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LOSS OF P2RX7 RECEPTOR ACTIVITY IN MRL lpr / lpr T LYMPHOCYTES CORRELATES WITH THE EXPRESSION OF B220 ANNE LAURE GRIGNON A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requiremen ts for the degree Bachelor of Arts Under the sponsorship of Dr. Amy Clore Sarasota, Florida December, 2008
ii Dedication "La science a t elle promis le bonheur ? Je ne le crois pas. Elle a promis la vrit, et la question est de sav oir si l'on fera jamais du bonheur avec de la vrit." [Emile Zola] (Did science promise happiness? I do not think so. Science promised the truth, and now the question is wherever we will be able to make happiness out of truth.) I would like to dedicat e this thesis to the three people who constitute my family: Yveta, Christophe and Alexandra Grignon. While I am out seeking truth you have helped me make happiness out of that truth. You have been my rocks through the past 21 years and for this I am foreve r thankful. Through the stress, the lack of sleep, you have always been by my side, no matter the distance between us. You have turned a child into a driven and strong woman because you have helped me everyday make the right choices in my life. Thank you for your strength, for your laughs, for your love. I love the three of you very much, and without you this work would have not come to exist.
iii Acknowledgements "The values of a human being is assessed by his capacity to give, and not his capacity to take." [Albert Einstein] I have been blessed to have found on my path so many people who have been willing to give in order to enrich both my academic and personal life, I am grateful to all of you and wouldn't be the person I am with out your support. Specifically, I would like to thank Dr. Clore, my thesis sponsor and academic advisor, for bringing out in me the best possible student, for your patience and understanding when I came to face obstacles, and for the long hours and inves tment in this thesis and all of my other academic projects. Dr. Walstrom, baccalaureate committee member, for encouraging me to pursue my projects when I was ready to quit, for working with my difficult schedules, and for your support in all of the classe s I have taken with you. Dr. Beulig, baccalaureate committee member, for reading and commenting on my work. Dr. Bob, for inspiring the work in this thesis, and for allowing me to work with his wonderful CNRS team. Dr. Julie Legrand, for working with me on this project, for her technical and personal help. Jeanne Ware, for the "power" talks, for believing in my work, and for being my "campus mom". Adam Schafer, for editing my many scientific papers, for flowers when sick, chocolate when stressed, an d an eternal shoulder to lean on. To my wonderful family, for always believing in me, no matter the obstacles and for your unconditional love. To my great roommates and friends for all of their support.
iv Table of Contents Pages Dedication ii Acknowledgements iii List of Tables & Figures vi Abstract vii 1. Introduction 1 1.1 Sustaining the Immune System's Homeostasis 3 1.2 The Fas/FasL Pathway 5 1.3 Autoimmunit y and the Autoimmune MRL lpr/lpr Mouse as a Model 7 1.3.1 lpr Mutation in the MRL lpr/lpr Mouse 7 1.3.2 FasL Over expression in MRL lpr/lpr Mice 10 1.3.3 Functions of Membrane and Soluble Forms of FasL 11 1.4 The Purinergic Receptor P2rx7 12 1.4.1 The Purino receptor Family 12 1.4.2 The P2rx7 Receptor 13 1.4.3 The P2rx7 Receptor and its Relation to Autoimmune Diseases 15 2. Materials and Methods 18 2.1 Mice 18 2.2 Cells 18 2.3 Fluorescent Pro bing for Subsequent Detection of Calcium Flux and of Pore 19 Formation 2.4 Flow Cytometry 20 2.4.1 Antibody Labeling 20 2.4.2 Analysis of P2rx7 Channel and Pore Formation 21 2.4.3 Fluorescence Activated Cellular Sorting 2 1 2.5 Real Time Reverse Transcriptase Polymerase Chain Reaction (RT PCR) 22 2.5.1 Total RNA Extraction 22 2.5.2 Complementary DNA Synthesis (cDNA) 23 2.5.3 Absolute Quantification of RT PCR 23 2.6 Western Blot 25 3. Res ults 28 3.1 Lymphocytic Populations of MRL +/+ and MRL lpr/lpr Mice 28 3.2 P2rx7 Activity in MRL +/+ and MRL lpr/lpr Mice Lymphocytic sub populations 29 3.2.1 P2rx7 Channel Activity 29 3.2.2 P2rx7 Pore Activity 31 3.2.3 Cleavag e of CD62L by P2rx7 33 3.2.4 Effects of ATP dosage on CD62L Cleavage and Pore Formation 35
v 3.3 P2rx7 Quantification of P2rx7 in MRL +/+ and MRL lpr/lpr Mice Lymphocytic Sub populations 36 3.3.1 Transcriptional analysis of P2rx7 36 3. 3.2 Translational analysis of P2rx7 38 3.4 Implication of B220/CD45RABC in P2rx7 Inactivation 39 3.4.1 Induction of the Expression of B220 40 3.4.2 Functional Inhibition of B220 43 4. Summary of the Present Research, Future Directions and Perspectives 48 5. Appendix 52 6. Bibliography 58
vi List of Equations, Figures & Tables Equation 3.1 Value of calcium flux Equation 3.2 Percentage of cells forming a pore Equation 3.3 Percent age of cells from which CD62L is cleaved Figure 1.1 Fas Receptor and its ligand FasL Figure 1.2 The apoptotic pathways via Fas/FasL interactions Figure 1.3 Physiopathology of the MRL lpr/lpr mouse. Figure 1.4 Structure of the protein receptor P2rx 7. Figure 1.5 Biological activities of the P2rx7 receptor in ATP or NAD presence. Figure 3.1 P2rx7 channel activity stimulated by ATP in T and B lymphocytic sub populations of MRL +/+ and MRL lpr/lpr mice. Figure 3.2 P2rx7 por e activity stimulated by ATP in T and B lymphocytic sub populations of MRL +/+ and MRL lpr/lpr mice Figure 3.3 CD62L cleavage activity by P2rx7 stimulated by ATP in T and B lymphocytic sub populations of MRL +/+ and MRL lpr/lpr mice. Figure 3.4 Transcriptional expression profile of P2rx7 and HGPRT in T and B lymphocytic sub populations of MRL +/+ ,MRL lpr/lpr C57BL/6 and C57BL/6 P2rx70/0 mice. Figure 3.5 Translational expression profile of P2rx7 in T and B lymphocytic sub populations in MRL +/+ and MRL lpr/lpr mice. Figure 3.6 Induction of B220 expression with ConA on T and B lymphocytes of MRL lpr/lpr mice. Figure 3.7 CD62L cleavage activity by P 2rx7 in T and B lymphocytes of MRL lpr/lpr mice stimulated by ATP and ConcA in vitro Figure 3.8 Functional inhibition of P2rx7 and B220 in vitro in MRL lpr/lpr lymphocytes. Figure 5.1 Diagram of a flow cy tometer Figure 5.2 Example of a two dimensional dot plot graph Table 2.1 Table of monoclonal antibodies (fluorescent tags in parenthesis) used during flow cytometry assays. Table 2.2 Composition of reactive mixture us ed for cDNA sysnthesis Table 2.3 Nucleotidic primers used for amplification through real time RT PCR. Table 2.4 Amplification conditions for real time RT PCR. Table 2.5 Polyclonal antibodies to probe the Western blo ts. Table 3.1 T and B lymphocyte distribution 30 32 34 5 6 9 13 15 30 32 34 37 38 41 42 44 53 55 20 23 24 25 27 29 Pages
vii Abstract During the immune response, T lymphocytes activated by a particular antigen undergo a very important expansion phase. These lymphocytes must be rapidly depleted at the end of the response in order to maintain the critical homeostasis governing the immune system (Hildeman et al. 2002). This negative regulation is effected, partly, by the apoptotic receptor Fas and of its ligand, FasL, expressed at the surface of activated T lymphocytes. In MRL lpr/lpr mice, as in patients affected by the autoimmune lymphoproliferative syndrome ( ALPS), the mutation of the fas gene is responsible for the development of systematic autoimmune pathologies such as systemic lupus erythematosus (SLE) and of secondary l ymphatic organ hyperplasia due to the accumulation of activated T lymphocytes of the abnormal immunophenotype CD90 + CD4 CD8 B220 + (double negative, T DN) (Lee et al. 2005). The B220 molecule is a differentiation marker for B lymphocytes. Its presence o n the surface of T DN lymphocytes indicates that the lymphocytes have received an apoptosis signal, but that they managed to escape the autoregulation process due to their fas gene mutation (Griffith et al. 1995). While 11 susceptibility loci for SLE have been characterized, the underlying causes of the disease remain unknown. In the MRL lpr/lpr mouse model, it has been previously demonstrated that the faults in the immune system's homeostasis pathway linked to the Fas receptor were one of the causes for t he disease's onset (Lee et al. 2005) In the course of the present work, we studied the possible role of the purinergic receptor, P2rx7, in SLE development in the MRL lpr/lpr mouse model using flow cytometry, western blotting and real time RT PCR. In bot h auto immune MRL lpr/lpr mice
viii and their wildtype counterparts, MRL +/+ the results indicate that the P2rx7 receptor is inactive in lymphocytic subpopulations expressing tyrosine phosphatase B220, such as in activated T lymphocyte that have received an ap optotic signal, as well as B lymphocytes. It appears that neither transcriptional or translational regulation, nor the presence of a mutation on the P2rx7 gene can explain the receptor's inactivity. The in vitro induction of B220 expression at the T lympho cytes surface in the MRL lpr/lpr mice diminishes P2rx7 receptor's activity. Reciprocally, inhibition of tyrosine phosphatase activity for CD45 isoforms including B220 lead to an augmentation of P2rx7 activity in lymphocytes expressing B220. Consequently, B 220 and/or other isoforms of CD45 are implicated in the inactivation of P2rx7 in T B220 + lymphocytes as well as in B lymphocytes, and this relationship may play a role in the development of the lupic phenotype. Dr. Amy Clore Division of Natural Scie nces
Grignon, 1 Chapter 1: Introduction The immune response to infectious agents is composed of two major components that are complementary to each other: the innate response that is immediately initiated, and the subsequent adaptive response, which is specific to the an tigen and allows for immunological memory. Innate immunity provides the first line of defense against antigens and consists of cellular and biochemical mechanisms that are in place before the infection occurrence which allows for a short response time. The se mechanisms will respond in the roughly same manner, and to the same extent, whether or not the antigen has been previously encountered (Martini, 2004). The innate immune system is thus composed of the following levels: the exterior physical and chemical barriers such as epithelia and the antimicrobial substances they produce, and the phagocytic cells (neutrophils, macrophages) and natural (NK) cells. Another vital component of the innate immune system is the complement system composed of small proteins ( C1q, C1r, and C1s), which normally circulate as inactive zymogenic forms (Carroll, 2004). When an antibody stimulates the C1 complex, proteases are released and will cleave specific proteins allowing for cytokines to enter the bloodstream. Cytokines, the m ain regulators of innate immunity, will in turn initiate a cascade of further cleavages which will lead to the activation of the cell killing membrane attack complex, thus inducing cellular swelling and ultimately, bursting (Carroll, 2004). Innate immunity mainly focuses on general structures common to many infectious agents, and is not able to recognize specific differences among diverse antigens (Martini, 2004). In contrast to innate immunity, the adaptive response increases with each subsequent exposur e to a particular antigen. This defense mechanism is particularly
Grignon, 2 specific due to its ability to recognize and remember antigens through its main components called lymphocytes, as well as the antibodies molecules secreted by a subset of cells. The two main types of adaptive responses are called humoral and cell mediated immunity (Delves & Roitt, 2000). Humoral immunity is partially accomplished by B lymphocytes that differentiate into plasma cells which secrete antibodies. These antibodies target and neutra lize microbes and the toxins they produce thus leading to their elimination by diverse effector mechanisms (Delves & Roitt, 2000). Only a particular subset of B cells, the memory B cells, will not subsequently be eliminated. These cells remain in the circu latory system, and will be carried by the organism throughout its lifetime (Delves & Roitt, 2000). If a pathogen the body has already encountered invades, memory B cells will recognize the pathogen and start to divide very quickly. The new generation of ce lls will kill the pathogen at such a pace that often the organism will not present outward symptoms of the pathogen's entry (Delves & Roitt, 2000). On the other hand, cell mediated immunity is mediated by T lymphocytes. Its main targets are viruses and som e bacteria that survive and resist destruction once inside of phagocytes, since these agents are inaccessible to antibodies (Delves & Roitt, 2000). T lymphocyte specificities are very restricted as they only recognize small antigenic peptides presented by major histocompability complex (MHC) molecules of antigen presenting cells (dendritic cells, macrophages, or B lymphocytes) via their T Cell Antigen Receptors (TCR) (Hildeman et al. 2002). T lymphocytes are a population of cells that are composed of both helper T cells and cytotoxic (or cytolytic) T lymphocytes (CTLs). When helper T cells are stimulated by the presence of a foreign antigen, they secrete cytokines which in turn stimulate the proliferation and differentiation of T lymphocytes,
Grignon, 3 as well as the activation of other cells such as B lymphocytes, macrophages and other leukocytes (Delves & Roitt, 2000). The cluster of differentiation (CD) markers are surface molecules used to identify and sort different types of leukocytes including diverse kinds of lymphocytes. CD molecules can play dual roles either as receptors or as ligands, often initiating signaling cascades that lead to alterations in the cell. In humans, the CD4 + helper T lymphocyte is an example of a cell responsible for B lymphocytes activat ion and differentiation, as well as macrophage activation. The CD8 + cytotoxic T lymphocyte is the agent responsible for the destruction of microbe infected cells as well as the killing of tumor cells (Weiss & Goodnough, 2005). CTLs will kill the antigen p roducing cells and once the infectious agent is eliminated, regulatory T cells suppress the immune response, as it is no longer needed (Martini, 2004). Once the threat posed by the pathogen has disappeared, the extremely large numbers of effector cells are no longer needed, and the apoptotic mechanism is initiated. 1.1. Sustaining the Immune System's Homeostasis T lymphocyte homeostasis is a vital mechanism to the immune system's balance. Antigen elimination in the body is followed shortly by a contracti on phase, during which a large majority of cells having responded to the antigen are eliminated either by passive apoptosis or activated T cell autonomous death (ACAD) or by activation induced cell death (AICD) ( Van Parijs et al 1998 ). This contraction ph ase occurs as a response to the lack of stimuli by antigens, costimulators and cytokines which, when not present, allow
Grignon, 4 for clonal expansion and differentiation of the lymphocytes into effector cells by inducing the expression of anti apoptotic proteins ma inly from the B cell leukemia/lymphoma 2 ( Bcl 2) family ( Van Parijs et al 1998 ). The activation of T lymphocytes by the antigen induces the expression of the growth factor, interleukin2 (IL 2) and of the high affinity IL 2 receptor responsible for the pr oliferation step ( Van Parijs et al 1998 ). It is thought that the ACAD path may result in cellular death of the activated T lymphocytes that survive following an IL 2 level drop, which would occur as a result of regulatory T cell activation and release of pro apoptotic members of the Bcl 2 family ( Van Parijs et al 1998 ). The presence of pro apoptotic factors then leads to the release of apoptosis inducing factor (AIF), Smac/DIABLO and cytochrome C by the mitochondria, and can be inhibited by the different T lymphocyte growth factors such as IL 2, IL 4, IL 7 or IL 15 (Ju et al. 1995) On the other hand, the AICD pathway (discussed in more detail in the section below) concerns T lymphocytes that are activated for a second time by their antigen and thus that did not die through the ACAD pathway (Hildeman et al. 2002). In autoimmune diseases, this homeostatic balance is misregulated and the apoptotic signals are not perceived by the effector cells thus leading to their accumulation in secondary lymphocytic org ans (Abbas et al. 2007). In the case of systemic lupus erythematosus (SLE), the T lymphocytes accumulate in ganglia and in the spleen causing inflammation and increased risks of infection and bleeding. Thus, one of the causes believed to trigger autoimmun e disease is the disruption of T lymphocyte homeostasis which is normally regulated by ACAD, a Fas/FasL mediated pathway (Ju et al. 1995)
Grignon, 5 1.2. The Fas/FasL Pathway The Fas receptor is constitutively expressed on the surface of numerous cellular types s uch as lymphocytes and hepatocytes. This type I transmembrane glycoprotein of 45kDa belongs to the tumor necrosis factor ( TNF) receptor family a group of cytokines able to induce apoptosis (Leonardo et al. 1999). Like TNFR1, Fas possesses in its intracel lular fraction a "death domain" implicated in the death signal transduction (Figure 1.1). Fas's ligand, FasL, is expressed in localized areas. In fact, the ligand is preferentially expressed in cells belonging to the immune system such as Natural Killer ce lls (NK) and activated T lymphocytes (Leonardo et al. 1999). This type II transmembrane glycoprotein is a homotrimer of 40 kDa belonging to the TNF family. Figure 1.1.: Tumor necrosis factor, and its receptor, TNF R1, and the Fas Receptor and its ligan d FasL, adapted from Marcel et al. 2002. The green color indicates the four (TNF R1) and three (Fas) cysteine rich pseudorepeats, the red the homotrimeric protein domains, the pink and yellow the transmembrane domains, and the blue the intracellular death domains (Figure by A.L. Grignon) The interaction between FasL and Fas (Figure 1.2) recruits caspase proteases that initiate a cascade of proteolytic events leading to cellular death for cells carrying the Fas receptor. Two types of caspases are implicated in these events. The initiation caspases (caspases 8 and 10) are at the origin of the transduction pathway, and the effector
Grignon, 6 caspases (e.g. caspase 3) are implicated in vital substrate cleavage in the cell (i.e. cleavage of lamin, Parp, etc) and in DNase CAD activation (Leonardo et al. 1999). Depending on the cell type, receptor Fas induced apoptosis is either independent (in lymphocytes) or dependent (for hepatocytes) on cytochrome c liberation by the mitochondria (Leonardo et al. 1999). Figure 1.2. : The apoptotic pathways via Fas/FasL interactions from Thome et al. 2001. Apoptosis can occur via either cell surface death receptor or mitochondrial dependent pathways. When CTLs are stimulated, they will express FasL, which will bind to the death recep tor Fas on a target cell. Fas will then recruit the Fas associated death domain (FADD) adapter protein to form the death inducing signaling complex. Once FADD is formed, a cascade of signaling events will follow: caspase 8 is activated and will then, in tu rn, activate downstream caspases, such as caspase 3, leading up to cellular apoptosis. The death signal can also be initiated due to internal cellular damages. In this case, mitochondrial cytochrome c is released and the apoptotic protease
Grignon, 7 activating facto r 1 (Apaf 1)/caspase 9 complex is then activated. Intracellular receptor proteins, such as Apaf 1, will be the agents stimulating caspase activation which will, once again, lead to apoptosis. Many of these interactions found in pro apoptotic signaling path ways are mediated by one of three related protein protein interaction motifs: death domains (DDs), death effector domains (DEDs) and caspase recruitment domains (CARDs). 1.3. Autoimmunity and the Autoimmune MRL lpr/lpr Mouse as a Model Autoimmune diseas es are the result of a defect, of unknown origin leading to self antibody tolerance. These pathologies can be specific to an organ, such as is the case in type 1 diabetes, or systemic, such as in the case of systemic lupus erythematosus (SLE). Autoimmune d iseases are multi factorial. In fact, they are often thought to have a complex genetic component (additional examples include Celiac disease, as reviewed in Dieterich et al. 1997 and Polyglandular Autoimmune Syndrome Type III as reviewed in Papadopoulos et al. 1994) There is often a hormonal factor (8 women suffer SLE for every man), and environmental factors can also play roles (e.g. infections as discussed by Lee et al. 2005). The study of autoimmune disease thus represents a real stake in public hea lth since over 40 autoimmune pathologies are in existence today, and they have been estimated to be the third leading cause of morbidity (Lee et al. 2005). For unknown reasons, SLE incidences have tripled over the course of the last twenty years (Lee et a l. 2005). 1.3.1. lpr Mutation in the MRL lpr/lpr Mouse The MRL lpr/lpr mouse is a model for systemic lupus erythematosus and rheumatoid arthritis and present lymphoproliferation due to the spontaneous insertion of a retrotransposon from the 5.3kb early t ransposable element (Etn) ( Rieux Laucat et al.
Grignon, 8 1995). This insertion into the second intron of the fas gene results in the premature termination of the fas transcript, leading to the production of a non functional Fas protein (Rieux Laucat et al. 1995). Nonetheless, the lpr mutation is leaky: approximately 2% of the mRNA is transcribed, and thus about 10% of Fas proteins are functional (Fisher et al. 1995). Through the use of the MRL lpr/lpr mouse model, the molecular nature of the formerly named Canale /Smith syndrome was further elucidated and the condition was renamed "APL syndrome" (Autoimmune Lymphoproliferative Syndrome) ( Berthold et al. 1989) In fact, the heterozygous mutation of the Fas receptor is thought to be largely responsible for lymphopro liferation and systemic type autoimmune pathologies in patients (Rieux Laucat et al. 1995). In mice, the autoimmune and lymphoproliferation pathologies start appearing at the age of two months, and the mice die around four to five months, generally of a g lomerulonephritis with immune complexes (Figure 1.3). While the MRL lpr/lpr mouse lineage is essentially isogenic, it is important to note that, as in the human APL syndrome, large phenotypic variability exists from one animal to another probably due to a strong epigenetic (hormonal and/or environmental) component (Fisher et al. 1995) In the same way as in human beings, female mice develop the autoimmune syndromes more rapidly and more severely, and consequently have a significantly shorter life span than males (Fisher et al. 1995)
Grignon, 9 A B Figure 1.3.: Physiopathology of the MRL lpr/lpr mouse. A. Hyperplasia of secondary lymphocytic organs (ganglia on top and spleen on bottom) of MRL lpr/lpr and the MRL +/+ mice. B. Characteristic cu taneous symptoms of systemic lupus erythematosus. Adapted from Bob et al. 2006. The inactivation of the apoptosis inducing receptor Fas leads to the accumulation of CD90 + CD4 + CD8 B220 + or CD90 + CD4 CD8 + B220 + phenotype T lymphocytes in secondary lymp hocytic organs (Reno et al. 1998). These activated T lymphocytes will progressively repress CD4 or CD8 expression in order to become double negative T lymphocytes (T DN cells) of an abnormal phenotype: CD90 + CD4 CD8 B220 + (Reno et al. 1998). Following their activation, the CD4 + and CD8 + T lymphocytes that will engage in the Fas/FasL apoptotic pathway will express a particular isoform of CD45 called B220 or CD45RABC (Reno et al. 1996). In the presence of the fas gene mutation, the B220 marker at the T D N cell surface indicates that these T lymphocytes have received a death signal, but that the cells have escaped the autoregulation process. In the oldest MRL lpr/lpr mice populations (four to five months), T DN cell populations can represent up to 85% of t otal T lymphocytes (Reno et al. 1998). Cutaneous attacks due to SLE MRL +/+ MRL +/+ MRL lpr/lpr MRL lpr/lpr
Grignon, 10 The CD45 molecule is the most abundant transmembrane glycoprotein (10%) at T and B lymphocytes' surfaces (Reno et al. 1996). CD45 exists as eight potential isoforms, five of which (including B220) have been found i n humans (Reno et al. 1996). This protein is a tyrosine phosphatase implicated in tyrosine residue dephosphorylation on diverse molecules such as Lck which plays a role in transduction of the activation signal of the antigen receptors TCR and BCR on T and B lymphocytes (Reno et al. 1996). 1.3.2. FasL Over expression in MRL lpr/lpr Mice In humans as in mice, Fas's ligand is one of the 11 known loci contributing to lupus susceptibility (Lee et al. 2005). The Bob laboratory, in which I performed my thesis research, previously produced work indicating that T lymphocytes of MRL lpr/lpr mice, and in particular T DN cells, over express the FasL molecule at the membrane level (Benihoud et al. 1997). This over expression confers upon them a strong spontaneous c ytotoxic power in vitro as well as in vivo compared to Fas expressing cells (Bonardelle et al. 2001). More surprisingly, in more recent work, the team further observed that activated B lymphocytes in MRL lpr/lpr mice also over express FasL in their membra nes thus conferring upon them the same strong cytotoxic power in vitro (Bonardelle et al. 2005) Consequently, the laboratory formulated the hypothesis that tissues originating from MRL lpr/lpr mice carrying functional Fas molecules (due to the leakiness o f the mutation) are the non specific targets of attack by lymphatic cells over expressing FasL (T and B lymphocytes and NK cells) (Adachi et al. 1995). These attacks would thus be responsible for the autoimmune pathology development in MRL lpr/lpr mice. T his hypothesis is reinforced by the absence of autoimmune pathologies in animals rendered
Grignon, 11 fully Fas receptor deficient by homologous recombination, even though they present a lymphoproliferation pathology (Adachi et al. 1995). The Bob team was able to va lidate this hypothesis by creating a murine model spontaneous for chronic and articulary hepatic pathologies solely depending on the interaction of Fas with its ligand (Bob et al. 1997). 1.3.3. Functions of Membrane and Soluble Forms of FasL Not only d oes one find the over expression of FasL in murine lymphocyte plasma membranes or the membranes of patients mutant for the Fas receptor, but an elevated concentration of the soluble form of FasL (sFasL) is also found in their serum (Tanaka et. al. 1996). In fact, similar to the case of the TNF family members, FasL can be released under a soluble form of 26 kDa through the action of metalloproteinases that cleave the extracellular domain of the protein in the juxtamembrane region (Tanaka et. al. 1996). In the case of TNF, the implicated protease is the metalloproteinase TNF converting enzyme ( TACE or ADAM17) but the one cleaving FasL remains unknown (Tanaka et. al. 1996). Along the same lines, sFasL's function still remains to be determined since pro an d anti apoptotic effects have both been documented for this serum molecule (Tanaka et. al. 1996). Patients suffering from NK type nasal lymphoma, a rare type of lymphoma characterized by angiocentricity (invasion and blockage of blood vessels by lymphoma cells resulting in marked necrosis of the normal and neoplastic tissues), present with elevated FasL concentrations in their serum (Cheung et al. 2002). This elevation is thought to be at the origin of autoimmune hepatitis in these patients since the elim ination by chemotherapy of the lymphoma triggers the disappearance of both sFasL and the autoimmune hepatitis (Sato et al. 1996). Conversely, sFasL plays a pro apoptotic role by competing with the membrane form of FasL for its binding to Fas.
Grignon, 12 1.4. The P urinergic Receptor P2rx7 When the lpr mutation is transposed into another murine genetic background, such as C57BL/6, the lymphoproliferation is present, but not the lupus type autoimmune pathologies (Elliot et al. 2005). The genetic background is thus essential to the autoimmune pathology development. The same mechanism is true for human patients, as a Fas mutation does not always induce the APL syndrome (Elliot et al. 2005). Other loci, such as human and murine SLEB4, another of the 11 loci characteri zed for lupus susceptibility, can play a large role. SLEB4 corresponds to a particular gene of interest, P2rx7, a member of the purinergic receptor sub family P2X whose implication in lupus onset has been only recently suggested (Elliot et al. 2005). 1.4 .1. The Purino receptor Family In 1978, Burnstock et al. characterized a receptor family capable of purine binding. This family of purinoreceptors was divided in two sub categories: the P1 sub family with high affinity preference for adenosine and the P2 sub family, which prefer ATP and ADP (Burstock et al. 1980). The P2 receptor family was then divided into two categories: metabolic P2Y receptors which, like P1 receptors, are coupled to G proteins, and P2X receptors, which are types of ion channels. Seve n P2Y receptors (P2Y 1 to P2Y 6 and P2Y 11 ) and seven P2X receptors (P2X 1 to P2X 7 ) have been cloned and identified in humans (Khakh et al. 2001). Nonetheless, the biological functions of these proteins still remain an open question. The basic structure of P2X family members is very conserved and composed of two hydrophobic transmembrane regions: an extracellular loop and two N and C
Grignon, 13 cytoplasmic terminal extremities (Figure 1.4). Depending on the family member, this structure forms homo or hetero oligomere s necessary for their biological function (Khakh et al. 2001). Figure 1.4: Structure of the protein receptor P2rx7. Residue 1 (methionine), 25 (asparagine), 46 (valine), 334 (valine), 355 (isoleucine) and 595 (tyrosine). Regions from 1 25, 47 33 4, 356 595 topological domains; 26 46 and 335 355 transmembrane domains. Adapted from: http://www.uniprot.org/uniprot/Q99572 1.4.2. The P2rx7 Receptor The P2rx7 receptor, initially called P2Z, was f irst described in 1980 at the surface of mastocytes (Cockcroft et al. 1980). It has since been identified on the surface of several other hematopoietic cells such as macrophages, lymphocytes, monocytes and dendritic cells, but also on the surface of vario us nervous system cells (Khakh et al. 2001). The P2rx7 receptor possesses a weak sensitivity to ATP since it responds to concentrations of approximately 100 M, while the levels necessary for the activation of other members of the P2X family are on a sca le from 0.5 to 5 M (Khakh et al. 2001). ART2.2 protein activation by NAD can also induce ADP ribosylation of the P2rx7 N ter C ter 1 25 4 6 334 355 595
Grignon, 14 receptor, and thus is also responsible for its activation (Figure 1.5) (Khakh et al. 2001). Depending upon the length of stimulation by ATP of the receptor, diverse biological activities are induced. A brief activation of P2rx7 (on the order of seconds) induces a cationic channel activity, notably responsible for intracellular calcium augmentation. A prolonged activation (on the order o f minutes) of the receptor will lead to pore formation, permitting an influx and an efflux of solutes with molecular weights lower than 900 Da (Khakh et al. 2001). The formation model of this pore remains a topic of debate in that the pore could be formed by a cluster of monomers or by the assembly of hetero oligomeres. The only established detail is the fact that the C terminal region possesses crucial amino acids needed for the formation of the pore (Smart et al. 2003). The P2rx7 also enables the acti vation of proteases such as the ones responsible for pro IL1 to IL1 maturation, or in CD62L cleavage (Figure 1.5). This last function allows lymphatic cells to leave secondary lymphatic organs, and to migrate towards the infection site (Auger et al. 2 003). In the four hours that follow the receptor's activation, apoptosis or aponecrosis phenomena are observed. In fact, characteristic apoptotic proteins such as caspase 3 and 9 are activated, but in the case of aponecrotic death, the classically observed apoptotic bodies are not formed (Auger et al. 2005). The cells instead die by necrosis, emptying their cellular contents in the extracellular media (Auger et al. 2005). For this reason and also for its implication in pro inflammatory cytokine maturation the P2rx7 receptor is referred to as a pro inflammatory receptor.
Grignon, 15 Figure 1.5.: Biological activities of the P2rx7 receptor in ATP or NAD presence. Depending upon the stimulation length, ATP stimulates the P2rx7 receptor to form a channel (after ~1s) allowing the passage of calcium into the lymphocyte, or a pore (after ~1min) allowing the passage of solutes of size smaller than 900 Da. The stimulation of the P2rx7 receptor also induces the cleavage of CD62L by a protease. Figure by A.L. Grignon. The downstream signal cascade of the receptor is still unknown, but the activation of certain proteins such as Src (avian sarcoma [Schmidt Ruppin A 2] viral oncogene homolog) phosphatidylinositol 3 kinase (PI3K) or the extracellular signal regulated kinases 1 and 2 (Erk1/2) has been observed following the death mechanism induction through P2rx7 (Auger et al. 2005). 1.4.3 The P2rx7 Receptor and its Relation to Inflammation and Autoimmune Diseases
Grignon, 16 In the case of all infections, due to bacteria, virus, paras ites or fungi, the first defense mechanism of an organism is the inflammatory response (innate response). More generally, inflammation is vascular tissue's response to a physical (from heat, cold, ionizing radiations etc) or chemical (composed acids or bas es, bacterial toxins, etc.) aggression (Abbas et al. 2007). It is also the consequence of tissue necrosis, itself being secondary to numerous causes, such as for example an arterial occlusion. Inflammation is not a synonym for infection, but an infection will generally have an inflammatory response (Ferrari et al. 1997). Whether the inflammation's cause is infectious or not, there will be macrophage stimulation that will in turn liberate pro inflammatory cytokines such as IL 1, IL 6, IL 8, IL 12 and TNF (Ferrari et al. 1997). Elevated levels of TNF or Interleukin 1 ( IL 1 ) are found in the serum of patients suffering from autoimmune pathologies (Ferrari et al. 1997). IL 1 is one of the key mediators of inflammation caused by infections. Bacterial lipopolysaccharide (LPS) and other inflammatory factors are inducers of the inactive form of IL 1 known as pro IL1. This pro cytokine must then undergo a maturation phase triggered by caspase 1, in order to acquire its activity. The LPS induced stimulus d oes not assure, by itself, IL 1 's maturation (Ferrari et al. 1997). ATP stimulation via P2rx7 receptor activation could play the role of a secondary signal in this secretion/maturation phase (Ferrari et al. 1997). Furthermore, recent studies suggest tha t P2rx7 plays a role in membrane TNF cleavage in microglia (Suzuki et al. 2004). Previous evidence for a possible role of P2rx7 in autoimmunity as discussed by Ferrari et al. in 1997 indicates the need to study its implications in autoimmune pathologies d evelopment.
Grignon, 17 As previously mentioned, the P2rx7 receptor could be a candidate for SLE development due to its localization in the SLE susceptibility locus, SLEB4. The main objective of this study was thus to investigate receptor P2rx7's activity in T and B lymphocytic sub populations of autoimmune MRL lpr/lpr mice. In order to evaluate this activity, we assayed the receptor's abilities (in presence of ATP) to form an ionic channel versus a pore allowing the passage of solutes of molecular weights smaller th an 900 Da and to activate the protease implicated in the cleavage of the adhesion molecule CD62L. In the course of this work, we also have evaluated P2rx7's expression in the diverse lymphocytic populations by r eal time polymerase chain reaction ( RT PCR) a nd by western blot. The obtained results lead to the research of a possible relationship between the CD45 phosphatase and the P2rx7 receptor.
Grignon, 18 Chapter 2: Materials and Methods 2.1. Mice The wildtype mice MRL +/+ and MRL lpr/lpr homo zygous for the lpr mutation originating from the Jackson Laboratory (Bar Harbor, ME) were maintained in the animal facility of the Institut Andre Lwoff, in Villejuif, France. Control C57BL/6P2rx7 0/0 mice, from which the expression of P2rx7 had been inactiv ated by homologous recombination, and the wildtype C57BL/6 mice, both originated from the Jackson Laboratory and were maintained in the animal facility of the Pasteur Institute, Paris, France. All experiments were performed in accordance with institutional Animal Research Committee guidelines. 2.2. Cells Mice underwent euthanasia prior to removal of the spleen, axillary and peritoneal lymphatic ganglia. Lymphatic cells were isolated from secondary lymphatic organs, filtrated and collected by centrifugati on at 1000g for five minutes. The cellular pellets were then resuspended in phosphate buffer (PBS, 0.01M phosphate buffer, 0.0027M potassium chloride and 0.137M sodium chloride pH 7.4) or in RPMI 1640 medium containing 10% fetal calf serum (FCS). For cellu lar sorting experiments, a purification step for lymphocytes was performed using Ficoll Hypaque (density = 1.17 g/liter) in order to eliminate the presence of erythrocytes, granulocytes and possible dead cells. For experiments involving stimulation with co ncavaline A (ConcA), lymphoid cells were suspended in a RPMI 1640 medium ( Espinel Ingroff et al. 1996) containing 10% FCS and 4 g/ml of ConcA, and were cultivated for three days at 37¡C in a 5% CO 2 atmosphere. For experiments involving inhibition of P2rx7 phosphatase activity with 1
Grignon, 19 [N, O bis (5 Isoquinolinesulfonyl) N methyl L tyrosyl] 4 phenylpiperazine (KN 62) or B220 phosphatase activity with N (9,10 Dioxo 9,10 dihydro phenanthren 2 yl0 2, 2 dimethyl propionamide (CD45I), lymphoid cells were incubated for 2 hours in presence of 200nM of each respective inhibitor in PBS buffer. 2.3. Fluorescent Probing for Subsequent Detection of Calcium Flux and of Pore Formation The activity of the P2rx7 channel was evaluated through the use of the Oregon Green 488 ( or 2', 7' difluorofluoresceine) probe originating from Invitrogen (Cergy Pontoise), which emits fluorescence in presence of intracellular calcium. The lymphocytes originating from the spleen and lymphatic ganglia of MRL +/+ and MRL lpr/lpr mice were marked with the calcium probe as well as fluorescent antibodies anti B220 PE Cy5.5 and anti CD90 APC At time t=0, the marked cells were stimulated with 500 M of ATP, and the intracellular calcium levels were monitored for 20 minutes by flow cytometry (see a descr iption of this technique in Appendix). The activity of the pore was visualized via the incorporation of the fluorescent probe YO PRO originating from Invitrogen (Cergy Pontoise) in the presence of 500 M of ATP. This probe of size 629 Da is incapable of p enetrating in cells through channels but can cross the membrane through the P2rx7 pore (permeable to solutes of MW<900 Da) and becomes fluorescent once it intercalates in between DNA bases. Lymphocytes of MRL +/+ and MRL lpr/lpr mice were incubated with th e YO PRO probe in presence of ATP. The cells were then marked with antibodies anti B220 PE Cy5.5 and anti CD90 APC and analyzed with flow cytometry, both described below.
Grignon, 20 2.4. Flow Cytometry 2.4.1. Antibody Labeling Cellular staining was performed using fluorescent antibodies, with an incubation time of 15 minutes at 4¡C and in total absence of light. All monoclonal antibodies were purchased from the PharMingen Society (BD Bioscience, San Diego, CA, USA) and were conjugated to fluorescent molecules such as fluorescein isothiocyanate (FITC), phycoerythrine (PE), phycoerythrine cyanine 5.5 (PE Cy5.5) or allophycocyanine (APC) as itemized in Table 2.1. Table 2.1.: Table of monoclonal antibodies (fluorescent tags in parenthesis) used during flow cytometry as says. Antibodies Function of antibody's protein target AcMn murine anti B220 (PE Cy5.5, APC) B220 or CD45RABC is a transmembranous protein specific to B lymphocytes AcMn murine anti CD90 (FITC, APC) CD90 is a transmembranous protein specific to murine T lymphocytes AcMn murine anti CD62L (FITC, APC) CD62L is a membrane adhesion protein of lymphocytes Monoclonal Antibodies AcMn murine anti RFcy (PE) RFcy is a membrane receptor specific to constant parts (Fc) of type G immunoglobulin. In order to reduce the nonspecific biding of antibodies through their constant region, Fc, the cells were preincubated with an antibody (Fc block) directed against the murine Fc for five minutes at 4¡C. After the cellular marking, cells were stored at 4¡C prior to the taking of flow cytometry readings. All antibodies used were diluted in PBS buffer containing 3% FCS, 2% HEPES and 0.1% sodium azide.
Grignon, 21 2.4.2. Analysis of P2rx7 Channel and Pore Formation The flow cytometer used was a FacsCalibur (Becton Dickinson) p ossessing two excitation sources: an Argon laser and a red diode with respective wavelengths of 488nm and 635nm (see Appendix for additional theory and figure captions detailed parameters of the cytometer's use). The signals transmitted by the flow cytomet er were analyzed through the Cell Quest program (Becton Dickinson) version 3.3. The total number of cells counted in each experiment was dependent of the chosen population: 15,000 to 20,000 cells for T lymphocytes CD90 + B220 and T lymphocytes CD90 + B220 + and 9,000 to 12,000 for B lymphocytes CD90 B220 + 2.4.3. Fluorescence Activated Cellular Sorting The cytometer used for cellular sorting prior to real time PCR (below) was a FacsVintage model with the Diva option from the Becton Dickinson company. Th is apparatus uses the same working principles as the flow cytometer, but is coupled to a mechanism that allows sorting of a heterogeneous mixture of cells into several containers based upon the specific light scattering and fluorescent characteristics of e ach cell ( Davey et al. 1996) In order to perform the above task, a controlled liquid fragmentation of 30,000 drops/sec leads to the formation of electrostatically charged droplet each containing a cell ( Davey et al. 1996) The droplets are then deviated from their initial fall by a constant electrical field, and are directed towards collecting flasks. Before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescence of each cell is measured ( Davey et a l. 1996) The apparatus permits the separation of up to four different populations through charge and partial charge apposition, based on the fluorescent cellular labels. In the performed experiments, the
Grignon, 22 following three cellular populations were sorted t hrough CD90 FITC and B220 PE antibody staining: CD90 + B220 T lymphocytes, CD90 + B220 + T lymphocytes, and CD90 B220 + B lymphocytes. 2.5. Real Time Reverse Transcriptase Polymerase Chain Reaction (RT PCR) 2.5.1. Total RNA Extraction The total RNAs of the di fferent T and B lymphocyte sub populations purified by flow cytometry were extracted with the RNeasy Mini kit (Qiagen; Valencia, CA, USA) following the general procedure (details as described in manufacturer's instructions). Cellular pellets were resuspend ed in lysis buffer containing guanidium thiocyanate (6.0 M), and § mercaptoethanol (140 nM), thus assuring protein denaturation, and particularly that of RNases. The genomic DNA was then broken up by the mechanical action of a syringe, and eliminated after being passed through a column. The solution was then placed on a silica column. After several washes, the RNA was eluted from the column by an RNAse free water wash, and a 0.1% diethyl pyrocarbonate (DEPC) treatment. The RNA was finally quantified and qua lity was assessed by absorption spectroscopy using 260nm and 280nm wavelengths (Systeme Nanodrop, Agilent; data not shown) and stored at 80C until its utilization. 2.5.2. Complementary DNA (cDNA) Synthesis cDNAs were synthesized from a 0.5 to 2 g tota l RNA through the use of reverse transcriptase activity at 42C. In order to promote cDNA synthesis based on mRNA, the primer used was an oligo dT 18 mer, permitting hybridization at the mRNA tails. The composition of the reaction mixture used (Invitrogen, Cergy Pontoise) is described in
Grignon, 23 Table 2.2. The elimination of secondary structures of total RNA was effectuated by incubation in a 70C bath for five minutes, and the cDNAs were stored at 20C until their utilization. Table 2.2.: Composition of reactive mixture used for cDNA synthesis RNA (1ng to 5 g) 10 L Oligo dT18 (500 g/ml) 1 L dNTP Mix (dATP, dGTP, dCTP, dUTP, each in a 10mM concentration) 1 L DTT (0.1M) 2 L Rnases Inhibitor (40 units, L) 1 L Reaction Buffer (5x) 4 L Reverse Transcripta se SuperScriptII RT 1 L (200 units) RNAse free sterile water 10 L Total Volume 30 L 2.5.3. Absolute Quantification by Real Time RT PCR Quantification of mRNAs corresponding to select gene was accomplished through quantitative PCR, using SYBR green as the fluorophore (LightCycler 2.0, Roche) and using the fact that the fluorescence emitted is proportional to the amount of double stranded DNA present. Amplification of cDNA target sequences followed the same steps as classical PCR: cDNA denaturation hybridization of primers (Table 2.3) and elongation through DNA Taq polymerase (see Table 2.4 for cycling parameters). Fluorescence upon binding to the resulting double stranded DNA was measured at the end of each elongation cycle. After the last cycle, the temperature was progressively elevated to 95C, during which fluorescence was continuously read for the denaturation curve (data not shown). Temperature augmentation induced the dissociation of double stranded DNA, and when
Grignon, 24 50% of DNA was disassociate d, the fluorescence reduction corresponded to synthesized product's melting temperature. This step was crucial as it permitted confirmation that a single PCR product was synthesized using each primer pair selected. Hypoxanthine Guanine Phosphoribosyl Trans ferase (HGPRT) was used as an endogenous control (Bob et al. 2006). Table 2.3.: Nucleotide primers used for amplification through real time RT PCR. Sense Primer Annealing Temperature Anti sense Primer Annealing Temperature Size of Amplified Fragment (bp) P2rx7 Gene 5' AGC ACG AAT TAT GGC ACC GT 3' 60C 5' CCC CAC CCT CTG TGA CAT TCT 3' 60C 213 bp HGPRT Gene 5' GTA ATG ATC AGT CAA CGG GGG AC 3' 62C 5' CCA GCA AGC TTG CAA CCT TAA CCA 3' 55C 176 bp *HGPRT: Hypoxanthine Guanine Phosphoribosyl Tran sferase Table 2.4.: Amplification conditions for real time RT PCR. Temperature Time (seconds) Number of Cycles Initial Denaturation 95C 600 1 Denaturation 95C 5
Grignon, 25 Primer Hybridization Primer Temperature 10 Elongation 72C 12 40 Quantification of transcripts in samples was performed by measuring the sample's threshold cycle (Ct) and by using a standard curve constructed by serial dilutions of RNA equivalent cDNA from 0.0064pg to 10pg into 1 RT buffer The standard curve was made by plotting th e obtained Ct values against the logarithms of the standard concentrations, and by applying a linear regression curve to the data. Standard curves were used to calculate the amounts of each RNA (P2Rx7 versus HGPRT) on a per cell basis and the P2rx7 values were then divided by the HGPRT values to determine the relative expression levels. The amplification product was also purified by gel electrophoresis on an agarose gel, and quantified through spectrophotometry at a wavelength of 260nm (data not shown). 2 .6. Western Blot Flow cytometry purified T and B lymphocyte sub populations were lysed in Radio Immuno Precipitation Assay (RIPA) buffer (1% Triton X 100, 1% deoxycholate, 150nM NaCl, 50mM Tris pH 8.0, 1% Nonidet NP40) containing 1% SDS which permitted pr otein solubilization and denaturation, as well as the lysing of cellular membranes. The homogenate underwent centrifugation (10000g at 4C for 20 minutes) in order to separate debris from cellular DNA. The protein concentration was determined through the B radford method (Pierce, Rockford, IL, USA). Equal amounts of total proteins from the different samples were separated on an acrylamide gel with a 4% to 12% gradient (Gel
Grignon, 26 NuPage 4 12%, Invitrogen) at 200mV and transferred with the Vertical Electrophoresis A pparatus V15.17 from (Gibco BRL) to a nitrocellulose membrane (X Cell II Blot Module, Invitrogen) for one hours and a half at 25mV. The membrane was then incubated for one and a half hour at 37C with TBS solution (10mM Tris HCl pH 7.4, 140mM NaCl, 5mM KCl ) containing 0.2% Tween and 3% low fat milk. The membrane was incubated overnight at 4C with either rabbit anti murine P2X7 diluted 1/1000 (Abcys) or murine anti actin diluted to 1/500 (Sigma) in the TBS and milk solution. Incubation with the secondary go at antibody anti rabbit IgG coupled to HRP (Pierce, diluted to 1/1500) in the TBS and milk solution was accomplished during an hour and a half incubation at ambient temperature. Anti mouse polyclonal antibodies (Ac) were purchased from Abcys (Paris, France ) while the anti rabbit antibody was purchased from Sigma (St Quentin Fallavier, France); a list of used antibodies and function of their target is provided in Table 2.5. Table 2.5.: Polyclonal antibodies to probe the Western blots. Antibodies Function of antibody's protein target Rabbit Ac anti mouse P2rx7 The P2rx7 receptor was identified on the surface of several hematopoietic cells such as macrophages, lymphocytes, monocytes and dendritic cells, and possesses a weak sensitiv ity to ATP (Khakh et al. 2001) Rabbit Ac anti mouse actin Actin is a globular protein, a monomeric subunit of microfilaments, and one of the three major components of the cytoskeleton. Polyclonal Antibodies Goat Ac anti rabbit IgG I mmunoglobulin G (IgG) is a
Grignon, 27 coupled to Horseradish Peroxidase (HRP) monomeric immunoglobulin, built of two heavy chains and two light chains. Each IgG has two antigen binding sites. The antibodies on the membrane were visualized by a peroxidase substrate inducing a bioluminescence (reagent ECL, Super Signal West Dura Extended Duration Substrate, Pierce) according to ma nufacturer's instructions. The photons emitted are then captured digitally using a Kodak Image Station 2000MM.
Grignon, 28 Chapter 3: Results and Discussion SLE4 is one of the 11 known loci for lupus susceptibility (Lee et al. 2005) Elliott et al. (2005) have recently suggested that in the New Zealand Black (NZB) mouse model, the P2rx7 a gene locus is implicated in the disease's development. Moreover, SLE (as in many of the autoimmune pathologies) is a disease characterized by chronic inflammation, a characteristic that might directly relate to P2rx7, as this purinergic receptor also has a pro inflammatory activity (Lee et al. 2005) Consequently, in the present study, P2rx7 activity was evaluated here in T and B lymphocytes of MRL lp r/lpr mice. 3.1. Lymphocytic Populations of MRL +/+ and MRL lpr/lpr Mice To briefly review, in autoimmune MRL lpr/lpr mice, a mutation in the Fas gene is responsible for the accumulation in secondary lymphoid organs of abnormal T lymphocytes that escaped the apoptosis mechanism (T DN cells) and that exhibit a CD90 + CD4 CD8 B220 + phenotype. The percentage of these cells rises with the age of the mice due to antigen recognition (Bob et al. 1997). For reasons discussed in the introduction, and according to the findings of Bob et al. (1997), we would expect that in healthy MRL +/+ mice, the population of T DN cells would be largely non existant, but that a very minor population of B220 + T lymphocytes with a CD4 + or CD8 + phenotype would exist, correspondin g to T lymphocytes that have received an apoptosis signal. An experiment was thus run in order to characterize T and B lymphocytic populations in MRL +/+ and MRL lpr/lpr mice (Table 3.1 ), which generally confirmed the above stated predictions since CD90 + B 220 + T lymphocytes accounted for 81% for the total population in MRL lpr/lpr mice.
Grignon, 29 Table 3.1.: T and B lymphocyte distribution. B lymphocytes CD90 B220 + T lymphocytes CD90 + B220 T lymphocytes CD90 + B220 + MRL +/+ mice 29% 62% 1.8% MRL lpr/lpr mice 7.5% 8.7% 81% Distribution of the different T and B lymphocytic sub populations in lymphocytic ganglia of MRL +/+ and MRL lpr/lpr mice, age of four months Because T DN lymphocytic population of MRL lpr/lpr mice escape apoptotic mechanism regulations, t his population was selected as the population of choice in order to study P2rx7 activity. This population was compared to the B220 T lymphocytes as well as to the B220 + B lymphocytes populations originating from MRL +/+ and MRL lpr/lpr mice. 3.2. P2rx7 A ctivity in MRL +/+ and MRL lpr/lpr Mice Lymphocytic Sub populations P2rx7 stimulation through ATP induces a number of activities such as cellular death or cytokine maturation through formation of either a channel or a pore (Budagian et al. 2003). In orde r to analyze P2rx7 activity, three criteria were used: 1) intracellular calcium flux through the channel, 2) pore formation for solutes of size smaller than 900 Da, and 3) cleavage of the CD62L adhesion molecule. 3.2.1. P2rx7 Channel Activity The activity of the P2rx7 channel was evaluated through the use of the Oregon Green 488 probe which emits fluorescence in intracellular calcium presence. At time t=0, the marked cells were stimulated with 500 M of ATP, and intracellular calcium levels were monitored b y flow cytometry. The three T and B lymphocytic populations were analyzed with a dot plot
Grignon, 30 ATP +ATP associating the CD90 and B220 markers (Figure 3.1A). Windows of analysis (Figure 3.1A: R2 B220 + T lymphocytes, R3 B220 T lymphocytes and R5 B220 + B lymphocytes) de limited the subpopulations in which the fluorescence intensity of the Oregon Green probe was measured in function of time, and this done through the displacement of the labeled R6 region on the time axis in Figure 3.1B. For each time t, a value of calcium flux was calculated using Equation 1. The flux of calcium was graphically represented in arbitrary units (AU) as a function of time (Figure 3.1C and 3.1D). Eq. 1: F(t) = Average of fluorescence at time t Average of fluorescence at time t Average of fluorescence at t=0 Average of fluorescence at t=0
Grignon, 31 Figure 3.1.: P2rx7 channel activity stimulated by ATP in T and B lymphocytic sub populations of MRL +/+ and MRL lpr/lpr mice. The cells were sti mulated by 500 M of ATP. A quantity of 10 6 lymphocytes of MRL +/+ and MRL lpr/lpr mice were then incubated with Oregon Green, after which the cells were labeled with anti B220 PE Cy5.5 and anti CD90 APC antibodies. Oregon Green's fluorescence intensity was an alyzed with flow cytometry in each T and B lymphocytic population (n=1). (A) Representative example of an obtained dot plot of flow cytometry trials; the R5 region represents B220 + B lymphocytes, the R2 region represents B220 + T lymphocytes and the R3 re gion represents B220 T lymphocytes. (B) Representative example of a dot plot obtained from the combination of the R2, R3 and R5 gated regions, as Oregon Green's fluorescence is monitored in function of time. The R6 gate is displaced along the time axis t o determine a measure of fluorescence in function of time, and these values are then graphically represented for the MRL +/+ and MRL lpr/lpr mice. The three populations for MRL +/+ mice (C) and MRL lpr/lpr mice (D) were extrapolated from (B) and the calcium fl ux (in arbitrary units) was plotted against time (in minutes).
Grignon, 32 A stimulation of 20 minutes by 500 M of ATP induced an increase of the levels of intracellular calcium in B220 T lymphocytes for both of MRL +/+ and MRL lpr/lpr mice up to almost 0.25 AU (Fi gure 3.1C and 3.1D), which indicates that P2rx7 is functional in this T lymphocyte sub population. However, in the B220 + T lymphocytes and B220 + B lymphocytes populations, the ATP stimulation did not induce significant variation in the intracellular calc ium levels (Figure 3.1C and 3.1D). It is interesting to note that no matter the mouse lineage used, the two cellular sub populations that express the membrane phosphatase B220 do not respond much to ATP stimulation, thus suggesting that P2rx7 channel activ ity is reduced, or perhaps even non existent in these sub populations. 3.2.2. P2rx7 Pore Activity The potential activity of the pore form of P2rx7 was visualized via the incorporation of the fluorescent probe YO PRO in the presence of 500 M of ATP (Bob et al., 1997). The cells were then marked with antibodies anti B220 PE Cy5.5 and anti CD90 APC and analyzed with flow cytometry. The three lymphocytic sub populations were delineated through analysis windows (Figure 3.2A: R5, R2, R3) in which the percentage of YO PRO + cells were determined by a histogram (Figure 3.2B). The percentage of cells forming a pore is represented by the bar graph C and D of Figure 3.2, and was calculated with Equation 2. Eq. 2: % of cells forming a P2rx7 pore = %(YO PRO + cells) +AT P %(YO PRO + cells) ATP
Grignon, 33 Figure 3.2.: P2rx7 pore activity stimulated by ATP in T and B lymphocytic sub populations of MRL +/+ and MRL lpr/lpr mice. Cells were stimulated with 500 M ATP in the presence of the Yo PRO. A quantity of 10 6 lymphocytes from MRL +/ + and MRL lpr/lpr mice were then labeled with anti B220 PE Cy5.5 and anti CD90 APC antibodies. The percentage of YO PRO positive cells was analyzed with flow cytometry (n=10). (A) Representative example of an obtained dot plot of flow cytometry trials; the R 5 region represents B220 + B lymphocytes, the R2 region represents B220 + T lymphocytes and the R3 region represents B220 T lymphocytes. (B) A two parameters histogram was obtained from the combination of the R2, R3 and R5 regions and the positive (fluor escent) cells were gated within a marker (M1) to miminize contributions of cells with background levels of staining. The three positive populations for MRL +/+ mice (C) and MRL lpr/lpr mice (D) were extrapolated from (B) and the percentage of cells forming t he P2rx7 pore was quantified using Eq. 2 in the text. Error bars= +SE
Grignon, 34 The stimulation with 500 M of ATP induced the formation of a pore in B220 T lymphocytes in MRL +/+ as well as MRL lpr/lpr mice. However only 14% of the cells were capable of forming this pore in MRL lpr/lpr mice compared to 40% in MRL +/+ mice (Figure 3.2C and 3.2D). In B220 + T lymphocytes originating from either MRL +/+ or MRL lpr/lpr mice, the percentage of cells forming a pore is reduced by roughly half compared to T lymphocytes t hat do not express B220 (Figure 3.2C: MRL +/+ : B220 T=40% 5 and B220 + =23% 6; in 3.2D MRL lpr/lpr : B220 T=14% 2 and B220 + =6% 1). B lymphocytes have little pore activity (Figure 3.2C and 3.2D). As it was the case with channel activity, expression of th e molecule B220 seems to be associated with low P2rx7 pore activity. 3.2.3. Cleavage of CD62L by P2rx7 As mentioned in Chapter 2, in ATP's presence, P2rx7 activates a transduction cascade of unknown nature leading to the proteolytic cleavage of the adhes ion molecule CD62L (Auger et al. 2003) This cleavage constitutes the focus of our third study criterion for P2rx7's activity. The percentage of T and B lymphocytes from which the CD62L surface molecule was cleaved was determined in MRL +/+ and MRL lpr/lp r mice by flow cytometry using anti CD90 FITC anti CD62L PE and anti B220 PE Cy5.5 as markers in presence of ATP. The three lymphocytic sub populations were delineated by analysis windows (Figure 3.3A: R2, R3, R5) in which the percentage of cells expressing CD62L at their surface is determined by a mono parametric histogram (Figure 3.3B). The percentage of cells from which CD62L is cleaved, represented by bar graphs C and D of Figure 3.3, was calculated using the Equation 3.
Grignon, 35 Eq. 3: % of cells from which CD 62L is cleaved = [(# of cells CD62L + ) +ATP / (# of cells CD62L + ) ATP ] x100 Figure 3.3.: CD62L cleavage activity by P2rx7 stimulated by ATP in T and B lymphocytic sub populations of MRL +/+ and MRL lpr/lpr mice. Cells were stimulated with 500 M ATP. A qua ntity of 10 6 lymphocytes from MRL +/+ and MRL lpr/lpr mice were then labeled with anti CD90 FITC anti CD62L PE and anti B220 PE Cy5.5 antibodies The percentage of CD62L+ cells was analyzed with flow cytometry (n=10). (A) Representative example of an obtain ed dot plot of flow cytometry trials; the R5 region represents B220 + B lymphocytes, the R2 region represents B220 + T lymphocytes and the R3 region represents B220 T lymphocytes. (B) A two parameters histogram was obtained from the combination of the R2 R3 and R5 regions and the positive (fluorescent) cells were gated within a marker (M1) to minimize contributions of cells with background levels of staining. The three positive populations for MRL +/+ mice (C) and MRL lpr/lpr CD62L Cleavage
Grignon, 36 mice (D) were extrapolated fro m (B) and the percentage of cells from which CD62L was cleaved was quantified using Eq. 3 in the text. Error bars= +SE Treatment with ATP stimulated a strong cleavage of CD62L at the surface of B220 T lymphocytes in MRL +/+ mice (Figure 3.3C: 86% 9 of cells have CD62L cleaved) as well as in MRL lpr/lpr (Figure 3.3D: 75% 11 of cells have CD62L cleaved). CD62L cleavage appears to be more efficient in the B220 T lymphocytes population of MRL +/+ mice than MRL lpr/lpr mice. However, only very modest signi ficant cleavage is found in B220 + T lymphocytes and in B lymphocytes in either MRL +/+ or MRL lpr/lpr mice (Figure 3.3C and 3.3D). 3.2.4. Effects of ATP dosage on CD62L Cleavage and Pore Formation In order to assure that the P2rx7 receptor is being activ ated, and not another pore from the P2X family, says as for CD62L cleavage and pore formation in presence of a range of low ATP concentrations from 0 to 200 M were conducted (data not shown). No CD62L cleavage or pore formation was observed with concentrat ions under 200 M ATP. The other members of the P2X family are activated by ATP concentrations in the 0 to 100 M range (Khakh et al. 2001) while P2rx7 requires higher levels, these observations suggest that the previous manipulations did, in fact, target specifically the purinergic receptor P2rx7. The above results indicate that P2rx7 is functional in B220 T lymphocytes of both mice lineages, even if its activity seems reduced in MRL lpr/lpr mice. However, the P2rx7 receptor is largely inactivated in ly mphocytes expressing the membrane phosphatase B220. Among the diverse hypotheses that could explain this inactivity, possible regulation at the transcriptional and/or translational level were first studied.
Grignon, 37 3.3. P2rx7 Quantification of P2rx7 in MRL +/+ and MRL lpr/lpr Mice Lymphocytic Sub populations The transcriptional and translational expression profiles of the P2rx7 gene were established based on extracted RNA or on proteins, respectively from T and B lymphocytic populations. These originated from s econdary lymphoid organs of MRL +/+ and MRL lpr/lpr mice and were purified with the cellular sorter prior to extraction of RNA (using anti CD90 and anti B220 fluorescent antibodies). 3.3.1. Transcriptional analysis of P2rx7 The transcriptional expression of P2rx7 was analyzed by real time RT PCR (SYBR Green method), in T and B lymphocytic sub populations of MRL +/+ MRL lpr/lpr C57BL/6 wildtype, and in C57BL/6 P2rx7 0/0 mice lacking P2rx7. The RNA levels coding for P2rx7 were reported on the basis of the n umber of cells utilized during the real time RT PCR, and compared to those coding for the ubiquinating gene hypoxanthine guanine phosphoribosyl transferase ( HGPRT) as shown in Figure 3.4 (Bob et al., 1997).
Grignon, 38 Figure 3.4.: Transcriptional expression prof ile of P2rx7 in T and B lymphocytic sub populations of MRL +/+ ,MRL lpr/lpr C57BL/6 and C57BL/6 P2rx70/0 mice. A quantity of 1.5x10 7 mouse lymphocytes were labeled with anti CD90 FITC and anti B220 PE antibodies. After the cellular sorting process, the RNAs were extracted from the three cellular populations obtained, cDNAs were synthesized and real time PCR was performed (n=1). Standard curves were used to calculate the amounts of each RNA (P2Rx7 versus HGPRT) on a per cell basis and the P2rx7 values were the n divided by the HGPRT values to determine the relative expression levels. (AU: arbitrary units) In the four lineages of mice studied, the P2rx7 gene was transcribed in all cell types but at variable levels. Figure 3.4 demonstrates that there is no clear relation between P2rx7 activity observed in a lymphocytic population and its transcription levels. In fact, while B220 + T lymphocytes do not have a P2rx7 activity (Figure 3.2 and 3.3) in the MRL +/+ mice, the quantity of the mRNA is roughly equivalent to that in B220 T lymphocytes. On the same note, while B lymphocytes of MRL +/+ mice have no P2rx7
Grignon, 39 activity, the relative messenger level is roughly twice that of B220 T lymphocytes that have a normal activity. Although overall, l evels in MRL lpr/lpr mice ( when calculated relative to HGPRT) are low, the levels of P2rx7 mRNA in B220 + vs. B220 T lymphocytes are comparable. The presence of transcripts in a mice model inactivated for the P2rx7 gene is not surprising because the primers were chosen upstream of t he inactivated exon. 3.3.2. Protein Blotting Levels of the P2rx7 protein were analyzed by a preliminary western blot through the use of a polyclonal rabbit anti mouse P2rx7 serum (data not shown). The T and B lymphocytic sub populations of both the MRL + /+ and MRL lpr/lpr mice lineages were purified by the cellular sorter. The output of the sorting is on the order of 10% (data not shown). Therefore, for a population as minor as the one of B220 + T lymphocytes in the MRL +/+ mice (1.8% of the total populati on), on average, only 15000 cells are obtained (Bob et al. 1997). The western blot results were generally in line with the results obtained with the transcriptional profile expression analysis of the P2rx7 gene. In fact, all populations studied express the P2rx7 protein. The only exception was in the B220 + T lymphocyte population of MRL +/+ mice where the protein quantity may have been too low as even actin was not detected. While these results seem to confirm that no major deficit in the transcriptiona l and/or translational expression of the P2rx7 gene explain the receptor's inactivation in lymphocytic sub populations expressing B220, more replicates of this experiment are needed to confirm the obtained results.
Grignon, 40 3.4. Implication of B220/CD45RABC in P2 rx7 Inactivation The alternative hypothesis to explain a deficiency in function of P2rx7 is the idea that the phosphatase B220 plays a role in the inactivation of P2rx7. In order to evaluate this implication, the expression of B220 was induced in vitro a t the surface of T lymphocytes by using a T lymphocyte polyclonal activator concavalin A (ConA). P2rx7 activity was then evaluated by the measure of CD62L cleavage. Instead of activating a specific clone of T lymphocytes like an antigen would, ConA activat es, in a non specific manner, a pool of T lymphocytes (Budagian et al. 2003). As it is the case as with a specific antigen activation, the T lymphocytes activated by ConA express B220 before entering the process of apoptosis. 3.4.1. Induction of the Expr ession of B220 MRL lpr/lpr mice lymphocytes were put in culture in the presence of 4 g/ml of ConA during three days. Each day, a sample of the culture was taken and the expression of B220 was followed at the surface of the different T lymphocyte sub popul ations by flow cytometry. ConA induced the formation of three populations of CD90 + T lymphocytes having different expression levels of B220 (Figure 3.6A): B220 High T lymphocytes, with a relative fluorescent intensity greater than 2x10 2 which is character istic of a strong B220 expression B220 Medium T lymphocytes, with a relative fluorescent intensity between 1.5x10 1 and 1.5x10 2 which is characteristic of a medium B220 expression B220 Low T lymphocytes, with a relative fluorescent intensity less than 1.5x10 1 which is characteristic of a low to null B220 expression
Grignon, 41 The evolution of the percentage of B220 "high" or "medium" B220 T lymphocytes is represented in Figure 3.6. Figure 3.6.: Induction of B220 expression with ConA on T and B lymphocytes of MRL lpr/ lpr mice. A quantity of 10 8 cells were cultured during three days in a RPMI 1640 media containing 10% FCS and 4 g/ml of ConA. Each day, 2x10 6 cells were labeled with anti CD90 FITC and anti B220 PE Cy5.5 The percentage of each T lymphocyte sub population e xpressing variable levels of B220 was analyzed by flow cytometry (n=6). (A) Representative example of an obtained dot plot of flow cytometry trials; the R5 region represents T B220 High lymphocytes, the R6 region represents T B220 Medium lymphocytes and th e R7 region represents T B220 Low lymphocytes. (B) Graph representing the percentage of T B220 High (red) and T B220 Medium (blue) lymphocytes, 24, 48 and 72 hours after the addition of ConA. The percentage (approximately 5%) of B220 High T lymphocytes d oes not vary significantly under the effects of ConA (Figure 3.6B). Also, since these T lymphocytes are activated cells and in their last steps of apoptosis, this population does not allow the study of the P2rx7 activity. On the other hand, T lymphocytes e xpressing low or moderate levels of B220 have received an apoptosis signal, but since their death is not immediate, P2rx7's activity can be studied in these. Furthermore, the stimulation through ConA allows this population to be amplified since on the firs t day of culture they represent 7% of the sample and 23% on the third day (Figure 3.6B).
Grignon, 42 The relationship between the P2rx7 receptor and the B220 phosphatase was studied through the cleavage of CD62L from samples taken each day from the culture and stimu lated with 500 M of ATP. For the "low" and "medium" B220 T lymphocytic populations, two parameters were simultaneously evaluated: B220's average fluorescence intensity, which allows the determination of B220 levels at the surface of cells, and the percenta ge of cells from which the CD62L molecule was cleaved under the effects of ATP (Figure 3.7B). Figure 3.7.: CD62L cleavage activity by P2rx7 in T and B lymphocytes of MRL lpr/lpr mice stimulated by ATP and ConcA in vitro Each day, 2x10 6 cells were label ed with anti CD90 FITC anti B220 PE Cy5.5 and anti CD62L PE and analyzed by flow cytometry (n=6). (A) Representative example of an obtained dot plot of flow cytometry trials; the R5 region represents T B220 High lymphocytes, the R6 region represents T B220 M edium lymphocytes and the R7 region represents T B220 Low lymphocytes. The red analysis window delineates the populations that were used in (B), and had to be extended to enclose a part of the R7 window, as more cells were needed to perform the analysis ( B) Graph representing the percentage of T B220 Medium lymphocytes (orange) and the percentage of CD62L cleaved cells (purple) 24, 48 and 72 hours after the addition of ConA. In the B220 Medium T lymphocyte population (Figure 3.6A: R6), the average intensi ty of fluorescence for the B220 rises with the number of days of ConA stimulation. As a result, ConA augments the percentage of cells expressing B220 (Figure 3.7B:
Grignon, 43 orange curve). Inversely to this augmentation, the percentage of these cells having a cleave d CD62L surface molecule diminishes. In fact, on the first day of culture, as was reported in the case of the MRL +/+ mice (Figure 3.6B), ATP induced cleavage in 90% of cells from which CD62L was cleaved, while only 60% cleavage was observed on the third d ay of culture. Consequently, the expression and/or augmentation of B220's expression is correlated with an inhibition of CD62L cleavage by P2rx7. 3.4.2. Functional Inhibition of B220 The best known function of B220 is its tyrosine phosphatase activity. T hus, in order to better comprehend B220's relationship with P2rx7, MRL lpr/lpr lymphocytes that strongly expressed B220 were stimulated by ATP in presence of an inhibitor of P2rx7, KN 62, or in presence of phosphatase activity inhibitor of CD45 called CD45 I. The formation of the pore and cleavage of CD62L were studied as in the previous section. KN 62, or 1 [N, O bis (5 Isoquinolinesulfonyl) N methyl L tyrosyl] 4 phenylpiperazine, is an agent commonly used for the inhibition of P2rx7 (Auger et al. 2005). However, this molecule inhibits P2rx7 in a non specific manner, since it is also capable of inhibiting CaM Kinase II (Budagian et al. 2003). CD45I, or N (9,10 Dioxo 9,10 dihydro phenanthren 2 yl0 2, 2 dimethyl propionamide, is an inhibitor of the tyrosin e phosphatase activity specific to the ensemble of CD45 isoforms (Budagian et al. 2003).
Grignon, 44 Figure 3.8.: Functional inhibition of P2rx7 and B220 in vitro in MRL lpr/lpr lymphocytes. A quantity of 10 6 mouse lymphocytes were incubated for 2 hours in presenc e of KN 62 or CD45I. The cells were then incubated for 40 minutes with YO PRO at 37C, for (A) then 30 minutes with 500 M of ATP at 37C, and finally for 15 minutes with anti CD90 APC and anti B220 PE Cy5.5 antibodies at 4 ¡ C; for (B) 30 minutes with 500 M of ATP at 37C, and finally for 15 minutes with anti CD90 FITC anti B220 PE Cy5.5 and anti CD62L PE antibodies at 4 ¡ C. The formation of the pore (A) and CD62L cleavage (B) were analyzed by flow cytometry (n=1). The inhibition of pore formation and of CD62L c leavage by KN 62 supports the experiments with a range of ATP concentrations (data not shown), indicating that it is in fact the activity of the P2rx7 receptor (or at least of CD45 isoform) that is being observed (Figure 3.8A and 3.8B). The treatment wit h CD45I appeared to induced an augmentation of the pore formation and of CD62L cleavage in comparison to the cells that were untreated for both B220 and B220 + T lymphocytes (Figure 3.8B). However, this augmentation is significantly higher in B220 + T lymph ocytes. These results suggest that other potential isoforms of CD45 could be implicated in P2rx7 inactivation. Concerning B220 + B
Grignon, 45 lymphocytes, only the pore activity is augmented in presence of the CD45 inhibitor. More replicates should be performed of the se inhibition assays before making any final determinations, however. Multiple hypotheses explaining the inhibition of P2rx7 in the presence of B220 can be formulated. The first hypothesis is that CD45 would have a steric blocking action that may be dire ct or indirect on P2rx7. The other hypothesis would be the possible functional inhibition of P2rx7 by tyrosine phosphatase activity of the CD45 protein These hypotheses will be further elaborated upon in the next chapter.
Grignon, 46 Chapter 4: Summar y of the Present Research, Future Directions and Perspectives Autoimmune pathologies are multi genetic diseases of uncertain molecular origin. In mice, the MRL background predisposes the organism to autoimmune diseases. The lpr mutation of the Fas recepto r potentializes the aberration and severity of pathologies in this genetic background (Rieux Laucas et al. 1995). Thus, the MRL lpr/lpr mouse model permitted the identification of Fas receptor's ligand (FasL) as a susceptible locus for SLE since one chara cteristic of SLE is the development of a strong inflammatory component (Rieux Laucas et al. 1995). Furthermore, a recent publication suggests that P2rx7 is a locus of susceptibility to SLE; the results seemed to indicate that the lupic mouse, NZB, is a ca rrier of an allele responsible for a lessened response of the P2rx7 receptor to ATP (Lee et al. 2005). Therefore, we further studied the implications of the pro inflammatory receptor in the etiology of this disease. Specifically, we studied the activit y of P2rx7 in T and B lymphocytic sub populations of MRL lpr/lpr and MRL +/+ mice, through the following three criteria: 1) intracellular calcium flux activation by the channel, 2) the formation of the pore and 3) the cleavage of the adhesion protein CD62L The obtained results show that for the three criteria studied, the P2rx7 receptor, stimulated by ATP, remains inactive in lymphocytic populations expressing the particular isoform of the CD45 membrane phosphatase B220. The experiments of CD62L cleavage c onducted in the presence of increasing ATP concentrations, as well as in the presence of an agent commonly used for the inhibition of P2rx7, KN 62, also confirmed that it is likely a defect in P2rx7's activity, and not of another receptor of the P2X family that potential isoforms of CD45 could be implicated
Grignon, 47 in P2rx7 inactivation. Finally, the lack of P2rx7 activation by NAD confirms that the receptor is inactive in lymphocytes expressing the B220 molecule (data not shown). The P2rx7 receptor's inactivatio n in lymphocytes expressing B220 does not appear to be linked to a defect in the transcriptional and/or translational expression of the protein as was demonstrated by the results obtained from the real time RT PCR and western blot. Research investigating t he possibility of a mutation did not evidence any polymorphisms that could explain the differences in activity (Kono et al. 2000). In fact, the P2rx7 sequences of both MRL lpr/lpr and MRL +/+ mice are identical to that in BALB/c mice. In turn, BALB/c lymp hocytes are more sensitive to ATP than the lymphocytes of the C57BL/6 mice that were used as controls in our experiments (Bob, personal communication). The lymphocytic subpopulations with the P2rx7 activity deficit are B lymphocytes that constitutively e xpress B220, and activated T lymphocytes that have received an apoptotic signal. This T lymphocyte population, which as we verified, is very minor in MRL +/+ mice (~2% of T lymphocytes) can represent up to 85% of the total T lymphocyte (T DN cells) in the MRL lpr/lpr mouse. It is, in fact, surprising that the pro inflammatory receptor P2rx7 is inactive in the T DN lymphocytic population, since the accumulation of these cells occurs in parallel to rises in levels of pro inflammatory cytokine such as IL 18, T NF IFN INF # etc (Ferrari et al. 2006). This defect of activity of P2rx7 is thus similar to a "loss of function" of the receptor in secondary lymphoid organs of MRL lpr/lpr mice. These findings pose the possibility of the existence of a link between t he P2rx7 receptor activation state and cytokine production in MRL lpr/lpr mice.
Grignon, 48 The hypothesis that we are currently favoring is the functional implication of B220 tyrosine phosphatase activity in P2rx7 receptor's inactivation. Two methodologies were use d in order to investigate this hypothesis: the first consisted in the induction of B220's expression in MRL +/+ mice lymphocytic populations; and the second consisted in the inhibition of the phosphatase activity of B220 in the MRL lpr/lpr mice lymphocytic populations. The induction of B220 expression through the use of concanavaline A on T lymphocytes led to a decrease in CD62L cleavage activity by P2rx7. These results were supported by the analysis of other study criteria of the receptor (pore formation, calcium flux etc), and seem to be consistent with the idea that B220 plays a role in the inactivity of P2rx7. It is, however, necessary to note that ConA does not activate T lymphocytes by strictly equivalent pathways to the ones triggered by antigen recon naissance (Smart et al. 2003). As a result, we are unable to clarify whether the cellular signaling pathway linking CD45ABC (B220) to P2rx7's inactivation is the direct pathway responsible for T lymphoctes accumulation in MRL lpr/lpr mice, or if the pathw ay activated by ConA is yet another regulation mechanism. Reciprocal experiments to B220 induction were the inhibition of the tyrosine phosphatase CD45 activity by the usage of a functional inhibitor, CD45I. The inhibition of CD45 induced a potentializat ion of CD62L cleavage activity and of the P2rx7 pore formation. However, this inhibitor is not uniquely specific to the phosphatase activity of B220, but also to the phosphatase activities of all CD45 isoforms. Consequently, these results must be confirmed by other P2rx7 criteria of analysis such as specific inhibition of CD45ABC since they may implicate B220 but also other CD45 isoforms as well. Nonetheless, the function of the tyrosine phosphatase site could be, in part, responsible
Grignon, 49 for the blockage of P2 rx7 activity. Recently, it has been demonstrated that cells expressing low levels of the CD45RB isoform have a limited display of phosphatidylserines in the outer leaflet of the plasma membrane under P2rx7 stimulation by ATP. An inhibitory role of CD45RB o n the flip flop mechanism of phosphatidylserines induced by P2rx7 was proposed (Elliot et al. 2005). However, no defined action mechanism has been described for B220's role in P2rx7. Two hypotheses are thus possible: either P2rx7 inhibition by steric blo ckage, or a functional inhibition by the dephosphorylation of the critical tyrosine. The first hypothesis is that CD45 would have a blocking action that may be direct or indirect on P2rx7 through the binding to its active tyrosine site. Such a steric block age theory has been discussed in the case of the soluble function of FasL (Griffith et al. 1995). This form can have a blocking function in apoptosis by entering in competition with the membrane form of FasL for binding to Fas. Bioluminescence resonance e nergy transfer (BRET) and/or Frster resonance energy transfer (FRET) experiments could be performed in order to research possible physical interaction between P2rx7 and B220. The other hypothesis would be the possible direct or indirect action of the ty rosine phosphatase activity of the CD45 protein on P2rx7. Kim et al. (2001) recently characterized a protein complex formed around the P2rx7 protein in Human Embryonic Kidney 293 lineage ( HEK293). The characterization of this complex by mass spectrometry e videnced the presence of PTPP phosphatase at tyrosine 343, the site of dephosphorylation which is responsible for the activity inhibition of P2rx7. Co immunoprecipitation experiments using P2rx7 and CD45 isoforms should be conducted in order to possibly p ull the entire protein complex out of solution and thereby identify
Grignon, 50 unknown members of the complex as should research on the phosphorylation state of tyrosine 343 of P2rx7 which is activated by B220, to further test the idea that the tyrosine phosphatase activity is an active player in P2rx7 inactivation. Flow cytometry usage for the calcium flux measurements through the P2rx7 channel gave promising results. These measures necessitate optimization in order to be able to observe rapid calcium phenomena. In fact, the experiments conducted with the use of the Oregon Green probe shows a significant calcium flux around 10 minutes. The use of a different probe for which the fluorescence is directly proportional to the intracellular calcium concentration along wi th a video microscopy detection would allow the visualization of microscopically localized ionic flux that could occur at an earlier stage. These studies are now occurring under the direction of Dr. JM. Cancela (NBCM, CNRS, Gif sur Yvette). In order to con firm and to simplify the study model, EL4 T lymphocytes (cell line from the C57BL/6N mouse) expressing P2rx7 could be transfected in a stable manner with B220, and HEK293 lymphoid cells with both P2rx7 and B220. In addition, in order to confirm our results in vivo MRL lpr/lpr and MRL +/+ mice could knocked out for either P2rx7 or B220 or both and observed for P2rx7 activity to verify if CD45ABC is the sole player in P2rx7 inactivation As previously mentioned, the active membrane FasL can be cleaved in it s extracellular domain to give a soluble form. A very elevated concentration of a secreted form of FasL was found in MRL lpr/lpr mice serum (Bob, personal communication). On the other hand, it was demonstrated that FasL can be released under a microvesicu lar form by certain tumoral cells by an unknown mechanism (Suzuki et al. 2004). Consequently, the molecular nature of the released FasL (trimeric soluble form or
Grignon, 51 microvesicular form) should be analyzed in MRL lpr/lpr mice serum. Furthermore, Ferrari et al in 1997 have also demonstrated that activated monocytes secrete active IL 1 in microvesicles which necessitates a second activation signal given by the P2rx7 receptor. The next step for Dr. Bob's laboratory will thus be the study of whether, as is the case for IL 1 P2rx7 plays a role in signaling the secretion of FasL. In conclusion, comprehension of the relationship between P2rx7 and B220 could lead to a better understanding of the progression of SLE and may lead to improved treatments and perhaps ul timately the discovery of a cure for the disease.
Grignon, 52 Chapter 5: Appendix Flow cytometry is a powerful technique for the analysis of multiple parameters of individual cells within heterogeneous populations. Flow cytometers are used in a range of applications from immunophenotyping, to ploidy analysis, to cell counting and GFP expression analysis (Herzenberg et al. 2006). The flow cytometer performs this analysis by passing thousands of cells per second through a laser beam and capturing the ligh t that emerges from each cell as it passes through. The data gathered can be analyzed statistically by flow cytometry software to report cellular characteristics such as size, complexity, phenotype and health (Herzenberg et al. 2006). The flow cytometer is composed of the following: the fluidic system which presents samples to the interrogation point and takes away the waste, the lasers which are the light source for scatter and fluorescence, the optics which gather and direct the light, the detectors whi ch receive the light and the electronics and peripheral computer systems which convert the signals from the detectors into digital data and perform the necessary analysis (Figure 5.1) (Herzenberg et al. 2006).
Grignon, 53 Figure 5.1: Diagram of a flow cytometer from http://www.ab direct.com/uploads/signal1.jpg Light is emitted from the 635nm red and 488 blue lasers, passes through a lens and hits the stream of cells coming from the fluidic system. Ref racted light is collected by forward scatter (FSC) and side scatter (SSC) detectors, while fluorescence is collected by a series of mirrors and filters in the photomultiplier tubes (PMT 1 4). The data are then collected electronically and can be graphicall y displayed. The interrogation point is the heart of the system, this is the point at which the laser and the sample intersect and the optics collect the resulting scatter and fluorescence. For accurate data collection it is important that particles or c ells are passed through the laser beam one at the time. Most flow cytometers accomplishes this though hydrodynamic focusing by injecting the sample stream containing the cells into a flowing stream of sheer fluid or saline solution (Tung et al. 2004). The sample stream thus becomes compressed to roughly one cell in diameter (Tung et al. 2004). Flow cytometers
Grignon, 54 can accommodate cells that span roughly three orders of magnitude in size. In most cases, flow cytometers will be detecting cells between 1 and 15 m icrons in diameter, although through the use of specialized systems, it possible to detect particles outside of this range. Laser light is used to detect individual cells through the stream (Roederer et al. 2004). As a cell passes through the laser, it wi ll refract or scatter light at all angles. Forward scatter, or low angle light scatter, is the amount of light that scattered in the forward direction as laser light strikes the cell (Tung et al. 2004). The magnitude of forward scatter is roughly proport ional to the size of the cell, and this data can be used to quantify that parameter. This scattered light is recorded as follows: light is quantified by a detector that converts intensity into voltage (Roederer et al. 2004). In most cytometers, a blocking bar called an obscuration bar is placed in front of the forward detection scatter detector. The obscuration bar prevents any of the intense laser light from reaching the detector (Tung et al. 2004). As a cell crosses the laser, light is scattered around the obscuration bar and is collected by the detector. The scattered light received by the detector is translated into a voltage pulse. Because small cells produce a small amount of forward scatter, and large cells produce a large amount of forward scatter, the magnitude of the voltage pulse recorded for each cell is proportional to the cell size. If a histogram of these data is plotted, smaller cells appear towards the left and larger cells appear toward the right (Herzenberg et al. 2006). A histogram of f orward scatter data is a graphical representation of the size distribution within a population, but such a graph presents only one dimensional data (Tung et al. 2004). Since a cell traveling through a laser beam will scatter light at all angles, light sc attering at larger angles, for example to the side, is caused by granularity and structural
Grignon, 55 complexity inside the cell. This side scattered light is focused through a lens system and is collected through a separate detector usually located 90 degrees from the laser's path (Herzenberg et al. 2006). The signals collected by the side scatter detector can be plotted on one dimensional histogram as in the forward scatter's case. Individually, these one dimensional scatter histograms do not necessarily show the complexity of the cell populations (Roederer et al. 2004). For example what appears to be a single population in the forward scatter histogram is in reality multiple populations that can only be discerned by looking at the data in a second dimension. This is done though the use of two dimensional dot or scatter plots (Tung et al. 2004). The peaks from the forward and side scatter histograms correlate with the colors of the dot plot as seen in Figure 5.2. Figure 5.2: Example of a two dimensional dot plot graph. Peaks from the forward and side scatter one dimensional histograms correspond to colors in the two dimensional dot plot graph. Through the use of two dimensional dot plots, multiple cellular populations can be characterized. Adapted from www.invitrogen.com Forward Scatter Histogram Side Scatter Histogram
Grignon, 56 Another parameter used to obtain information about a cell's structure and function is fluorescence. Fluorescence is the term used to describe the excitation of a fluorophore to a higher energy le vel followed by the return of this fluorophore to its ground state with the emission of light (Herzenberg et al. 2006). The energy in the emitted light depends on the energy level to which the fluorophore is excited, and it has a specific wavelength, and consequently a specific color. In order to study cellular characteristics, fluorescent molecules, such as fluorophore labeled antibodies, are added to the cell sample (Roederer et al. 2004). The antibody binds to a specific molecule on the cell surface or inside the cell, and when laser light of the right wavelength strikes the fluorophore, a fluorescent signal is emitted and detected by the flow cytometer. The fluorescent light emitted from the labeled cells as they pass through the laser then travels thr ough the same path as the side scatter signal, and as the light travels down, it is directed through a series of mirrors and filters, so that particular wavelength ranges are delivered to the appropriate detectors (Roederer et al. 2004). Fluorescence dat a are collected in generally the same way are forward and side scatter data. In a population of labeled cells, some will be brighter than others. As each cell crosses the path of the laser, a fluorescent signal is generated and the fluorescent is then dire cted to the appropriate detector where it is translated into a voltage pulse proportional to the amount of fluorescence emitted (Roederer et al. 2004). All of the voltage collected can then be displayed graphically. The FacsCalibur (Becton Dickinson) is capable of detecting four different fluorochromes permitting the analysis of six parameters counting cellular size and granularity. When several fluorescence detectors are used simultaneously, the signal must
Grignon, 57 be electronically compensated due to the partia l overlapping of the fluorochromes emission spectra (Tung et al. 2004).
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