IL-4 stimulation comparing P62 KO and WT mice (data not shown), so the lack of the P62 protein does not affect these B cells functions induced by IL Differentiation of naïve T cells in vitro CD4 positive T helper cells can be classified into two groups, namely TH1 and TH2 cells based on their pattern of cytokine secretion [100]. Naïve T cells defined as CD4 +, CD62L + can differentiate into either of these two subsets, depending on the cytokine milieu present during antigen stimulation [1, 128]. TH2 differentiation takes place in the presence of IL- 4 and is characterized by secretion of IL-4, IL-5 and IL-13. Since intact IL-4R signal transduction is required for the efficient development of TH2 cells, P62 knock out mice were analyzed for their capacity to develop this phenotype.

44 40 CHAPTER 3. RESULTS Figure 3.11: Cytokine production by in vitro differentiated T cells. Slenocytes of P62 genetargeted or WT mice were enriched for CD4 +, CD62L + double positive naïve T cells and were differentiated either towards TH1 (IL-12 plus αil-4) or TH2 phenotype (IL-4 plus αifn-γ). Cells were restimulated with medium (C=Control), αcd3 (CD3) or PMA/IONO (P/I) for 24 h, after which supernatants were analyzed by ELISA for cytokine production. (A) IL-4 secretion, (B) IL-5 secretion, (C) IL-13 secretion, or (D) IFN-γ secretion. Data are representative of three independent experiments. Naïve CD4 + T cells were isolated by FACS from spleens of P62 gene-targeted mice and WT littermates and were differentiated in vitro into TH1 or TH2 cells, respectively. After 72h of stimulation, cells were rested for 48h and restimulated with either PMA/IONO or αcd3 for 24 h. Secreted cytokines were analyzed by ELISA, with IFN-γ being considered the major TH1 cytokine and IL-4, IL-5 and IL-13 being TH2 cytokines. Cells of both WT and P62 gene-deficient mice displayed a robust IFN-γ secretion of up to 20 ng/ml by TH1 differentiated cells, whereas TH2 differentiated cells showed a residual level of up to 2 ng/ml (see figure 3.11). The TH2 cytokines IL-4 and IL-13 were not detectable in supernatants of TH1 differentiated cells and averaged 4000 pg/ml and 1500 ng/ml in supernatants of TH2 differentiated cells; IL-5 was detectable in minor concentrations of up to 100 pg/ml in supernatants of both TH1 and TH2 differentiated cells. Taken together, both WT and P62 gene-deficient T cells showed secretion of the expected key cytokines for either TH1 or TH2 differentiated cells. In this setting of in vitro differentiation, no difference was detectable between P62 gene-targeted or WT mice, indicating that P62 is dispensable for this IL-4 function.

45 3.2. INVOLVEMENT OF P62 IN IL-4R SIGNALING Infection with Leishmania major Experimental leishmaniasis is a well established in vivo model in which the generation of a TH2 immune response is fatal for the host organism. The parasites (Leishmania major) reside intracellularly in macrophages and can be controlled by a cellular immune response employing CD4 + T helper cells. In this setting protection is mediated by a TH1 type response, where IFN-γ activates macrophages to produce reactive oxygen- and nitrogenintermediates like the inducible nitric oxide synthetase (INOS) that acts leishmanicide. On the contrary, generation of a TH2 immune response results in the production of cytokines such as IL-4, IL-6 and IL-10, inhibiting the activation of macrophages and rendering the host unable to control the infection [9, 55, 86]. We infected P62 gene-targeted and WT mice on a susceptible Balb/c genetic backgound with L. major promastigotes in the right hind foot pad and monitored footpad swelling over time (see figure 3.12 A). Both groups of mice exhibited footpad swelling starting on d 14 and getting worse until mice were sacrificed on d34 as a regression in the inflammatory response no longer seemed likely. P62 gene-deficient mice showed no resistance towards this infection, thereby indicating that the development of a TH2 immune response was also possible in this in vivo experiment, supporting the data from the in vitro differentiation studies. Additionally, the parasite burden in the infected foot, the draining lymph node and the spleen was monitored (data not shown), and the immune response was characterized by measuring immunoglobulin levels in sera of infected mice. Levels of IgE, which correlate with the failure to control the infection, are shown in figure 3.12 B. Again, no difference Figure 3.12: Infection of mice with Leishmania major. P62 gene-targeted and WT mice on a susceptible Balb/c background were immunized on d 0 with 2 x 10 6 promastigotes in the right hind foot pad. (A) Footpad swelling was monitored over time and is expressed as ratio of footpad thickness of the infected foot vs. the uninfected foot. Data are expressed as mean ± SEM (n=5). (B) Serum levels of IgE 27 d after infection. Data are expressed as mean ± SEM (n=5)

46 42 CHAPTER 3. RESULTS was detected between P62 gene-targeted and WT mice, supporting the observation that P62 protein is not required for mounting an immune response against Leishmania or is at least redundant in this setting Fibroblast functionality In the human sytem, IL-4 is described to be involved in eotaxin secretion by fibroblasts, which is of importance in atopic diseases and asthma [94, 96]. A recent publication by Moscat et al. [32] connected the upregulation of eotaxin in mouse embryonal fibroblasts (MEFs) upon IL-4 stimulation with the atypical protein kinase C, which prompted us to analyze P62 gene-deficient mice for this IL-4 phenotype. Short term primary cell lines of MEFs were generated from P62 gene-targeted and Balb/c WT mice and were stimulated in vitro with recombinant IL-4. For quantification of eotaxin transcription, total RNA was isolated at time points 12, 24 and 48 h and was subjected to a light cycler PCR for CCL11. Supernatants collected after 24 and 48h were analyzed by ELISA. Quantification of mrna levels showed a pronounced upregulation of eotaxin in WT MEFs after 48 h, whereas samples from P62 gene-targeted mice exhibited a significantly reduced upregulation of mrna levels ( 200 fold vs. approximately 20 fold as compared to unstimulated controls; see figure 3.13). Also, direct measurement of the protein levels displayed a high concentration of eotaxin in supernatants of IL-4 stimulated WT fibroblasts (3000 pg/ml), whereas in P62 gene-targeted fibroblasts less than 1000 pg/ml were secreted. This observation supported the observation made on transcriptome level. Figure 3.13: Upregulation of eotaxin in MEFs upon IL-4 stimulation. MEFs were prepared from three different donors for P62 gene-targeted or Balb/c WT mice, respectively. Cells at passage 1 or 2 were stimulated with 20 ng/ml IL-4 or were left unstimulated. (A) Total RNA was prepared after 48h of stimulation and eotaxin1 mrna levels were determined with normalization to the house keeping gene HPRT. Data are expressed as mean ± SEM (n=6)., p vs WT. (B) Supernatans were collected after 48 h and analyzed by ELISA. Data are expresed as mean ± SEM (n=4)., p 0.05 vs WT.

47 3.2. INVOLVEMENT OF P62 IN IL-4R SIGNALING 43 These data indicate a non-redundant role of the P62 protein in the release of eotaxin by primary mouse fibroblasts upon IL-4 stimulation. This observation constitutes the first phenotype of P62 gene-targeted mice in response to IL-4, thereby supporting the initial assumption that P62 protein contributes to IL-4R signal transduction in some cell types OVA induced allergic asthma Based on our observations in vitro (see ), we assessed eotaxin functionality in P62 gene-targeted mice in vivo. Rothenberg et al. reported that eotaxin is important for early recruitment of eosinophils in a murine model of asthma [118], which was later confirmed by knock out studies in mice deficient for eotaxin or its receptor CCR3 [111]. Therefore we established a model of OVA induced allergic airway disease in our laboratory (in cooperation with the group of PD Dr. A. Pahl, Department of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen) [21]. Mice on a susceptible Balb/c background, either P62 gene-deficient or WT, were sensitized by i.p. administration of 200 µg OVA on days 1, 14 and 20. Mice were challenged by intranasal application of 100 µg OVA in saline on days 26 to 29, two times a day. 18 h after the last challenge, mice were sacrificed, and lungs were rinsed three times with 1ml HBSS-buffer. Samples were centrifuged, supernatants were collected as broncheoalveolar lavage fluid (BALF) for measurement of cytokines and cells were differentially counted for composition and absolute numbers. Levels of IL-4, IL-5, IL-13 and the chemokine eotaxin1 were determined in BALF of OVAchallenged mice by ELISA, whereat no significant difference was detected between P62 gene-targeted mice and WT controls. Likewise, both absolute numbers of cells recovered from the lungs, and absolute numbers of eosinophils did not differ between groups of mice (see figure 3.14). Taken together, no abnormalities in eosinophil recruitment could be detected in this animal model of late phase eosinophilia. Figure 3.14: OVA induced allergic airway disease. (A) Absolute eosinophil numbers in the BALF of OVA-challenged mice were determined by differential cell count after hematoxylin/eosin staining. (B) Levels of IL-4 in BALF were determined by ELISA. Data are expressed as the mean ± SD (n=6) and are representative of two independent experiments.

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