A Thesis Entitled Identification of Fcγriia STAT6 Interaction and The

A Thesis

Entitled

Identification of FcγRIIA STAT6 Interaction and the Subsequent Effects

Elizabeth P. Beil

Submitted to the Graduate Faculty in partial fulfillment of the requirements for the

Masters of Science in Biomedical Science

______Randall Worth, Ph.D. (Major Advisor/Committee Chair)

______R. Mark Wooten, Ph.D. (Committee Member)

______Z. Kevin Pan, Ph.D. (Committee Member)

______Patricia Komuniecki, Ph.D. (Dean, College of Graduate Studies)

The University of Toledo June, 2012

This document is copyright material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Identification of FcγRIIA STAT6 Interaction and the Subsequent Effects

Elizabeth P. Beil

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science in Biomedical Science

The University of Toledo January 2012

FcγRIIA, a transmembrane receptor for immunoglobulin G (IgG), triggers phagocytosis, endocytosis and oxidative burst in leukocytes. Signal transduction via FcγRIIA leading to these outcomes has been relatively well studied. To identify additional signaling partners, a yeast two- hybrid analysis employing the cytoplasmic domain of FcγRIIA was performed. Interestingly, the

SH2 domain of signal transducer and activator of transcription 6 (STAT6) appeared in our analysis. STAT6 traditionally interacts with interleukin-4 (IL-4) receptors to induce expression of IL-4 and IL-13 in T cells. Macrophages have also been observed to produce IL-4 in response to pharmaceutical and pathogenic stimuli and to respond to IL-4. However, production of IL-4 or IL-13 subsequent to FcγR ligation has not been observed. Therefore, to better define the specific interaction of FcγRIIA with STAT6, we engineered Chinese hamster ovary cells to express FcγRIIA and RFP-STAT6 to determine if specific ligation of FcγRIIA induces STAT6 nuclear translocation. This resulted in lack of consistent expression of the RFP-STAT6 causing us to perform our subsequent experiments in the human macrophage-like cell line, THP-1. To

ii further elucidate this interaction, FcγRIIA immunoprecipitation followed by western blotting of

FcγRIIA and the whole cell lysates from THP-1 macrophages, was performed. We observed the constitutive interaction of non-phosphorylated STAT6 with FcγRIIA. We subsequently sought to examine STAT6 activation after stimulation of THP-1 macrophages with various FcγR ligands.

Using IL-4 as a positive control, we observed heat-aggregated IgG induces significant amounts of STAT6 translocation compared to untreated cells (p<0.05). We then sought to determine if IL-

4 was produced from THP-1 macrophages after FcγR ligation. We were unable to detect IL-4 from THP-1 macrophages, but were able to detect IL-4 from Jurkat cells, a human T cell line, that were stimulated with ionomycin and phorbol myristal acetate. We then examined the effects of IL-4 on FcγR-mediated activities. We observed that transient exposure of THP-1 macrophages to IL-4 had no significant effect on endocytosis or phagocytosis.

Our current observations indicate an interaction exists between FcγRIIA and S TAT 6 through the SH2 domain of STAT6 producing nuclear translocation of non-phosphorylated

STAT6 without induction of IL-4 gene expression. This observance may signal an important avenue of research in understanding the mechanisms of phosphorylation of STAT6 and STAT6 induced gene expression.

iii

Dedication

Proverbs 3:5,6 “Trust in the Lord with all thine heart; and lean not unto thine own understanding.

In all thy ways acknowledge Him, and He shall direct thy paths.”To Jared, Wesley, and Nelsa thank you for making me into the woman I am today; and to my nephew and niece, Joshua and

Emma, who are a constant reminder to live each day as if it were your last.

Acknowledgements

There are many people who without their support this work would not have been

possible. I would first like to thank my husband for loving me, standing by me, encouraging me,

and pushing me to accomplish more than I ever thought possible. I would next like to thank my

family for all of their love and encouragement they have given me through this journey. I am incredibly grateful for the constant love from my husband and my family as I would not be where I am today if it were not for them.

I would next like to thank Dr. Randall Worth, my major advisor. He has been an advisor, a mentor, a teacher, and a friend. I owe him a debt of gratitude for allowing me to be a student in his lab and to work on this project. I consider it a privilege to work with him.

Finally, I would like to thank the other Worth lab members. Thank you, Joshua Vieth and

Martha Fernstrom for the discussions, encouragement, and laughter over the past few years. I look back on my experience in the Worth lab with fond memories.

Table of Contents

Abstract ii

Table of Contents vi

List of Figures viii

List of Abbreviations x

Chapter 1 Literature Review 1

1.1 Aspects of Immunity………………………………………………………………...... 1

1.2 Macrophages…………………………………………………………………………..3

1.3 Fc Receptors…………………………………………………………………………..5

1.4 Fcγ Signaling………………………………………………………………………….9

1.5 Signal Transducer and Activator of Transcription…………………………………...12

1.6 Interleukin-4 and Interleukin-4 Receptor……………………………………………15

Chapter 2 Materials and Methods 18

2.1 Reagents and Antibodies…………………………………………………………….18

2.2 Yeast Two–Hybrid System…………………………………………………………..19

2.3 Chinese Hamster Ovary Culture…………………………………………………….20

2.4 Red Fluorescent Protein-Signal Transducer and Activator of Transcription 6……...20

2.5 THP-1 Culture……………………………………………………………………….21

2.6 Bead Preparation…………………………………………………………………….21

2.7 Heat-Aggregated Immunoglobulin G Preparation…………………………………..22

2.8 Monomeric Immunoglobulin G Preparation………………………………………...22

2.9 Stimulation, Immunoprecipitation, Western Blotting………………………………22

2.10 Stimulating, Staining, Fluorescence Microscopy………………………………….24

2.11 Jurkat Culture………………………………………………………………………25

2.12 Stimulation and RNA Extraction…………………………………………………..25

2.13 Real Time Quantitative Reverse Transcriptase Polymerase Chain Reaction………26

2.14 Erythrocyte Preparation…………………………………………………………….27

2.15 Phagocytosis………………………………………………………………………..27

2.16 Endocytosis…………………………………………………………………………28

2.17 Statistics…………………………………………………………………………….29

Chapter 3 The Interaction of FcγR I I A w it h S TAT 6 30

3.1 The SH2 Domain of STAT6 Interacts with the Cytoplasmic Domain of FcγRIIA……………………………………………………………………………..30

Chapter 4 The Role of FcγR Ligation in STAT6 Signaling 35

4.1 Nuclear Translocation of STAT6 Following FcγR Ligation…………………………35

4.2 Ligation of FcγRs Does Not Produce IL-4 mRNA………………………………….38

4.3 FcγR-Mediated Activities Are Not Affected by IL-4………………………………..41

Chapter 5 Conclusions and Discussion 43

References 48

vii

List of Figures

3-1 SH2 sequence of human STAT6…………………………………………………….31

3-2 Construct of RFP-S TAT 6 …………………………………………………………...32

3-3 Representative fluorescence images of RFP-STAT6 transfection into CHO-IIA

cells…………………………………………………………………………………33

3-4 Representation of constitutive interaction of S TAT 6 with FcγRIIA………………..34

3-5 Representation of constitutive interaction of STAT 6 w it h Fc γRs…………………..34

4-1 Fluorescence micrographs of STAT6 nuclear translocation upon FcγR ligation……36

4-2 Quatification of STAT6 nuclear translocation in THP-1 macrophages……………...37

4-3 Representation of IL-4 production from stimulated Jurkat cells…………………....39

4-4 Representation of the fold change in IL-4 from stimulated THP-1

macrophages………………………………………………………………………...40

viii

4-5 Quantification of phagocytosis using epifluorescence microscopy analysis of

THP-1 macrophages stimulated with 50ng/mL IL-4………………………………41

4-6 Trend of endocytosis analyzed through flow cytometric analysis of THP-1

macrophages stimulated with 50ng/mL IL-4………………………………………42

List of Abbreviations

Ab…………………Antibody

CHO………………Chinese Hamster Ovary Cells CHO-IIA………….Chinese Hamster Ovary Cells transfected with human FcγRIIA CHO-IIAGFP……..Chinese Hamster Ovary cells transfected with FcγRIIA-GFP CXCL8……………Chemokine Interleukin-8

EA…………………IgG-opsonized erythrocytes

FcR………………...Fc Receptor FcαR……………….Fc Receptor for IgA FcδR……………….Fc Receptor for IgD FcεR……………….Fc Receptor for IgE FcγR……………….Fc Receptor for IgG FcµR………………Fc Receptor for IgM FITC……………….Fluorescein Isothiocyanate FITC-haIgG……….FITC-conjugated haIgG

γc………………….Common Gamma Chain

H131……………….High-responder FcγRIIA isoform HaIgG……………..Heat-Aggregated IgG Complexes

IC…………………..Immune Complex IFN………………...Interferon IL…………………..Interleukin IL-4R………………Interleukin-4 Receptor Ig…………………...Immunoglobulin IgA…………………Immunoglobulin A IgE………………….Immunoglobulin E IgG………………….Immunoglobulin G IP3………………….Inositol triphosphate ITAM……………….Immunoreceptor Tyrosine-based Activation Motif ITIM………………..Immunoreceptor Tyrosine-based Inhibitory Motif kDa…………………Kilodalton

M1………………….Classically Activated Macrophages M2………………….Alternatively Activated Macrophages MHC……………….Major Histocompatability Complex

MR…………………Regulatory Macrophages

NK………………….Natural Killer Cells

PE…………………..Phycoerythrin PI3K………………..Phosphoinositide 3-kinase PIAS………………..Protein Inhibitors of Activated Stats PKC………………...Protein kinase C PLC………………...Phospholipase C PMA………………..Phorbol Myristal Acetate PMN………………..Polymorphonuclear neutrophilic leukocyte

ROS………………...Reactive Oxygen Species R131…………………Low-responder FcγRIIA isoform

SH2…………………Src Homology 2 Domain SHIP………………..SH2 Domain-containing Inositol 5’-Phosphatase SLE…………………Systemic Lupus Erythematosus SOCS……………….Suppressors of Cytokine Signaling

TH…………………..T Helper Cell TNF…………………Tumor Necrosis Factor TLR…………………Toll-like Receptor

WASp……………….Wiskott-Aldrich Syndrome Protein

Chapter 1

Literature Review

1.1 Aspects of Immunity

Phagocytes

The immune system is made up of two main branches of immunity: innate and adaptive. Innate immunity is made up of phagocytes, pattern recognition receptors, and the complement system. The first element are the phagocytes: macrophages and polymorphonuclear neutrophilic leukocytes

(PMNs). Macrophages reside in the host tissues while PMNs are generally found in the blood.

Pathogens opsonized with immunoglobulin (Ig) ligate Fc receptors (FcR) (Ravetch and Kinet 1991), certain sugar moieties ligate mannose receptors (Apostolopoulos and McKenzie 2001), and anionic polymers and acetylated low-density lipoproteins ligate scavenger receptors (Gough and Gordon 2000) leading to phagocytosis and death of the pathogen. Phagocytes also contain membrane-bound granules

(lysosomes) capable of fusing to phagosomes and releasing their contents to aid in the killing of the microbe (Mayorga, Bertini et al. 1991). Phagocytes also produce reactive oxygen and nitrogen metabolites (Babior 1978; Babior 1978; Klebanoff 1980) upon phagocytosis to aid in killing of the pathogen. Macrophage phagocytosis produces an abundance of inflammation that aids in resolution of infection. Macrophages become activated to release cytokines, chemokines, and various mediators of inflammation such as prostaglandins, leukotrienes, and platelet-activating factor after interaction with a pathogen (Janeway 2005).

Pattern Recognition

The second element of innate immunity is pattern recognition. The innate immune system 1 recognizes pathogens through receptors that bind to regular patterns present on the pathogens. This is evident with the Toll-like receptors (TLR) present on phagocytes. Each TLR binds a specific pattern on a pathogen, producing the pro-inflammatory cytokines interleukin-1 β (IL-1β), IL-6, IL-12, and TNF-

α, and chemokines that recruit monocytes, neutrophils, and effector cells to the infection such as chemokine IL-8 (CXCL8) (Heine and Lien 2003). Innate immunity is also involved in the eradication of viruses through the production of interferon (IFN) α and IFNβ from the infected cell binding to the interferon receptor and signaling through the JAK/STAT pathway (Hervas-Stubbs, Perez-Gracia et al.).

Natural killer cells can then become activated by IFNα, IFNβ, and IL-12 to contain the viral infection

(Biron, Nguyen et al. 1999). Another component of innate immunity is the complement system. This system opsonizes pathogens allowing phagocytes that express complement receptors to phagocytose the pathogen and forms the membrane attack complex to eradicate the pathogen (Frank and Fries

1991).

Adaptive Immunity

Whereas phagocytes are involved in innate immunity, antigen presenting cells, T cells, and B cells are integral to adaptive immunity. Macrophages, dendritic cells (Guermonprez, Valladeau et al.

2002), and B cells (Rodriguez-Pinto 2005) are the antigen presenting cells that function to present antigen to naïve T cells producing effector T cells. T cells exist as two main subpopulations: cytotoxic

CD8+ T cells and helper CD4+ T cells. Through strength of the TCR-co-receptor signal and interaction of CD4 with major histocompatability complex (MHC) II or CD8 with MHC I expressed on antigen- presenting cells, effector T cells are produced (Germain 2002). Cytotoxic CD8+ T cells function by recognizing viral peptides presented on MHC I of infected antigen presenting cells and trigger the cell to undergo apoptosis through the release of perforin, granzymes, and granulysin contained in lytic granules (Russell and Ley 2002; Lieberman 2003). CD4+ T cells are further subdivided into the T helper cells (TH), TH1 and TH2. The primary effector function of TH1 cells is macrophage activation

2 through cell contact and secretion of IFN-γ, which aids in pathogen destruction (Stout and Bottomly

1989). TH2 cells act primarily on B cells both to stimulate naïve B cells to produce IgM and to cause isotype switching of the B cell (Snapper and Paul 1987; Noelle, Roy et al. 1992). Antibodies have three contributions to immunity: neutralization of pathogens, opsonization of pathogens, and complement activation (Janeway 2005). Neutralization of pathogens by antibodies inhibits viruses and intracellular bacteria from attaching and entering cells. Opsonization of pathogens by antibodies allows FcRs to bind the Fc portion of the antibody and ultimately clear the bacteria. Finally, complement activation signals a cascade of events which results in the death of the pathogen through loss of integrity of the pathogen’s outer membrane and through opsonization. Therefore, production of antibodies is an integral member of the adaptive immune response.

1.2 Macrophages

M1 Macrophages

Macrophages have more recently been divided into two main categories: classically activated macrophages (M1) and alternatively activated macrophages (M2). These subsets of macrophages differ in their activation, cytokine production, and function. Macrophages become classically activated upon exposure to two signals. The first signal is IFN-γ from natural killer cells, which primes the macrophage for activation (Nathan 1991). The second signal is from TNF, which is endogenously produced from the macrophage upon TLR ligation. M1 macrophages produce a variety of pro- inflammatory cytokines including IL-1, IL-6, and IL-23 (Edwards, Zhang et al. 2006). These cytokines aid in the production of inflammation with subsequent resolution of infection. The functions of M1 are extensive. They include the ability to migrate to sites of inflammation, phagocytose pathogens

(Ezekowitz and Gordon 1984), produce reactive oxygen intermediates and nitrogen species (Babior

1978; Babior 1978), restrict tryptophan from vacuolar organisms limiting the pathogen’s intracellular growth (Carlin, Borden et al. 1989), and induce apoptosis of damaged cells, which lacks the inflammation involved with phagocytosis partially due to the production of transforming growth factor

β (TGF-β) thus protecting host tissues (Fadok, Bratton et al. 1998).

M2 Macrophages

The second class of macrophages consists of the M2 macrophages. M2 were first discovered in murine macrophages that were exposed to IL-4 with subsequent upregulation of mannose receptor activity (Stein, Keshav et al. 1992). Traditionally, this type of macrophage is induced when exposed to

IL-4 or glucocorticoids. M2 primarily produce the anti-inflammatory cytokine, IL-10 (Gerber and

Mosser 2001). The novel chemokine, alternative macrophage activation-associated CC-chemokine-1

(AMAC-1), a secretory product distinctive of M2 has been discovered to aid in the development of effector TH1 cells (Adema, Hartgers et al. 1997; Kodelja, Muller et al. 1998). M2 macrophages also have a suppressive activity as they inhibit the proliferation of lymphocytes and TH2 cells, which could aid in attenuating inflammation in allergic and autoimmune diseases (Schebesch, Kodelja et al. 1997).

The main function of murine alternatively activated macrophages appears to be in wound healing through the stimulation of arginase activity, allowing them to convert arginine to ornithine, a precursor to collagen (Hesse, Modolell et al. 2001; Rutschman, Lang et al. 2001). It has been reported that human macrophages may aid in the production of fibronectin and extracellular matrix (Gratchev, Guillot et al.

2001). Therefore, M2 macrophages could be fundamental to the tissue remodeling process.

MR Macrophages

As mentioned above, traditionally macrophages are divided according to classical or alternative activation. Numerous groups have concluded that this causes immense confusion, as all other types of macrophages except M1 are placed in the M2 category. A third group has been named either Type-II activated macrophages or regulatory macrophages (MR). MR have been studied extensively in murine

4 systems. Like M1 macrophages, these macrophages require two signals for activation. The first signal is from lipopolysaccharide inducing M1 macrophages while the second signal is ligation of FcγRs

(Sutterwala, Noel et al. 1997). Once these signals are received, the macrophage produces abundant amounts of IL-10 while IL-12 is essentially abrogated (Sutterwala, Noel et al. 1998). It has been proposed that the ratio of IL-10 to IL-12 production could differentiate the M1 macrophages from MR.

Due to the upregulation of IL-10, the MR produce a potent anti-inflammatory effect, which could protect the host from extensive tissue damage. Upon presentation of antigen by this sub-category of macrophages, T cells were induced to produce high levels of IL-4 (Anderson and Mosser 2002).

Subsequently, mice vaccinated with ovalbumin in the presence of MR made significant amounts of

IgG1 (Anderson and Mosser 2002). Therefore, it has been proposed that ligation of FcγR on murine

MR by antigen-IgG complexes induces T cells to produce IL-4, which induces B cell isotype switching and production of IgG1. Numerous clinical implications exist for this production of IgG through MR.

Leishmania spp have been observed to bind host IgG and ligate the FcγR inducing the development of

MR that are permissive for intracellular growth of Leishmania (Miles, Conrad et al. 2005). Another example involves African trypanosomes changing their main surface antigen allowing a robust antibody response to develop resulting in formation of immune complexes. The immune complexes bind the macrophage FcγR, induce MR, which inhibit a future immune response to clones of trypanosomes (Baetselier, Namangala et al. 2001).

1.3 Fc Receptors

Receptors are also important players in both innate and adaptive immunity. FcRs are one group of receptors found on innate immune cells that are necessary for adaptive immunity to function. The

FcRs can be divided into five groups: FcαR, FcδR, FcεR, FcγR, and FcµR (Gessner, Heiken et al.

1998). The FcRs bind their respective immunoglobulins: FcγR binds immunoglobulin G (IgG), FcµR binds IgM, FcεR binds IgE, FcδR binds IgD, and FcαR binds IgA. FcγRs can be further divided into three categories: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) (Ravetch and Kinet 1991; van de

Winkel and Capel 1993; Hulett and Hogarth 1994; Joshi, Butchar et al. 2006). A fourth FcγR, FcγRIV, has recently been discovered on murine monocytes, macrophages, and neutrophils (Nimmerjahn,

Bruhns et al. 2005). FcγR are located on macrophages, monocytes, neutrophils, eosinophils, mast cells, basophils, platelets, and dendritic cells (Perussia, Dayton et al. 1983; Ravetch and Kinet 1991; Takai

2005). FcγR are encoded by at least eight genes found on chromosome 1, with individual isoforms generated through alternative splicing (Ravetch and Kinet 1991; Hogarth, Hulett et al. 1992). FcγR are typically classified as activating or inhibitory receptors, both of which drive phagocytic mechanisms.

Both activating and inhibitory receptors are found co-expressed on the surface of the same cell and are coengaged by the same IgG ligand, functioning in concert to keep a balanced immune response.

Therefore, FcRs are a link between innate and adaptive immunity because they are adaptive receptors found on innate cells.

IgG is the most abundant immunoglobulin in the human body, existing at concentrations of up to 10mg/mL in normal sera (Fahey and Lawrence 1963; Stiehm and Fudenberg 1966; van der Giessen,

Rossouw et al. 1975). In humans IgG exists as four monomeric isotypes: IgG1, IgG2, IgG3, and IgG4.

All four isotypes are involved in neutralization of pathogens. Of the IgG isotypes, IgG2 is traditionally not involved in opsonization of pathogens. IgG4 lacks the capability to activate the complement system, unlike the other three isotypes. IgG1 and IgG3 have the highest affinity for the FcγRs making them an integral facet for FcγR function (Bruhns, Iannascoli et al. 2009).

FcγRI

FcγRI is primarily expressed on monocytes and macrophages (Ravetch and Kinet 1991;

Hogarth, Hulett et al. 1992; Schreiber, Rossman et al. 1992; van de Winkel and Capel 1993). FcγRI can be expressed on neutrophils when in the presence of IFN-γ and G-CSF, triggering phagocytosis, antigen presentation, and endocytosis at sites of inflammation (Perussia, Dayton et al. 1983; Kerst, van de

Winkel et al. 1993). FcγRI binds monomeric IgG with high affinity (~10-7 M), and binds the sublcasses of IgG with an order of IgG3>IgG1>IgG4, while not binding IgG2 (van de Winkel and Capel 1993;

Bruhns, Iannascoli et al. 2009). It is composed of three Ig-like domains, and it has been proposed that the third domain may account for the high-affinity binding of IgG1 and IgG3 by the receptor (Allen and Seed 1989; Ravetch and Kinet 1991). As with other FcγR, FcγRI is involved in phagocytosis.

FcγRII

FcγRII consists of three isoforms: FcγRIIA, FcγRIIB, and FcγRIIC (Ravetch and Kinet 1991;

Schreiber, Rossman et al. 1992; van de Winkel and Capel 1993). They are low-affinity receptors, though FcγRIIA (~10-6 M, binding order of IgG3>IgG1>IgG2>IgG4) has a higher binding affinity than

FcγRIIB and FcγRIIC (~10-5 M, binding order of IGg1>IgG4>IgG3>IgG2) (Bruhns, Iannascoli et al.

2009). The two alleles of FcγRIIA encode two variants which differ at one residue and are named high- responder (H131) and low-responder (R131) (Warmerdam, van den Herik-Oudijk et al. 1993). The H131

-5 -4 exhibits a higher affinity for binding IgG2 (~4X10 M) than does the R131 (~8X10 M) (Bruhns,

Iannascoli et al. 2009). It has been suggested that homozygosity for R131 variant is associated with increased susceptibility for autoimmune diseases such as systemic lupus erythamatosus (Dijstelbloem,

Bijl et al. 2000; Moens, Van Hoeyveld et al. 2006). FcγRIIA, an activating receptor, consists of an extracellular domain to which the Fc portion of immunoglobulin G (IgG) binds, a transmembrane region, and a cytoplasmic tail, which is necessary for signaling (Huang, Hunter et al. 2004). Activation of FcγRIIA results in actin filament reorganization resulting in the formation of a phagosome and the initiation of signaling events leading to killing of pathogens and release of pro-inflammatory cytokines

7 and reactive oxygen species (Hunter, Kamoun et al. 1994; Joshi, Butchar et al. 2006). Several monoclonal antibodies have been raised to FcγRIIA, namely IV.3 (Jones, Looney et al. 1985; Looney,

Abraham et al. 1986) and KuFc79 (Vaughn, Taylor et al. 1985). Theses monoclonal antibodies have proven invaluable to studying the function of FcγRIIA.

FcγRIIB, an inhibitory receptor, dominates a response through higher expression or higher affinity for ligand as compared to FcγRIIA (Kalergis and Ravetch 2002; Nimmerjahn 2006). As a regulatory mechanism, the termination of an immune response through FcγRIIB is accomplished by signaling through binding and coaggregation of inhibitory receptors, and not through the actual loss of activating signals (Billadeau and Leibson 2002). Therefore, co-expression of both activating and inhibitory FcγR determines the threshold for cellular activation. FcγRIIC has been observed on natural killer (NK) cells and possesses the extracellular portion of FcγRIIB and the cytoplasmic portion of

FcγRIIA resulting from an unequal crossover event (Warmerdam, van den Herik-Oudijk et al. 1993).

FcγRIII

FcγRIII is a 50-80kDa glycoprotein encoded by two genes. FcγRIIIA is found on natural killer cells, and FcγRIIIB is found predominantly on neutrophils (Salmon, Millard et al. 1995; Gessner,

Heiken et al. 1998). FcγRIII binds aggregated immunoglobulins or antibody-antigen complexes with low affinity (~10-6 M), but high avidity (Ravetch and Kinet 1991). FcγRIIIA binds IgG sublcasses with an order of IgG3>IgG2>IgG4>IgG1. Alternatively, FcγRIIIB binds IgG subclasses with an order of

IgG1>IgG3. FcγRIIIB is attached to the outer surface of the cell membrane by a glycosyl phosphatidylinositol linkage (Huizinga, Kleijer et al. 1990; Ory, Clark et al. 1991). FcγRIIIA, but not

FcγRIIIB, is involved in phagocytic signaling as with the other FcγRs.

1.4 FcγR Signaling

With few exceptions, signaling through FcγR is achieved through association with the common gamma chain (γc), which contain a cytoplasmic ITAM signaling domain (Cambier 1995; Carlsson,

Candeias et al. 1995). Experiments involving a knock-out of the γ-chain in mice established the importance of this signaling molecule required for macrophage responses to Ig, such as phagocytosis and antibody-mediated cellular toxicity, as well as the initiation of the Arthrus reaction, despite the mice exhibiting a normal complement system (Sylvestre and Ravetch 1994; Takai, Li et al. 1994).

These findings established an important role for FcγR in immune and inflammatory signaling.

Phagocytic signaling occurs through the association of FcγRI with the γc chain. The cytoplasmic tail of FcγRI lacks intrinsic signaling function; therefore, phagocytosis and endocytosis are modulated via intracellular sequences (Edberg, Yee et al. 1999) or through association with periplakin, an intracellular protein (Beekman, Bakema et al. 2004).

FcγRIIA is a single chain protein that does not require the γc to signal. Instead, it uses the cytoplasmic tail that consists of an immunoreceptor tyrosine-based activation motif (ITAM) to signal.

The conventional ITAM consists of a YXXL/6-8amino acids/YXXL, whereas the ITAM-like region of

FcγRIIA was discovered to be YMTL/12amino acids/YMTL (Huang, Hunter et al. 2004). The tyrosines of the FcγRIIA’s ITAM-like region have been found to be phosphorylated by the protein tyrosine kinase family Src, which creates a Src homology 2 (SH2) docking site (Strzelecka, Kwiatkowska et al. 1997;

Joshi, Butchar et al. 2006) for Syk, a protein that contains an SH2 domain, to bind (Agarwal, Salem et al. 1993; Ghazizadeh, Bolen et al. 1994; Ghazizadeh, Bolen et al. 1995; Huang, Hunter et al. 2004).

Syk can interact with various other proteins to produce downstream events such as calcium release from the endoplasmic reticulum, actin rearrangement to form the pseudopods of the phagocytic cup, and production of reactive oxygen species (Kiefer, Brumell et al. 1998; Huang, Hunter et al. 2004;

Joshi, Butchar et al. 2006). Syk signaling is necessary for FcR phagocytosis and FcγR-triggered ROS production, as both of these processes decrease in Syk’s absence (Indik, Park et al. 1995; Indik, Park et al. 1995; Matsuda, Park et al. 1996). Mutation of either tyrosine residue of the ITAM-like region disrupts phagocytosis by 80% while mutation of both tyrosines abolishes phagocytosis completely

(Daeron, Malbec et al. 1994; Darby, Geahlen et al. 1994; Mitchell, Huang et al. 1994; Strzelecka,

Kwiatkowska et al. 1997). The first leucine residue has been shown to be essential for endoctyosis, while the L-T-L motif is required for phagolysosome fusion (Worth, Mayo-Bond et al. 2001; Kim,

Huang et al. 2003).

Internalization of a target mediated through FcγRs requires multiple steps. The first step entails actin rearrangement and phagocytic cup formation, which requries Syk binding to Rac and CDC42

(GTPases) through an unknown adaptor molecule (Hackam, Rotstein et al. 1997; Caron and Hall 1998;

Chimini and Chavrier 2000). CDC42 associates with the Wiskott-Aldrich Syndrome protein (WASp) at the phagocytic cup, where WASp is also able to bind CDC42 and Rac (Castellano, Montcourrier et al.

1999). WASp is subsequently able to interact with the Arp2/3 complex to mediate actin nucleation, pseudopod formation, and phagocytic cup formation (Welch, Iwamatsu et al. 1997; Machesky and Way

1998). It has also been shown that phosphatidylinositol 3-kinase (PI3K) accumulates at the phagocytic cup, and can be found in the membrane within seconds of FcγR cross-linking (Araki, Johnson et al.

1996; Vossebeld, Homburg et al. 1997; Marshall, Booth et al. 2001). Evidence shows that PI3K is important for closing off the plasma membrane thereby creating the phagosome (Cox, Tseng et al.

1999). Wortmannin, an inhibitor of PI3K, allows the formation of pseudopods, but inhibits membrane closure (Araki, Johnson et al. 1996). Phospholipase C (PLC) also accumulates at the phagocytic cup and is able to hydrolyze diacylglycerol (DAG) and inositol triphosphate(IP3), two phagocytic signaling molecules (Azzoni, Kamoun et al. 1992; Liao, Shin et al. 1992; Shen, Lin et al. 1994). PLC appears to be necessary for phagocytosis as inhibition of PLC disrupts phagocytosis (Botelho, Teruel et al. 2000).

DAG is able to transiently activate protein kinase C (PKC) (Nishizuka 1984), which along with PI3K interacts with myosin to begin retraction. This retraction occurs through ERK activation of myosin light-chain kinase (Mansfield, Shayman et al. 2000; Kamm and Stull 2001). The phosphorylated myosin associates with actin, stimulating phagosome entry into the cell (Ryder, Niederman et al. 1982;

Chavrier 2002; Araki, Hatae et al. 2003). Dynamin, a cytoskeleton molecule stimulated by PI3K, creates the phagosomal vesicle by “pinching off” at the plasma membrane opening (Araki, Johnson et al. 1996; Gold, Underhill et al. 1999). Through the creation of IP3 from PLC, IP3 is able to ligate the

IP3 receptor present on the endoplasmic reticulum with subsequent release of calcium (Mignery,

Sudhof et al. 1989; De Camilli, Takei et al. 1990; Mignery and Sudhof 1990). Release of calcium from the phagosome following FcγRIIA-mediated phagocytosis has been observed with the highest calcium concentrations being present at the phagosome or phagocytic cup (Sawyer, Sullivan et al. 1985;

Lundqvist-Gustafsson, Gustafsson et al. 2000). It has been observed that calcium encircles phagosomes in FcγRIIA-transfected CHO cells with abolishment of phagolysosome fusion through disruption of the

L-T-L signaling motif of the ITAM (Kindzelskii and Petty 2003; Worth, Kim et al. 2003). Therefore, phagolysosome fusion possibly relies on IP3-released calcium. Also, closing of the phagosome and fusion of the phagosome with lysosomes is mediated through soluble NSF attachment receptor proteins

(SNARE) (Hay and Scheller 1997; McNew, Parlati et al. 2000; Luzio, Pryor et al. 2005).

FcγRIIB, an inhibitory receptor, signals through an immunoreceptor tyrosine inhibitory motif

(ITIM) similar to the ITAM, and has been shown to recruit SH2 domain-containing inositol 5’- phosphatase (SHIP) and SHP-2, a tyrosine phosphatase. Phagocytosis has been observed to be enhanced in the absence of SHIP in FcγR-mediated phagocytosis (Muta, Kurosaki et al. 1994; Ono,

Bolland et al. 1996; Isakov 1997; Cox, Dale et al. 2001). SHP-2 dephosphorylates tyrosine residues of adaptor molecules, such as Cbl, SLP-76, and CRKL, causing inhibition of Rac activation thereby inhibiting phagocytic signaling (Binstadt, Billadeau et al. 1998; Kant, De et al. 2002).

FcγRIIIA, a transmembrane receptor, requires association with the γc chain for receptor expression and phagocytic signaling (Wirthmueller, Kurosaki et al. 1992; Park, Isaacs et al. 1993; Park,

Murray et al. 1993). FcγRIIIB does not associate with the γc chain and lacks cytoplasmic signaling capability (Wirthmueller, Kurosaki et al. 1992). FcγRIIIB is capable of inducing leukocyte effector functions such as antibody dependent cell mediated cytotoxicity and phagocytosis. There are several possible explanations: its interaction with FcγRIIA induces signaling, complement receptor 3 acts as a signaling partner, or it is localized in membrane rafts. Both FcγRIIA and FcγRIIIB move to membrane rafts after crosslinking, allowing efficient interaction with signaling molecules (Zhou, Lublin et al.

1995).

1.5 Signal Transducer and Activator of Transcription (STAT)

Seven signal transducers and activator of transcription (S TATs ) have been identified to date.

The STATs have a similar structural arrangement, which includes an amino terminus that aids in dimerization, a coiled coil domain involved in interactions with other proteins, a central DNA binding domain, a Src homology 2 (SH2) domain, a conserved tyrosine residue that is phosphorylated in response to various stimuli, and a carboxyl transcriptional activation domain (Levy and Darnell 2002;

Reich 2007). Canonically, Jaks phosphorylate STATs, t he S TATs dimerize, and translocate to the nucleus, where they bind to specific DNA patterns to regulate gene transcription (Schindler, Kashleva et al. 1994; Ihle 1995).

Jaks

Jaks are a family of tyrosine kinases important for STAT signaling consisting of Jak1, Jak2,

Jak3, and Tyk2. Jaks contain a tandem kinase, which allows for phosphorylation capabilities, and a pseudokinase domain, which is the docking site for STATs (Frank, Gilliland et al. 1994; Luo, Rose et al. 1997) (Fujitani, Hibi et al. 1997). The amino-terminus confers binding of the Jak to the appropriate 12 cytokine receptor (Frank, Gilliland et al. 1994; Frank, Yi et al. 1995; Zhao, Wagner et al. 1995;

Kohlhuber, Rogers et al. 1997). Jak 1 and Jak3 are activated by cytokine receptors that use the γc, which include IL-2, IL-4, and IL-7 (Musso, Johnston et al. 1995) (Miyazaki, Kawahara et al. 1994).

Binding of cytokines to their receptors has been suggested to initiate signaling through homodimerization or heterodimerization of the receptor subunits, which leads to cross-phosphorylation thereby activating the Jaks (Ortmann, Cheng et al. 2000). Jaks then phosphorylate tyrosines on the cytoplasmic domain of the receptor, enabling the recruitment of proteins with SH2 binding domains, which are phosphorylated by Jaks. As mentioned previously, STATs contain an SH2 domain to allow interaction with cytokine receptors and subsequent tyrosine phosphorylation via Jaks.

STAT6

S TAT 6 is an important member of the STAT family. The gene for STAT6 has been mapped to

12q13.3-q14.1 (GenBank accession no. NM_003153) and is in proximity to STAT2. STAT6 is 19kb in length and contains 23 exons and 22 introns (Patel, Keck et al. 1998). Monomeric human STAT6 is

94kD (Hou, Schindler et al. 1994; Quelle, Shimoda et al. 1995). The structure of STAT6 is similar to the other STATs. The SH2 domain o f S TAT 6 serves two functions: a docking site for binding to phosphorylated tyrosine residues on the cytoplasmic tail of the receptor and allowing for dimerization of actived S TAT 6 via the phosphorylated tyrosines at position 641. The boundaries and critical residues of the SH2 domain of STAT6 were determined through mutational analyses (Mikita, Daniel et al.

1998). The transactivation domain constitutes the proline-rich carboxy-terminal portion of STAT6 and is relatively large, a feature it shares with STAT2 (Hoey and Schindler 1998). Interestingly, removal of the transactivation domain produces a dominant-negative mutant leading to suppressed STAT6 activity

(Lu, Reichel et al. 1997; Moriggl, Berchtold et al. 1997). The relevance of this continues to be questioned as an isoform of STAT6 displaying a truncated amino terminus appears to be functional

(Patel, Pierce et al. 1998). The DNA binding domain is the central part of the STAT6 molecule, and

13 allows for DNA binding when STAT6 exists as a dimer (Mikita, Campbell et al. 1996). S TAT 6 ha s recently been shown to translocate to the nucleus in the absence of phosphorylation, but lacks nuclear accumulation presumably due to the lack of phosphorylation (Chen and Reich 2010).

STAT6 plays an essential role in IL-4 signaling (Takeda, Tanaka et al. 1996). It has been observed that IL-4 interaction with IL-4R uses Jak1 and Jak3 to phosphorylate STAT6 (Kotanides and

Reich 1993; Hou, Schindler et al. 1994; Quelle, Shimoda et al. 1995). Previous studies have shown that the IL-4R is phosphorylated in response to the binding of IL-4 (Izuhara and Harada 1993; Smerz-

Bertling and Duschl 1995). Through studies relying on IL-4R mutants, it was determined that the three central tyrosines of the IL-4Rα, namely Y575, Y603, andY631, are important for the activation of

S TAT 6 (Hebenstreit, Wirnsberger et al. 2006). These tyrosines become phosphorylated once the receptor is ligated and are the docking sites for STAT6 monomers. STAT6 is subsequently phosphorylated by the activated Jak1 and Jak3 (Johnston, Kawamura et al. 1994; Witthuhn,

Silvennoinen et al. 1994). The process of STAT6 activation is rapid, as STAT6 phosphorylation reaches a plateau within 3 minutes of IL-4 stimulation (Quelle, Shimoda et al. 1995). S TAT 6 t he n homodimerizes through the interaction of the SH2 domains and rapidly translocates to the nucleus.

Nuclear translocation has been found to rely on the sequence of amino acids from 135-140 and nuclear import is through importin-α-importin-β1 receptors (Chen and Reich 2010). The DNA binding domain of STAT6 dimers allows binding to DNA (Mikita, Campbell et al. 1996), while gene transcription of

IL-4 and IL-13 is dependent on serine phosphorylation (Levy and Darnell 2002).

STAT6 has numerous functions in various types of cells. For example in T cells STAT 6 is involved in the development of TH2 cells and the production of TH2 cytokines including IL-4, IL-5, and

IL-13 (Kaplan, Schindler et al. 1996; Shimoda, van Deursen et al. 1996; Takeda, Tanaka et al. 1996).

As mentioned previously, STAT6 promotes immunoglobulin class switching to IgE in B cells. In macrophages STAT6 aids in the IL-4-induced differentiation of M2 and mediates the IL-13 induced

14 expression of MHC class II (Takeda, Kamanaka et al. 1996; Martinez, Helming et al. 2009).

Interestingly, suppression of T-cell proliferation from STAT6 activity in M2 has been observed, which could aid in attenuating inflammation in allergic and autoimmune diseases (Huber, Hoffmann et al.

2010). STAT6 has also been shown to facilitate transcription mediated by PPARγ receptor in macrophages and dendritic cells thus regulating lipid metabolism (Szanto, Balint et al. 2010).

Attenuation of STAT signaling occurs through multiple mechanisms. It has been suggested that the tyrosine phosphatase SHP-1 can interact with the IL-4R to downregulate its function (Haque,

Harbor et al. 1998). Suppressors of cytokine signaling (SOCS) also contribute to attenuation by completing a negative feedback cycle: activated STATs stimulate transcription of the SOCS genes and the resulting SOCS proteins bind phosphorylated JAKs and their receptors to terminate the pathway

(Alexander 2002). Lastly, the protein inhibitors of activated stats (PIAS) bind to the activated STAT dimers and prevent them from binding DNA (Greenhalgh and Hilton 2001).

1.6 Interleukin-4 and Interluekin-4 Receptor

Interleukin 4

IL-4 is a type I cytokine produced by TH2 cells, basophils, mast cells, eosinophils, macrophages, and PMNs (Brown, Pierce et al. 1987; Bradding, Feather et al. 1992; Arock, Merle-Beral et al. 1993; Seder and Paul 1994; Moqbel, Ying et al. 1995; Brandt, Woerly et al. 2000; Pouliot, Turmel et al. 2005). IL-4 has numerous functions. For example IL-4 causes naïve T cells to develop into cells producing IL-4, IL-5, IL-10, and IL-13 (Hsieh, Heimberger et al. 1992; Seder and Paul 1994). A second function is B cell class switching to express IgE and IgG4 (Gascan, Gauchat et al. 1991). A third function of IL-4 is clearing helminth infections. IL-4 signaling via IL-4R and STAT6 are involved in clearing Trichinella spiralis as mice lacking IL-4Rα could not expel the worm while mice with intact

STAT6 had increased gut motility (Vallance, Galeazzi et al. 1999; Urban, Schopf et al. 2000). IL-4 has numerous functions in macrophages and monocytes. Interestingly, in human monocytes IL-4 inhibits the expression of the activating FcγRs (FcγRI, FcγRIIA, and FcγRIIIA) (te Velde, Huijbens et al. 1990), and upregulates the expression of FcγRIIB causing a downregulation in FcγR-mediated activities

(Pricop, Redecha et al. 2001). Monocytes and macrophages have also been found to upregulate the mannose receptor and endocytic activity in response to stimulation with IL-4 and IL-13 (Montaner, da

Silva et al. 1999). Another profound effect on monocytes/macrophages is inhibiting the production of pro-inflammatory cytokines (TNF) and other mediators (cyclooxygenase-2, prostaglandin E2, and superoxide) resulting in an anti-inflammatory population (Chomarat and Banchereau 1998). MHC class

II has been found to be upregulated as well in macrophages allowing for more antigen presentation (de

Waal Malefyt, Figdor et al. 1993; Hart, Bonder et al. 1999). Finally, recent studies have focused on elucidating the effect of IL-4 in determining the production of M2 and their function, and have shown that IL-4 is critical for establishing the M2 population (El Chartouni, Schwarzfischer et al. ; Var in,

Mukhopadhyay et al. ; Weisser, McLarren et al. ; Van den Bossche, Bogaert et al. 2009).

Interleukin 4 Receptor

The IL-4 receptor (IL-4R) partially consists of the 140kDa IL-4Rα chain that binds IL-4 with high affinity (20-300 pM) (Park, Friend et al. 1987). IL-4Rα is composed of an extracellular domain of four conserved cysteines, a WSXWS motif, and fibronectin III modules, while the intracellular domain has proline-rich box regions that are necessary for Jaks to bind (Hebenstreit, Wirnsberger et al. 2006).

The IL-4R complex is traditionally found on hematopoietic, endothelial, epithelial, muscle, fibroblast, hepatocyte, and brain tissues (Ohara and Paul 1987; Lowenthal, Castle et al. 1988). IL-4R can exist as two different types, one composed of the common γc and α chain (Leonard, Noguchi et al. 1994;

Russell, Johnston et al. 1994). While the second IL-4R is a high-affinity heterodimer composed of IL-

4Rα chain and the IL-13Rα1 chain capable of binding IL-13 with subsequent activation of Jak1 and 16

Tyk2 a nd S TAT 6 (Obiri, Debinski et al. 1995; Miloux, Laurent et al. 1997; Obiri, Leland et al. 1997)

(Keegan, Johnston et al. 1995; Welham, Learmonth et al. 1995). IL-4R is capable of signaling through two different pathways. The first pathway involves activation of the insulin receptor substrate-1/2 (IRS-

1/2) in response to IL-4Rα ligation modulating cellular proliferation (Wang, Keegan et al. 1992; Wang,

Keegan et al. 1993). The second pathway involves STAT6 and was discussed previously.

Based on this knowledge, we sought to determine if an interaction between STAT6 and FcγRIIA existed. To accomplish this, we performed yeast two-hybrid analysis involving the ITAM of FcγRIIA with STAT6 and also performed western blotting of whole cell lysates and immunoprecipitation of

FcγRIIA. To determine the downstream affects of this interaction, we used epifluorescence microscopy to obser ve S TAT 6 nuclear translocation and to observe the affect of IL-4 on phagocytosis and endocytosis. We also performed RT-PCR to observe IL-4 production after interaction of FcγRIIA with

S TAT 6 .

Chapter 2

Materials and methods

2.1 Reagents and Antibodies

Ham’s F-12 was purchased from Biowhittaker (Walkersville, Maryland). 10% fetal bovine serum was purchased from Gibco (Invitrogen Corp, Grand Island, New York). G-418, RPMI 1640, and

2.05mM L-glutamine was purchased from HyClone, Thermo Scientific (Logan, UT). FuGene6 was purchased from Roche (Basel, Switzerland). Qiagen miniprep was purchased from Qiagen

(Germantown, Maryland). Recombinant human Interleukin-4 was purchased from BD Pharmingen (BD

Biosciences). Polybead polystyrene 4.5 micron microspheres were purchased from Polysciences

(Warrington, Pennsylvania). Rabbit anti-human STAT6 polyclonal antibody and rabbit anti-human Syk polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, California).

PhosphoDetect rabbit anti-human STAT6 polyclonal antibody against the phosphorylated tyrosine at position 641 was purchased from Calbiochem (Philadelphia, Pennsylvania). LPS from Salmonella typhimurium was purchased from List Biological Laboratories, Inc. (Campbell, Ccalifornia). DAPI was a gift from Dr. Robert Blumenthal (The University of Toledo, Toledo, Ohio). Protease Inhibitor

Cocktail Kit was purchased from Thermo Scientific (Pierce Biotechnology, Rockford, Illinois). Protein

G agarose beads were purchased from Upstate (Millipore, Temecula, CA). Phorbol 12-myristate 13- acetateand human IgG FITC conjugate was purchased from Sigma-Aldrich (Saint Louis, Missouri).

Fluorescein (FITC)-conjugated AffiniPure F(ab’)2 fragment Goat Anti-Rabbit IgG and Rhodamine

(TRITC)-conjugated AffiniPure F(ab’)2 fragment Goat Anti-Rabbit IgG (H+L) were purchased from

Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pennsylvania). The 5 Prime Perfect Pure 18

RNA Cell and Tissue Kit were obtained from QAIGEN (Gaithersburg, Maryland). Sheep erythrocytes and rabbit anti-sheep IgG were purchased from Rockland (Alsever; Rockland Scientific, Gilbertsville,

Pennsylvania). pCMV-SPORT6 and THP-1 (ATCC TIB-202) were purchased from ATCC (Manassas,

Virginia.). pERFP-C1 was provided as a gift from Dr. Alan Schreiber’s laboratory (University of

Pennsylvania, Philadelphia, Pennsylvania). Novex Sharp Protein Standard, anti-rabbit alkaline- phosphatase conjugated secondary antibody, and Novex AP Chemiluminescent substrate were purchased from Invitrogen (Grand Island, NY). KpnI and XhoI were purchased from New England

BioLabs (Ipswich, MA). The Rapid DNA Ligation Kit was purchased from Roche Applied Science

(Mannheim, Germany). ECOS 101 Competent Cells were purchased from Yeastern Biotech (Taipei,

Taiwan). The Wizard Plus SV Minipreps DNA Purification System and dNTP were purchased from

Promega (Madison, Wisconsin). The Jurkat cell line (ATCC TIB-152) was a gift from Dr. Mark Wooten

(The University of Toledo, Toledo, Ohio). Human IL-4 primers (Forward sequence: 5’ TTT CTC CTG

GAA GAG AGG TGC TGA 3’. Reverse sequence: 5’ AGG TGA TAT CGC ACT TGT GTC CGT 3’.) and human GAPDH primers (Forward sequence: 5’ GGG AAG GTG AAG GTC GGA GT 3’. Reverse sequence: 5’ TCC ACT TTA CCA GAG TTA AAA GCA G 3’.) were purchased from Integrated DNA

Technologies, Inc. (Coralville, Iowa).

2.2 Yeast Two-Hybrid System

The ITAM of FcγRIIA was expressed as a fusion to the GAL4 DNA-binding domain in the pGBKT7 fusion vector. The cDNA library was expressed as a fusion to the GAL4 activation domain in the pGADT7 fusion vector. Interactions were determined based on selective growth requirement and the expression of βGAL resulting in blue-hued colonies. The positive clones were picked and transformed into competent Escherichia coli, which was then plated on Luria Bertani agar. The use of

19 ampicillin allowed for bacterial selection. Sequencing was performed at the University of Michigan.

Through the use of BLAST on Pubmed, it was discovered that the particular STAT6 sequence corresponded to the SH2 domain of STAT6 (see Figure 3.1).

2.3 Chinese Hamster Ovary Culture

Chinese hamster ovary (CHO) cells expressing human FcγRIIA (CHO-IIA) were generated by

Dr. Alan Schreiber’s laboratory (University of Pennsylvania, Philadelphia, Pennsylvania). The CHO-

IIA cells were maintained in Ham’s F-12, supplemented with 10% fetal bovine serum, and expression was maintained by selection with G-418.

The FcγRIIA-GFP (IIA-GFP) was constructed by swapping the FcγRIIA cDNA sequence from pCDNA3.1 to pEGFP-N1 with HindIII and SacII flanking sites. The Liga-Fast kit (Promega, Fitchburg,

WI) was used to allow for ligation to occur. Competent Escherichia coli was subsequently transformed and grown overnight on Lysogeny Broth agar plates containing kanamycin (25µg/mL). Individual colonies were selected and analyzed for insertion of FcγRIIA sequence. Plasmids were purified using

Qiagen miniprep. The plasmids were transfected into the CHO cells using FuGene6. FcγRIIA-GFP positive cells were selected via neomycin resistance and enriched by fluorescence sorting.

2.4 Red Fluorescent Protein-Signal Transducer and Activator of Transcription 6

The three reading frames of pERFP and pCMV-SPORT6 were transformed into competent

Escherichia coli cells by incubating at 42°C for 45 seconds. The competent cells were plated on separate kanamycin plates and incubated at 37°C for 24 hours. Three colonies from each plate were obtained and placed in 5mL Lysogeny broth and 5µL kanamycin (30mg/mL), and incubated at 37°C at 20

225 revolutions per minute for 36 hours. The DNA was purified using the Wizard Plus SV Minipreps

DNA Purification System. Enzymatic digestion was obtained through the use of KpnI and XhoI. The samples and the Novex Sharp protein standard were then run on agarose gel, and the 4.4kb band corresponding to the vector and the 3.9kb band corresponding to STAT6 were obtained and quantified.

The vector and STAT6 were ligated through the use of the Rapid DNA Ligation Kit. To confirm proper ligation the samples were subsequently enzymatically digested through the use of EcoRI and XhoI and run on agarose gel.

2.5 THP-1 Culture

The human monocytic leukemia THP-1 cell line was maintained in RPMI 1640 with 2.05mM

L-glutamine added and supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

This cell line was maintained at 37ºC in a humidified incubator with 5% saturated CO2. Cells were passaged every 2 or 3 days by taking a fraction of the suspended cells and transferring them to a new flask with fresh media.

2.6 Bead Preparation

100µL of 4.5µm Polystyrene beads were placed into a microcentrifuge tube. 1mL of buffered saline without calcium and magnesium (DPBS --) was added to the tube and then vortexed. The beads were centrifuged for 5 minutes at 10,000 x g and the supernatant was aspirated off. The beads were opsonized by incubation in a 10mg/mL solution of human IgG for 1 hour at 37°C, followed by two consecutive washes in DPBS -- at 10,000 x g for 10 minutes. The beads were resuspended for a final volume of 900µL in DPBS--. The beads were added to the cells at a 10:1 ratio.

2.7 Heat-Aggregated IgG Preparation

FITC-conjugated human IgG (20mg/mL) was diluted in buffered saline with calcium and magnesium (DPBS++) to a final concentration of 10mg/mL. The FITC-conjugated human IgG was incubated at 62ºC for 20 minutes to induce aggregation. Finally, the FITC-conjugated human IgG was centrifuged at 10,000 x g for 5 minutes and put on ice.

2.8 Monomeric IgG Preparation

FITC-conjugated human IgG (20mg/mL) was diluted in DPBS++ to a final concentration of

10mg/mL, vortexed, and placed on ice.

2.9 Stimulation, Immunoprecipitation, and Western Blotting

THP-1 cells were plated at a density of 5x106 cells per 100mm plate in 10 mL of complete media (total volume was 15mL) and 125µM PMA was added to induce differentiation. The THP-1 cells were incubated for 60 hours to ensure complete differentiation. The complete media was aspirated off and 2mL of new complete media was added to each plate. Five different conditions were set up. The negative control plate consisted of unstimulated THP-1 cells in 2mL of media. The positive control plate consisted of THP-1 cells in the presence of 50ng/mL IL-4 and 2mL of media. The third plate consisted of THP-1 cells in the presence of 4.5µm beads coated with human IgG at a ratio of 10:1. The fourth plate consisted of THP-1 cells in the presence of 10mg/mL heat-aggregated FITC-conjugated human IgG. The fifth plate consisted of THP-1 cells in the presence of 10mg/mL of monomeric FITC-

22 conjugated human IgG. The plates were incubated in a 37ºC waterbath for 30 minutes. The plates were then placed at 4°C, the media was aspirated, and 1.3mL of lysis buffer (protease inhibitors, TritonX

100, and buffer) was added to each plate. The cells were in lysis buffer for 15 minutes while at 4°C and then the cells were scraped off the plate using a cell scraper. The 1.3mL of lysis buffer and cells were transferred to a 1.5mL eppendorf tube, vortexed, and put in a 4ºC rotator for 45 minutes. The cells were spun in a microcentrifuge at 10,000 x g at 4ºC for 15 minutes. The whole cell lysates were obtained and transferred to fresh eppendorf tubes containing 0.45mg/mL IV. 3 when immunoprecipitating FcγRIIA.

Rabbit anti-human polyclonal STAT6 at a concentration of 200µg/mL was added to each tube when immunoprecipitating STAT6. The tubes were put back in the rotator in 4ºC for 2 hours. Protein G sepharose beads (60µL that were washed one time at 10,000 x g for 5 minutes in 1mL of lysis buffer) were added to each tube and the tubes were put back in the 4ºC rotator for 1 hour. The tubes were briefly centrifuged at 4ºC at 10,0000 x g. The whole cell lysates were transferred to fresh eppendorf tubes when immunoprecipitating FcγRIIA. The pellet was washed one time with 500µL of lysis buffer and spun briefly at 10,000 x g. Approximately 50µL of lysis buffer was left in each tube. 20µL of

NuPAGE LDS running buffer (4X) and 8uL of NuPAGE sample reducing agent (10X) were added to each tube. The tubes were boiled for 5 minutes and then centrifuged in 4ºC for 3 minutes at 10,000 x g.

Protein (35µL) was loaded in each lane and separated by SDS-polyacrylamide gel (4-12% Bis-Tris) electrophoresis. Fractionated proteins were electrophoretically transferred onto a PVDF membrane.

The prepared membranes were probed with specific primary antibodies (2µg/mL IV.3, or 1:1000 rabbit anti-human polyclonal phosphorylated-STAT6, or 1:1000 rabbit anti-human polyclonal STAT6, or

1:2000 rabbit ant-human polyclonal SYK) for 1 hour followed by anti-rabbit alkaline-phosphatase conjugated secondary antibody, visualized by Novex AP Chemiluminescent substrate, and film exposure. The membrane was stripped with stripping buffer (2% SDS, 50mM Tris, and 100mM beta- mercaptoethanol) after detection with the specific primary antibodies to reblot the membranes with

23 separate primary antibodies. Each experiment was repeated at least three times.

2.10 Stimulation, Staining, and Fluorescence Microscopy

THP-1 cells were plated at a density of 8x105 cells per well and grown on coverslips in a 6 well plate and 2mL of media was added. 125nM of PMA was added to each well for three days to induce differentiation. The first well was the unstimulated control, the second well was stimulated with

50ng/mL IL-4, the third well was stimulated with 10:1 ratio of 4.5µm human IgG coated beads, the fourth well was stimulated with 10mg/mL heat-aggregated FITC-conjugated human IgG, and the fifth well was stimulated with 10mg/mL monomeric FITC-conjugated human IgG. The stimulated cells were placed in a 37ºC water bath for 30 minutes. The cells were washed two times with 1mL of

DPBS++. 1mL of DPBS ++ and 1mL of 4% paraformaldehyde was added to each well for 30 minutes at 32 C to fix the cells. The fluid was aspirated off and 1mL of TritonX 100 was added to each well for

5 minutes 32 C to permeabilize the cells. The cells were washed two times with 1mL DPBS++. Rabbit anti-human polycloncal antibody (200µg/mL) was added to each well at a 1:100 dilution for 30 minutes at room temperature. Each well was washed two times with 1mL wash solution DPBS++ at 32°C.

Rhodamine (TRITC)-conjugated F(ab’)2 fragment goat anti-rabbit IgG in DPBS++ was added to each well at a 1:300 dilution for 30 minutes at 32°C. Each well was washed two times with 1mL wash solution. 1µg/mL DAPI was added to each well and incubated for 5 minutes at 32°C. Each well was washed two times with 1mL wash solution. The coverslips with cells on them were mounted in phosphate buffered saline and placed on an Axiovert 200 fluorescence microscope (Carl Zeiss,

Thornwood, NY) utilizing mercury illumination. Cells were visualized using differential interference

(brightfield) contrast or epifluorescence microscopy. Optical filters for fluorescein excitation and emission were 480DF22 and 530DF30, respectively (Chroma, Rockingham, VT). Images were

24 observed using an Orca ER-AG (Hamamatsu, Japan) CCD camera connected to a Dell Optiplex 620

Workstation (Round Rock, TX). Metamorph software (Molecular Devices, Downingtown, PA) was used to acquire and process images. When possible the two or three pictures were overlayed to indicate nuclear location and, when necessary, to indicate that the macrophage did bind FITC-conjugated human IgG. The procedure was repeated for different regions to determine the percentage of cells that showed STAT6 translocation. Each experiment was repeated at least three times and ≥50 cells for each test sample were assessed.

2.11 Jurkat culture

The Jurkat human T cell line was maintained in RPMI 1640 with 2.05mM L-glutamine added and supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 25µg/mL amphotericin B. Jurkat cells were passaged every 2 or 3 days by taking a fraction of the suspended cells and transferring them to a new flask with fresh media.

2.12 Stimulation and RNA extraction

THP-1 cells were plated at 5x106 cells per 100mm plate. RPMI media (10mL) supplemented with 10% fetal bovine serum, 10,000U/mL Penicillin G, 10,000U/mL Streptomycin, and 25µg/mL

Amphotericin B was added for a final volume of 15mL. PMA (125nM) was added to each 100mm plate. The cells were allowed to differentiate for 60 hours and then 10mL of media was aspirated off the plates for a final volume of 5mL. Four plates were set up: the first plate was unstimulated, the second plate was stimulated with 50ng/mL IL-4, the third plate was stimulated with 1mg/mL LPS, and the fourth plate was stimulated with 10mg/mL heat-aggregated FITC-conjugated human IgG. The plates

25 were stimulated for 8 hours, 24 hours, or 48 hours. After stimulation the media was aspirated and the cells were washed with 10mL DPBS++ one time, which was then aspirated. The cells were placed directly in the -80°C freezer for further use. Jurkat cells were also cultured in the complete RPMI media to a concentration of 2 X107 cells. The Jurkat cells were then centrifuged at 700 x g for 5 minutes and resuspended in antibiotic-free RPMI. To stimulate production of IL-4, the Jurkat cells were treated with PMA at a concentration of 5ng/mL plus ionomycin at a concentration of 750ng/mL for 6 hours. The mRNA from the THP-1 and the Jurkat cells was obtained using the 5 Prime

PerfectPure RNA Cell and Tissue Kit from QIAGEN. The purified RNA was stored in the -80°C freezer. Each experiment was repeated at least three times.

2.13 Real Time Quatitative Reverse Transcription Polymerase Chain Reaction

RNA was diluted to 1µg/5uL with nuclease-free water into PCR tubes while on ice. 2µL of primer (oligo dT, IDT 10uM) was added to each sample and then incubated at 70°C for 5 minutes in the

Eppendorf Mastercycler. The samples were then chilled at 4°C for 5 minutes and then remained at 4°C.

For each 20µL reaction 4.6µL nuclease-free water, 4µL 5X buffer, 2.4µL MgCl2, 1µL dNTP were mixed and then vortexed. Finally 1µL ImProm-IIRT was added. This 13µL was added to each sample and then mixed by pipetting. All reagents were from Promega ImProm kit #PR-A3802 except where noted. The samples were then placed in the Biorad iCycler and one cycle at 95°C for 10 minutes occurred, which allowed for melting. The samples then underwent forty cycles of amplification, which consisted of 95°C for 15 seconds to allow for denaturation, then 50°C for 30 seconds to allow for annealing, and finally 72°C for 45 seconds to allow for extension. The samples were then placed in the

Roche LightCycler 1.5 and allowed to anneal at 25°C for 5 minutes, then the first strand was extended for 60 minutes at 42°C, and finally heat inactivated by incubating at 70°C for 15 minutes. The samples

26 were then chilled and stored in -20°C. The housekeeping gene GAPDH was used as an internal control for mRNA integrity and yield. The DNA was quantified using the accompanying software from the

LightCycler. Each experiment was repeated at least three times.

2.14 Erythrocyte Preparation

100µL of sheep erythrocytes was obtained from the stock of erythrocytes purchased from

Rockland. DPBS ++ (900µL) was added to the erythrocytes and then mixed. The erythrocytes were washed twice by centrifuging at 1230 x g for 5 minutes and aspirating the supernatant. The erythrocytes were resuspended in 1mL DPBS ++ and 720uL were removed then the erythrocytes were placed in a fresh microfuge tube. Rabbit anti-sheep IgG (10mg/mL) was added to the erythrocytes and then vortexed. The erythrocytes were incubated for 30 minutes at 37°C water bath and then centrifuged at 1230 xg for 5 minutes and the supernatant was aspirated off. The erythrocytes were resuspended in

720µL of DPBS ++. Erythrocytes (100µL) were added to each well of a 6 well plate.

2.15 Phagocytosis

THP-1 cells were plated at a concentration of 8x105 cells per well in a 6 well plate. Complete media (2mL) and 125nM PMA was added to each well. The cells were allowed to differentiate for 60 hours. IL-4 (50ng/mL) was added to 5 of the 6 wells for a timecourse. IL-4 timepoints ranged from 30 minutes to 24 hours. Opsonized sheep erythrocytes were added at a 10:1 dilution. Cells were placed at

4°C and then the erythrocytes were added and allowed to bind for 1 hour. Cells were washed three times with 1mL DPBS++ and placed in a 37°C water bath for 30 minutes to allow the beads to internalize. Cells were placed at 4°C and non-internalized sheep erythrocytes were labeled with 27 rhodamine (TRITC) goat anti-rabbit IgG F(ab)2. Cells were fixed with 2% paraformaldehyde and visualized using epifluorescence microscopy. All cells in a field of view were counted. Binding was assessed by counting the number of bound erythrocytes per cell as well as the number of erythrocytes internalized. The internalized erythrocytes were determined by the lack of staining with the secondary antibody. Each experiment was repeated at least three times assessing ≥200 cells for each test sample

2.16 Endocytosis

THP-1 monocytes were placed in 2 separate 6 well plates at a concentration of 8 x105 cells per well and differentiated using 125nM PMA. IL-4 (50ng/mL) was added to four separate wells for a total of 30 minutes and 60 minutes. Plates were put on ice for 15 minutes. Macrophages were washed twice by adding 1mL cold DPBS ++ to each well and aspirating. DBPS ++ (1mL) was added back to each well. FITC-conjugated heat-aggregated human IgG (100µg/mL) and 100µg/mL human IgG were added to six individual wells. Plates were then incubated at 4°C covered with aluminum foil for 60 minutes to allow binding to occur. Macrophages were washed twice as described above. One plate was then warmed to 37°C to allow for internalization and one plate was kept at 4°C for 30 minutes. The warmed plate was then cooled at 4°C for 10 minutes. DPBS (1mL) was aspirated off. Non-internalized IgG complexes were labeled with 5µg/mL solution of goat anti-human IgG F(ab)2 fragments conjugated with phycoerythrin (PE) and then plates were incubated at 4°C for 30 minutes. Cells were washed twice with 2mL cold DPBS (--) and then 2mL cold DPBS (--) was added to each well the cells were incubated at 4°C for 15 minutes and then pipetted to detach the cells. 4% paraformaldehyde (2mL) for

60 minutes at 4°C was added to fix the cells and they were transferred to a tube and place in 4°C to store overnight. Cells were centrifuged for 5 minutes at 700 x g and resuspended in 500µL DPBS ++.

Samples were filtered through 100µm nylon mesh and analyzed using BD-FACSCalibur and using Cell

Quest software. Percent total binding and internalization were calculated by comparing the Mean

Fluorescence Intensity (MFI) of either FITC (total cell-associated immune complex, binding value) or

PE (external immune complex only, internalization value) at each timepoint of IL-4 and normalizing to untreated control samples. Each experiment was repeated three times, and 10,000 cellular events were assessed through flow cytometry for each test sample.

2.17 Statistics

Significance values determined by the two tailed Student’s t-test for the nuclear translocation, phagocytosis, and endocytosis studies. Significance was determined as p<0.05.

Chapter 3

The Interaction of FcγRIIA with STAT6

To ascertain the interaction of FcγRIIA with STAT6, we performed a yeast two-hybrid analysis and western blotting of whole cell lysates and FcγRIIA immunoprecipitation. We report the discovery of a constitutive interaction of non-phosphorylated STAT6 with FcγRIIA.

3.1 The SH2 Domain of STAT6 Interacts with the Cytoplasmic Domain of FcγRIIA

Initially, the potential interaction of the ITAM of FcγRIIA and STAT6 was identified through a yeast two-hybrid analysis. The yeast two-hybrid analysis is one method of determining novel protein interactions. Initial yeast two-hybrid experiments displayed few interactions between the ITAM of

FcγRIIA and the SH2 domain of STAT6. The cDNA sequence of the SH2 domain of STAT6 is shown in

Figure 3.1.

Figure 3.1 SH2 sequence of human STAT6. DNA sequence obtained through the yeast two-hybrid analysis, translated into the protein sequence and then Blasted with Pubmed. Representation of STAT6 amino acid sequence corresponding to 847 amino acids. Representation of the ITAM of FcγRIIA, where the phosphorylated tyrosines create the SH2 binding domain.

To further study the interaction between FcγRIIA and STAT6, FcγRIIA-GFP and RFP-S TAT 6 constructs were made (Figure 3.2). Chinese hamster ovary cells previously transfected with FcγRIIA-

GFP were used for the experiments (Worth, Mayo-Bond et al. 1996). Colocalization of the fluorescent proteins was not observed after RFP-STAT6 transfection into CHO cells due to lack of consistent expression of RFP-S TAT 6 (Figure 3.3).

Figure 3.2 Construct of RFP-STAT6. A) Enzymatic digestion of both pCMV-SPORT6 and the three reading frames of pERFP. B) Enzymatic digestion of C1-S TAT 6 , C 2 -S TAT 6 , C 3 -STAT6 to ensure proper ligation.

Figure 3.3 Representative fluorescence images of RFP-STAT6 transfection into CHO-IIA cells. A) Brightfield microscopy image of CHO-IIA epithelial cells stably transfected with FcγRIIA. B) Epifluorescence microscopy image of CHO-IIA cells transiently transfected with RFP-S TAT 6 .

To confirm the interaction of STAT with FcγRIIA, western blotting of whole-cell lysates from

THP-a macrophages and immunoprecipitation of FcγRIIA was performed. STAT6 was observed at similar levels in whole-cell lysates from THP-1 cells treated with IL-4, opsonized beads, haIgG, and monoIgG. However, phosphorylated STAT6 was only observed in response to IL-4 treatment (Figure

3.4). Additionally, immunoprecipitation of FcγRIIA showed that interaction with STAT6 occurs regardless of ligand presented, and appears to be phosporylation-independent (Figure 3.5).

Alternatively, immunoprecipitation of FcγRIIA located on macrophages with IL-4R ligation showed that FcγRIIA interacted with phosphorylated-S TAT 6 . This suggests that phosphorylated-S TAT 6 obtained through IL-4R ligation constitutively interacts with FcγRIIA. SYK, a protein containing an

SH2 domain known to interact with FcγRIIA, was observed at similar levels in whole-cell lysates and immunoprecipitation of FcγRIIA. These data suggest that the interaction of STAT6 with FcγRIIA is phosphorylation-independent.

Figure 3.4 Representation of constitutive interaction of STAT6 with FcγRIIA. Immunoprecipitation of FcγRIIA was loaded into each well after stimulation of THP-1 macrophages with IL-4, opsonized beads (Beads), heat-aggregated IgG (HaIgG), or monomeric IgG (MonoIgG) for 30 minutes. Samples were immunoblotted with anti-phosporylated-STAT 6 ( P-STAT6) and then anti- STAT6 to confirm constitutive interaction of STAT6 with FcγRIIA. Samples were then immunoblotted with anti-SYK as an internal control. Each experiment was performed three times.

Figure 3.5 Representation of constitutive interaction of STAT6 with FcγRs. Whole-cell lysates were loaded into each lane after THP-1 macrophages had been stimulated with IL-4, opsonized beads (Beads), heat-aggregated human IgG (HaIgG), or monomeric IgG (MonoIgG) for 30 minutes. Samples were immunoblotted with anti-phosphroylated-S TAT 6 ( P -STAT6) and then anti-STAT6 to confirm the presence of STAT6. Samples were immunoblotted with anti-SYK as an internal control. Each experiment was performed three times.

Chapter 4

The Role of FcγR Ligation in STAT6 Signaling

To ascertain the role of FcγR ligation in STAT6 signaling, experiments were performed to observe nuclear translocation of STAT6, the production of IL-4, and the affect of IL-4 on FcγR- mediated activities. By ligating FcγRs with heat-aggregated IgG, we report that STAT6 nuclear translocation occurred in a phosphorylation-independent manner based on particle size ligation of

FcγRs. We also report that phosphorylation of STAT6 is necessary for IL-4 expression. Finally, we report that IL-4 does not affect FcγR-mediated activities.

4.1 Nuclear Translocation of STAT6 Following FcγR Ligation

In order to determine the activation of STAT6, nuclear translocation experiments were performed. The presence of non-phosphorylated STAT6 in both the cytoplasm and the nucleus (Figure

4.1, Non-stimulated) was observed in the unstimulated THP-1 macrophages, which is consistent with previous data (Chen and Reich 2010). IL-4R ligation produced significant phosphorylated-S TAT 6 nuclear translocation (Figure 4.1, IL-4) confirming the canonical IL-4 signaling pathway. FcγR ligation with the various IgG-derived ligands did not produce significant STAT6 nuclear translocation when

(Figure 4.1 Beads and MonoIgG). FcgR ligation with heat-aggregated IgG produced significant nuclear translocation of non-phosphorylated STAT6 (Figure 4.1 HaIgG).

Figure 4.1 Fluorescence micrographs of STAT6 nuclear translocation upon FcγR ligation. The Non-stimulated row represents THP-1 macrophages without stimulation signifying no STAT6 nuclear translocation. 30 minutes of stimulation with either IL-4 or heat-aggregated human IgG (haIgG) induced significant nuclear translocation as evidenced by the TRITC micrographs. 30 minutes of stimulation with 4.5µm opsonized beads or human monomeric IgG did not produced significant nuclear translocation as evidenced by the TRITC micrographs. The FITC column represents FITC- conjugated IgG while the DAPI column represents nuclear staining. Each experiment was performed at least three times with observation of ≥50 cells.

Quatification of phosphorylated STAT6 nuclear translocation upon IL-4R ligation displayed significant translocation (82% ± 4), again indicating STAT6 signaling through the canonical pathway.

Quantification of non-phosphorylated STAT6 nuclear translocation displayed statistically significant translocation upon ligation of FcγRs with heat-aggregated IgG (23% ± 12) as compared to the nonstimulated control (Figure 4.2). The data was quantified through the use of epifluorescence microscopy by counting the number of nuclei positive for STAT6 translocation and then dividing by the total number of cells in that field. These data indicate significant non-phosphorylated STAT6 nuclear translocation upon FcγR ligation with heat-aggregated human IgG.

Figure 4.2 Quatification of STAT6 nuclear translocation in THP-1 macrophages. THP-1 macrophages stimulated with IL-4, Beads, HaIgG, or MonoIgG were analyzed through fluorescence microscopy to determine STAT6 nuclear translocation. STAT6 nuclear translocation was minimally affected by ligation of FcγRs with 4.5 µm opsonized beads (Beads) and monomeric human IgG (MonoIgG). Significant STAT6 nuclear translocation upon ligation of IL-4R (82% ± 4) and FcγRs (23% ± 12) with heat-aggregated IgG (HaIgG) suggesting STAT6 translocation dependent on particle sized ligation of FcγRs. N=3 individual experiments for each data point, and statistical significance was determined by two-tailed Student’s t-test. Significance was defined as *p>0.05 as compared to the control. 37

4.2 Ligation of FcγRs Failed to Produce IL-4

We next decided to look at the production of IL-4 from stimulated THP-1 macrophages, the production of which could downregulate FcγR-mediated activities. Jurkat T cells were observed to produce IL-4 mRNA (Figure 4.3), which was expected as T cells produce abundant amounts of IL-4

(Seder and Paul 1994). The production of IL-4 in the Blank sample (water, Figure 4.3) was observed on numerous occasions while using different nuclease free water samples possibly due to lack of purity of the reagent. FcγR ligation with heat-aggregated IgG or TLR4 ligation with LPS in THP-1 macrophages failed to produce significant fold change in IL-4 (Figure 4.4). These data suggests non-phosphorylated

STAT6 lacks the ability to induce expression of IL-4.

Figure 4.3 Representation of IL-4 production from stimulated Jurkat cells. Pretreatment of Jurkat cells with phorbol myristol acetate and ionomycin for 6 hours to induce IL-4 production. IL-4 mRNA was detected beginning at cycle number 5. Data are representative of one experiment performed on three separate days.

1.000000

0.800000 -4 -4 0.600000

0.400000 Fold Change in in IL Change Fold

0.200000

0.000000 Blank NSC LPS haIgG IL4

Figure 4.4 Representation of the fold change in IL-4 from stimulated THP-1 macrophages. Pre- treatment of THP-1 macrophages with 50ng/mL IL-4, 1mg/mL lipopolysaccharide (LPS), or 1mg/mL heat-aggregated human IgG (haIgG) for 8 hours resulted in lack of IL-4 mRNA production as compared to the nonstimulated control (NSC). Data are representative of one experiment performed on three separate days.

4.3 FcγR-Mediated Activities Are Not Affected by IL-4

In order to determine the affect of IL-4 on FcγR-mediated activities, phagocytosis and endocytosis experiments were performed. Phagocytosis occurred with a phagocytic index of 0.8 in unstimulated THP-1 macrophages (Figure 4.5). Exposure to IL-4 for various time points plus IgG- opsonized sheep erythrocytes (EA) did not produce a significant downregulation of phagocytosis

(Figure 4.5). This suggests that S TAT 6 signaling does not affect phagocytosis.

1.2

0.8

0.6

0.4 Phagocytic Index Index Phagocytic

0.2

0 EA 30min IL-4 + EA 1 hour IL-4 + EA 2 hour IL-4 + EA 4 hour IL-4 + EA

Figure 4.5 Quantification of phagocytosis using epifluorescence microscopy analysis of THP-1 macrophages stimulated with 50ng/mL IL-4. THP-1 macrophages pretreated with IL-4 for various timepoints and then stimulated with IgG-opsonized sheep erythrocytes (EA) to determine phagocytic efficiency. Data represented display phagocytic index, or the number of internalized targets per cell. No significant downregulation of phagocytosis upon IL-4 stimulation. Data are representative of one experiment performed on three separate days. Data was compared to IgG-opsonized sheep erythrocytes (untreated control). Statistical significance was determined by the two-tailed student’s t test and defined as *p<0.05.

To determine the effects of IL-4 on FcγR-mediated activities based on particle size, endocytosis experiments, utilizing heat-aggregated IgG, were performed. Endocytosis occurred at 80% in THP-1 macrophages stimulated with heat-aggregated IgG (Figure 4.6). Transient IL-4 stimulation of THP-1 macrophages did not significantly affect endocytosis (Figure 4.6). These data indicate transient IL-4 stimulation does not affect FcγR-mediated activities.

Figure 4.6 Trend of percentage of endocytosis analyzed through flow cytometric analysis of THP- 1 macrophages stimulated with 50ng/mL IL-4. THP-1 macrophages pretreated with IL-4 for various timepoints were then stimulated with haIgG to determine endocytic efficiency. No significant difference in endocytosis upon stimulation with IL-4. Data are representative of one experiment performed on three separate days. Data was compared to 0min IL-4 + haIgG (untreated) control. Statistical significance was determined by the two-tailed student’s t test and defined as *p<0.05.

Chapter 5

Discussion and Conclusions

Through a yeast two-hybrid analysis, the interaction of STAT6 with FcγRIIA was discovered. It was found that the SH2 domain of STAT6 interacted with the ITAM of FcγRIIA. Interestingly, the tyrosines of the ITAM-like region used for the yeast two-hybrid analysis were not phosphorylated.

Upon phosphorylation of the tyrosines of the ITAM, an SH2 binding domain is created. The SH2 binding domain was presumably how the SH2 domain of STAT6 interacted with FcγRIIA. Lack of tyrosine phosphorylation suggests a complete lack of SH2 binding domain or an altered SH2 binding domain. This indicates STAT6 does not require the normal SH2 binding domain to associate with

FcγRIIA. Therefore, the interaction of STAT6 with the ITAM-like region of FcγRIIA is possibly phosphorylation independent.

We chose to further elucidate this interaction through the use of a hybrid cell line. Chinese hamster ovary cells, an epithelial cell line, which had been transfected with green fluorescent protein

(GFP) tagged FcγRIIA (CHO-IIA) were obtained from the Petty laboratory (Worth, Mayo-Bond et al.

2001). This cell line was chosen specifically because FcγRIIA is the only FcγR present on this particular epithelial cell. The RFP-STAT6 construct was made and transfected into the CHO-IIA cells.

The CHO-IIA cell line allowed for testing of the interaction of RFP-S TAT 6 w it h GFP-FcγRIIA directly through colocalization of epifluorescence signal. There was lack of consistent expression of both the

GFP-FcγRIIA and the RFP-STAT6. The lack of consistency led to the use of THP-1 cells for subsequent experiments.

THP-1 monocytes, an acute human leukemia cell line, were established and characterized in

1980 by Tsuchiya (Tsuchiya, Yamabe et al. 1980). 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was shown to differentiate the monocytes into macrophages, which resulted in increased phagocytic capabilities, a change in the size and morphology of the cell, and differences in the makeup of intracellular vacuoles (Tsuchiya, Kobayashi et al. 1982). They also express the IL-4R, which allowed for a positive control for our experiments when stimulated with IL-4 only. These monocytes also express all three families of FcγRs. The interaction between STAT6 and FcγRIIA would be obtained, but the various FcγRs would be ligated as well. STAT6 could potentially interact with all three members of the FcγR family as they signal either through the ITAM of the γc or the ITAM-like region, both which possess SH2 binding domains. Monoclonal antibodies against all three FcγR families have been generated (Rumpold, Kraft et al. 1982; Jones, Looney et al. 1985; Dougherty, Selvendran et al.

1987). The monoclonal antibody for FcγRII is IV.3, which has been shown to bind nonspecifically to all three types of FcγRII. FcγRI and FcγRIII could have been blocked through the use of their respective monoclonal antibodies to better delineate the interaction. The effects of FcγRIIB would still have been present due to the lack of heterogeneity of IV.3. Lack of the effect of FcγRIIB on STAT6 signaling would be interesting to observe as FcγRIIB modulates cellular signaling together with

FcγRIIA. Previous evidence indicates that the ITIM of FcγRIIB signals through SHIP, and SHP-1,

SHP-2 to inhibit phagocytic signaling (Muta, Kurosaki et al. 1994; Ono, Bolland et al. 1996; Isakov

1997; Cox, Dale et al. 2001). Interestingly, STAT6 attenuation is through SHP-1 (Haque, Harbor et al.

1998). The ligation of FcγRIIB with subsequent signaling and activation of the SHP family could dephosphorylate the tyrosine of STAT6. FcγRIIB ligation could be the reason for lack of phosphorylated STAT6 in the whole cell lysate and immunoprecipitation of FcγRIIA when ligated with ha IgG. SHP-1 activation could have also dephosphorylated S TAT 6 , thereby reducing translocation to the nucleus. Recent evidence shows that non-phosphorylated STAT6 can effectively translocate to the

44 nucleus, but tyrosine phosphorylation appears to be necessary for nuclear accumulation (Chen and

Reich). It remains to be determined if the S TAT 6 observed in our experiments has been dephosphorylated versus non-phosphorylated. Mutation analysis of the tyrosine at position 641 of STAT6 with subsequent transfection would aid in further determining if the interaction of FcγRII A w it h STAT 6 is phosphorylation independent.

Western blot of the whole cell lysates and immunoprecipitants yielded the observation that

STAT6 constitutively interacts with FcγRIIA. Phosphorylated STAT6 is produced in the whole cell lystates when IL-4 ligates IL-4R suggesting activation of the canonical pathway of STAT6 signaling.

Phosphorylated STAT6 was also produced when THP-1 macrophages were stimulated with IL-4 with subsequent immunoprecipitation of FcγRIIA. This indicates the canonical pathway of STAT6 activation is occurring with subsequent constitutive interaction of phosphorylated STAT6 with FcγRIIA. This suggests STAT6 association with the ITAM-like domain of FcγRIIA is possibly phosphorylation independent.

It has been shown previously that the tyrosines present in the ITAM-like region of FcγRIIA do not become phosphorylated upon the endocytosis of small particles (Mero, Zhang et al. 2006).

Therefore, during endocytosis there could be either a lack of or an altered SH2 binding domain as both tyrosines lack phosphorylation. It is interesting that ligation of FcγRIIA with haIgG induced a significant amount of non-phosphorylated STAT6 nuclear translocation suggesting STAT6 interacts with an absent or altered SH2 binding domain. Consequently, our experiments suggest that S TAT 6 do e s not require phosphorylation to translocate to the nucleus, which again denotes that the interaction of

S TAT 6 w it h Fc γRIIA is phosphorylation independent.

The production of IL-4 from THP-1 macrophages when FcγRIIA is ligated with haIgG was then observed. Production of IL-4 was unable to be detected after quantification by RT-PCR. It was recently shown that murine macrophages produce minute amounts of IL-4 upon stimulation with LPS for 48 45 hours (Mukherjee, Chen et al. 2009). The THP-1 macrophages from our experiments were stimulated with IL-4 for up to four hours, which could explain the lack of IL-4 production. Another group discovered the production of IL-4 from dendritic cells when exposed to IL-4 during their development from bone marrow stem cells (Maroof, English et al. 2005). They conclude that the small amount of IL-

4 produced could be due to the rapid uptake of IL-4 as dendritic cells express the IL-4R, a process that has been described in T cells (Friedrich, Kammer et al. 1999; Ewen and Baca-Estrada 2001). This could also be occurring in the THP-1 macrophages as they also express the IL-4R. Monoclonal antibodies to IL-4R do exist and could be used to block this effect. The short half-life and relative instability of IL-4 mRNA previously noted in T cells (Umland, Razac et al. 1998; Butler, Monick et al.

2002) has been proposed to also play a role in the minute amount of IL-4 production from alveolar macrophages (Pouliot, Turmel et al. 2005). This could be true in our experiments as well. IL-4 has also been shown to be produced in minute amounts from neutrophils in the presence of Brefeldin A (Brandt,

Woerly et al. 2000), from unstimulated peripheral blood eosinophils (Moqbel, Ying et al. 1995; Bjerke,

Gaustadnes et al. 1996; Nakajima, Gleich et al. 1996; Brandt, Woerly et al. 2000), from normal human bone marrow-derived basophils stimulated with calcium ionophore or FcεRI ligation(Arock, Merle-

Beral et al. 1993), and from normal and transformed mast cells (Brown, Pierce et al. 1987; Bradding,

Feather et al. 1992). FcγRs are present on eosinophils, basophils, and mast cells. Performance of our experiments in these cells could produce a significant amount of IL-4. TH2 appear to produce the most abundant amounts of IL-4 (Mosmann, Cherwinski et al. 1986; Romagnani, Maggi et al. 1991).

Macrophages and TH2 cells are in frequent contact, and this production of IL-4 from TH2 cells could effectively alter the phenotype and phagocytic effector functions of the macrophage producing M2 with decreased FcγR-mediated activities.

The effect of IL-4 on the phagocytic effector mechanisms was also observed. It was determined that neither phagocytosis nor endocytosis was inhibited. Evidence shows that pretreatment of mouse

46 macrophages with IL-4 to for 12 hours through 48 hours causes a significant decrease in phagocytosis

(Varin, Mukhopadhyay et al.). Another group observed that human macrophages when stimulated with

IL-4 for extended time periods (6 days) showed no significant decrease in phagocytosis (Gratchev,

Kzhyshkowska et al. 2005). It is difficult to ascribe changes in phagocytosis specifically to the effect of

IL-4 signaling or IL-4 induced gene expression after prolonged periods of IL-4 exposure as the phenotype of the cell might have changed during that time. A transient exposure to IL-4 (1 hour) was recently shown to possibly aid in pathogen survival when phagocytosed by murine macrophages through phosphotidylinositol 3-kinase dependent signaling (de Keijzer, Meddens et al.). THP-1 macrophages used during our experiments were exposed to IL-4 for up to one hour. Therefore, early change in the phagosome leading to more optimal survival of pathogens could be occurring. Also, extended stimulation with IL-4 in human macrophages could inhibit phagocytosis as most evidence involves murine macrophages.

In conclusion we have demonstrated that there is an interaction between FcγRIIA and S TAT 6 , and appears to be phosphorylation independent. This interaction does not cause phosphorylation of

S TAT 6 , but does produce nuclear translocation of S TAT 6 . Once in the nucleus, the possibility exists that S TAT 6 causes gene expression of IL-4 and IL-13 in undetectable amounts. Subsequently, IL-4 stimulation of macrophages did not significantly affect phagocytosis or endocytosis. Further research into the effect of non-phosphorylated STAT6 nuclear translocation after interaction with FcγRIIA and where STAT6 specifically interacts with FcγRIIA should be performed to determine the influence on gene expression and protein production.

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