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Cannabinoids induce immunoglobulin class switching to IgE in B lymphocytes

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Title:
Cannabinoids induce immunoglobulin class switching to IgE in B lymphocytes
Physical Description:
ix, 92 leaves : ill. ; 28 cm.
Language:
English
Creator:
Agudelo, Marisela
Publication Date:

Subjects

Subjects / Keywords:
Cannabinoids -- pharmacology   ( mesh )
Receptor, Cannabinoid, CB2 -- drug effects   ( mesh )
Immunoglobulin Class Switching   ( mesh )
Immunoglobulin E   ( mesh )
B-Lymphocytes   ( mesh )
Flow Cytometry   ( mesh )
Th2 Cells -- immunology   ( mesh )
Mice   ( mesh )
CP55940
CB
IL-4
IgE
TLR4
CSR
Dissertations, Academic -- Molecular Medicine -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Cannabinoid treatment increases Th2 activity and previous reports showed B cells express the highest level of CB₂ mRNA relative to other immune cells suggesting that cannabinoids play a critical role in B cell activation and maturation. To examine the direct effect of cannabinoids on B cell antibody class switching, mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB₁ selective agonist, methanandamide, or the CB₂ selective agonist, JW015. The cultures were then analyzed at different times by flow cytometry for expression of B cell surface markers, such as CD19, CD138, CD40, MHCII, CD23, CD80, CD45R, immunoglobulins produced such as IgM, IgE, IgD, and IgG1, and Toll-like receptors such as TLR 2 and 4. Cells treated with CP55940 showed an increase in surface expression of IgE by day 5 in culture; methanandamide had no effect.CP55940 also induced an increase in secreted IgE in culture supernatants analyzed by ELISA. In addition, CB₂ receptors were increased on B cells following stimulation with IL-4 and anti-CD40 and the class switching effect of CP55940 was attenuated by the CB₂ antagonist, SR144528. We also observed that cannabinoid treatment of B cells modulates cell functions other than antibody class switching such as surface marker and TLR expression. CP55940 caused a significant increase in surface expression of TLR 4, but had no effect on other markers. Additional experiments with cannabinoid receptor selective agonists and antagonists suggested both CB₁ and CB₂ receptors were involved in the TLR effect.Receptor involvement and Gsubfield i coupling was supported by our findings that cannabinoids inhibit intracellular cAMP levels in forskolin stimulated B cells, and increasing intracellular cAMP with forskolin suppressed IgE antibody class switching in activated B cell cultures. These results suggest cannabinoids negatively regulate cAMP in B cells resulting in increased IgE. In conclusion, cannabinoids can directly affect the function of B cells by inducing antibody class switching to IgE and TLR4 expression through mechanisms involving CB₁ and CB₂ receptors suggesting the endocannabinoid system may be an important regulator of humoral immunity and the allergic response.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
Additional Physical Form:
Also available online.
Statement of Responsibility:
by Marisela Agudelo.
General Note:
Includes vita.

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University of South Florida
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Resource Identifier:
aleph - 002067993
oclc - 601624609
usfldc doi - E14-SFE0003014
usfldc handle - e14.3014
System ID:
SFS0027331:00001


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ABSTRACT: Cannabinoid treatment increases Th2 activity and previous reports showed B cells express the highest level of CB mRNA relative to other immune cells suggesting that cannabinoids play a critical role in B cell activation and maturation. To examine the direct effect of cannabinoids on B cell antibody class switching, mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB selective agonist, methanandamide, or the CB selective agonist, JW015. The cultures were then analyzed at different times by flow cytometry for expression of B cell surface markers, such as CD19, CD138, CD40, MHCII, CD23, CD80, CD45R, immunoglobulins produced such as IgM, IgE, IgD, and IgG1, and Toll-like receptors such as TLR 2 and 4. Cells treated with CP55940 showed an increase in surface expression of IgE by day 5 in culture; methanandamide had no effect.CP55940 also induced an increase in secreted IgE in culture supernatants analyzed by ELISA. In addition, CB receptors were increased on B cells following stimulation with IL-4 and anti-CD40 and the class switching effect of CP55940 was attenuated by the CB antagonist, SR144528. We also observed that cannabinoid treatment of B cells modulates cell functions other than antibody class switching such as surface marker and TLR expression. CP55940 caused a significant increase in surface expression of TLR 4, but had no effect on other markers. Additional experiments with cannabinoid receptor selective agonists and antagonists suggested both CB and CB receptors were involved in the TLR effect.Receptor involvement and G[subfield i] coupling was supported by our findings that cannabinoids inhibit intracellular cAMP levels in forskolin stimulated B cells, and increasing intracellular cAMP with forskolin suppressed IgE antibody class switching in activated B cell cultures. These results suggest cannabinoids negatively regulate cAMP in B cells resulting in increased IgE. In conclusion, cannabinoids can directly affect the function of B cells by inducing antibody class switching to IgE and TLR4 expression through mechanisms involving CB and CB receptors suggesting the endocannabinoid system may be an important regulator of humoral immunity and the allergic response.
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Cannabinoids Induce Immunoglobulin Class Sw itching to IgE in B Lymphocytes by Marisela Agudelo A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: T homas W. Klein, Ph.D. Raymond Widen, Ph.D. Shyam Mohapatra, Ph.D. Jun Tan, Ph.D. Date of Approval: May 18 2009 Keywords: CP55940, CB2, IL-4, IgE, TLR4, CSR. Copyright 2009, Marisela Agudelo

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DEDICATION This dissertation is dedicated to my Family for all their love and support throughout my career. Specially to my husba nd, Jerry Robles, for all his patience; my dad, Francisco Agudelo, for all his gui dance; my brother Daniel Agudelo, for his encouragement to see his sister finally graduating; to my mother, Alba Sierra, for all her love and care. In her loving memory; she will always be within us. And last, but not least to my dog, Chico, fo r being my faithful companion in my graduate school quest. Thank you all!

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ACKNOWLEDGEMENTS Special Thanks to…. My mentor, Dr. Thomas Klein: for all his support and guidance. Dr. Friedman: for his contagious pa ssion and charisma for science. Colleagues and friends, for all their tec hnical support and friendship on a daily basis: Cathy Newton Kellie Larsen Ping Jen Chou (Joe) Tracy Sherwood Izabella Perkins Dissertation Committee, for a ll their time and advice: Dr. Raymond Widen Dr. Shyam Mohapatra Dr. Jun Tan Examining Committee Chairperson, for her time and acceptance to serve in my committee: Dr. Gayle Cocita Baldwin

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i TABLE OF CONTENTS LIST OF TABLES ...............................................................................................................v LIST OF FIGURES ........................................................................................................... vi ABSTRACT ..................................................................................................................... viii INTRODUCTION ...............................................................................................................1 Cannabis and Cannabinoids .....................................................................................1 Cannabinoids and Medicine .....................................................................................1 Cannabinoid Receptors ............................................................................................3 Endocannabinoids and Synthetic Cannabinoids ......................................................4 B Lymphocytes ........................................................................................................6 Effects of Cannabinoids on the Immune System .....................................................7 Effects of Cannabinoids on B Lymphocytes ...........................................................7 Cannabinoids and Class Switching ..........................................................................8 Cannabinoids and Toll-like Receptors (TLRs) ........................................................9 Cannabinoid Receptor Mechanisms ........................................................................9 Project Significance ...............................................................................................10 OBJECTIVES .................................................................................................................... 12 MATERIALS AND METHODS .......................................................................................16 Mice .......................................................................................................................16

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ii Cannabinoid Drugs ................................................................................................16 Preparation of Purifi ed B Lymphocytes ................................................................17 B Cell Proliferation Assay .....................................................................................18 Cell Viability ..........................................................................................................18 Cannabinoid Treatment of Pu rified B lymphocytes ..............................................19 Forskolin and Isobutylmethylxanthine (IBMX) Treatment of Purified B Lymphocytes ....................................................................................................19 Immunoglobulins, Cell Surface Markers, and Toll like Receptors Analysis by Flow Cytometry ...............................................................................................20 Intracellular CB2 Receptor Analysis by Flow Cytometry ......................................20 Mass Spectrometry Analysis of CB2 Peptide .........................................................21 Detection of Secreted IgE in Culture Supernatants by ELISA ..............................21 cAMP Detection in Forskolin a nd Cannabinoid Treated B Cells ..........................22 Activation-Induced Cytidine Deaminase RT-PCR ................................................23 Statistical Analysis .................................................................................................24 RESULTS ....................................................................................................................... ...25 Aim 1. To Determine the Extent of Ca nnabinoid Receptor 2 Expression in B Cell Activation .........................................................................................................25 B Cell Number ...........................................................................................25 B Cell Purity and Viability ........................................................................27 B Cell Differentiation into Plasma Cells ...................................................29 B Cell Phenotype .......................................................................................31 CB2 Immunoreactive Protein Expression in B cells ..................................34

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iii Cross-Reactivity of CB2 Antibody .............................................................37 CB2 Specific Immunoreactivity Incr eases in Stimulated B Cells ..............40 Aim 2. To Determine the Effects of Cannabinoids on B Cell Activation and Antibody Class Switching................................................................................42 Effects of Cannabinoids on B cell Proliferation a nd Viability ..................42 Effects of Cannabinoids on Surface Marker Expression ...........................47 Effects of Cannabinoids on Class Switching .............................................51 Aim 3. To Determine the Molecular Mechanisms Involve in Cannabinoid Regulation of B Cell Antibody Class Switching .............................................55 CB2 Antagonist Attenuates CP55940 Effect on Surface IgE Expression ............................................................................................55 CP55940 Effect on IgE is Partially Inhibited in CB2 KO B cells ..............57 CP55940 Inhibits Intracellular cAMP .......................................................59 Forskolin and Isobutylmethylxant hine (IBMX) Inhi bit IgE Surface Expression ............................................................................................61 Forskolin Inhibits CP55940-Induced IgE Surface Expression on B Cells .....................................................................................................63 Cannabinoids Enhance Toll-Like Receptor 4 Surface Expression ............65 Both CB1 and CB2 are Involved in TLR4 Enhancement ...........................67 Forskolin Inhibits CP55940-Indu ced TLR4 Surface Expression on B Cells ..................................................................................................69 Activation-Induced Cytidine D eaminase (AID) Gene Expression Increases after B Cell Activation .........................................................71

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iv DISCUSSION .................................................................................................................... 73 B Cells are Potential Ta rgets of Cannabinoids ......................................................73 CB2 Specific Immunoreactivity Incr eases in Stimulated B Cells ..........................74 Cannabinoids Enhance IL-4-Induced IgE Production ...........................................75 Molecular Mechanisms Involved in the Cannabinoid Effect on IgE .....................77 Gi/o Protein-coupled Mechanisms .............................................................77 Other Possible Mechanisms .......................................................................81 Cannabinoids Induce TLR4 Surface Expression ...................................................81 Possible Mechanism of cAMP Regulation of TLR4 Expression ...........................83 SUMMARY ....................................................................................................................... 85 LIST OF REFERENCES ...................................................................................................87 ABOUT THE AUTHOR ....................................................................................... End Page

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v LIST OF TABLES Table 1. Cannabinoid Receptor Ligands ................................................................... 5

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vi LIST OF FIGURES Figure 1. C57BL/6 WT vs. CB2 KO Cell Number. ...................................................26 Figure 2. B Ce ll Purity and Viability .........................................................................28 Figure 3. B Cell Diffe rentiation into Plasma Cells ....................................................30 Figure 4. B Cell Surface Markers ..............................................................................32 Figure 5. B Cell Surface Markers in WT vs. CB2 KO Mice ......................................33 Figure 6. CB2 Immunoreactive Protein Expression in B Cells. .................................35 Figure 7. CB2 Receptor Expression in the B Cell Lines, 18.81 and K46u. ...............36 Figure 8. CB2 Specific Peptide Blocks CB2 Antibody Binding.................................38 Figure 9. Mass Spectrometry Analysis of CB2 Peptide .............................................39 Figure 10. CB2 Specific Immunoreactivity Increases over Time ................................41 Figure 11. B Cell Prolif eration in response to IL-4. ....................................................44 Figure 12. No Effect of Cannabinoids on B Cell Proliferation. ...................................45 Figure 13. No Effect of Cannabinoids on B Cell Viability ..........................................46 Figure 14. Cannabinoids Have no Eff ect on Surface Expression of CD19, MHCII, and B220 ....................................................................................................49 Figure 15. Effects of Cannabinoi ds on Surface Expression of CD23, CD80, and CD138 ........................................................................................................50 Figure 16. CP55940 Enhances Anti body Class Switching from IgM to IgE ..............53

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vii Figure 17. CP55940 Enhances IgE Secretion ..............................................................54 Figure 18. Treatment with Antagonists Shows CB2 Involvement ...............................56 Figure 19. CP55940 Effect on IgE is Partially Inhibited in CB2 KO B cells ..............58 Figure 20. CP55940 Inhibits Intracellular cAMP in B cells ........................................60 Figure 21. Forskolin and IBMX Inhibit IgE Surface Expression on B Cells ..............62 Figure 22. Forskolin Inhibits CP55940 -Induced IgE Surface Expression on B Cells .64 Figure 23. Cannabinoids Enhance Toll-Like Receptor 4 Surface Expression .............66 Figure 24. TLR4 Enhancement is Mediated by CB1 and CB 2 ....................................68 Figure 25. Forskolin Inhibits CP 55940-Induced TLR4 Surface Expression on B Cells70 Figure 26. AID Expr ession in Mouse B Cells .............................................................72 Figure 27. G Protein-Coupled Mechanisms .................................................................80 Figure 28. Possible Mechanisms Invol ved in Regulating Canna binoid Effects on IgE and TLR4 Surface Expression ...................................................................84

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viii Cannabinoids Induce Immunoglobulin Class Switching to IgE in B Lymphocytes Marisela Agudelo ABSTRACT Cannabinoid treatment increases Th2 activity and previous reports showed B cells express the highest level of CB2 mRNA relative to other imm une cells suggesting that cannabinoids play a critical role in B ce ll activation and maturation. To examine the direct effect of cannabinoids on B cell an tibody class switching, mouse splenic B cells were purified by negative selec tion and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective ca nnabinoid agonist, CP55940, or the CB1 selective agonist, methanandamide, or the CB2 selective agonist, JW015. The cultures were then analyzed at different times by flow cytome try for expression of B cell surface markers, such as CD19, CD138, CD40, MHCII, CD23, CD80, CD45R, immunoglobulins produced such as IgM, IgE, IgD, and IgG1, and Toll-like receptors such as TLR 2 and 4. Cells treated with CP55940 showed an increase in surface expression of IgE by day 5 in culture; methanandamide had no effect. CP55940 also induced an increase in secreted IgE in culture supernatants an alyzed by ELISA. In addition, CB2 receptors were increased on B cells following stimulati on with IL-4 and anti-CD40 and the class

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ix switching effect of CP55 940 was attenuated by the CB2 antagonist, SR144528. We also observed that cannabinoid treatme nt of B cells modulates cell functions other than antibody class switching such as surface marker and TLR expression. CP55940 caused a significant incr ease in surface expression of TLR 4, but had no effect on other markers. Additional experiments w ith cannabinoid receptor selective agonists and antagonists suggested both CB1 and CB2 receptors were involve d in the TLR effect. Receptor involvement and Gi coupling was supported by our findings that cannabinoids inhibit intracellular cAMP levels in forskolin s timulated B cells, and increasing intracellular cAMP with forskolin suppressed IgE antibody class switching in activated B cell cultures. These re sults suggest cannabinoids negativ ely regulate cAMP in B cells resulting in increased IgE. In conclusion, cannabinoids can dir ectly affect the function of B cells by inducing antibody class switc hing to IgE and TLR4 expression through mechanisms involving CB1 and CB2 receptors suggesting the endocannabinoid system may be an important regulator of humora l immunity and the allergic response.

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1 INTRODUCTION Cannabis and Cannabinoids Cannabis is one of the oldest psychotr opic drugs known to humanity (1). It has been used in China since the Neolit hic period, around 4000 BC (41). Even though cannabis has been widely known and used for centuries, it was not until the end of the 19th century that the psychoactive chemicals in marijuana began to be defined and purified. This started with the isolation of cannabinol from a red oil extract of cannabis. It’s chemical structure was elucidated in the early 1930s by R.S. Cahn, and its synthesis first achieved in 1940 in the labo ratories of R. Adams in the U.S.A. and Lord Todd in the U.K (55). Cannabis sativa contains more than 460 known chemicals and over 60 cannabinoid alkaloids (1 ); however, the main psychoactive component is 9tetrahydrocannabinol, commonly known as THC, the structure and purification of which was accomplished in Raphael Mechoul am's laboratory in 1964 (55). Cannabinoids and Medicine Cannabis use is widely known to offer an algesic, appetite stimulant, antiemetic, muscle relaxant, and anticonvulsant benefit (1). According to th e National Institute on

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2 Drug Abuse, since 1970, marijuana has been classified as a Schedule I controlled substance. This means that the drug, at least in its smoked form, has no commonly accepted medical use. However, in spite of the controversies in the U.S, Marinol (Dronabinol), a synthetic version of TH C, has been manufactured by Solvay Pharmaceuticals since the 1980s as an appr oved appetite stimula nt for HIV/AIDS patients and as an antiemetic to alleviate the nausea and vomiting asso ciated with cancer chemotherapy. To date, cannabinoid-based drugs are gaini ng political and public support to be used for the treatment of ch ronic diseases (31). GW Ph armaceuticals has undertaken a major research program in the United Kingdom to develop and market distinct cannabisbased prescription medicines in a range of medical conditions. Their research includes investigations in the relief of pain in mu ltiple sclerosis (MS), spinal cord injury, peripheral nerve injury, central nervous system damage, neur oinvasive cancer, cerebral vascular accident, as well as for the relie f of pain and inflammation in rheumatoid arthritis. Along with these investigations, new and better methods for delivery of these compounds are being studied such as inhala tion of THC aerosols ( 49) and therapeutic skin patch applications (64). Most recent c linical trials have also been conducted with preparations of 9-THC hemisuccinate administered in a rectal suppository, with inhaled cannabis and with cannabis extrac ts administered either in capsules (Cannador) or by a pump-action oromucosal spray (Sativex). Th e main cannabinoid constituents of both Cannador and Sativex are 9-THC and the non-psychoactive plant cannabinoid, cannabidiol (54). Even though the medicinal use of mariju ana and other cannabinoids is gaining

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3 worldwide support, a lot more research needs to be done on cannabinoids side effects and other potential medical uses. Therefore, unde rstanding the effects of these drugs on the immune system is critical. Cannabinoid Receptors It is now recognized that the brain and i mmune system have a cannabinoid system of receptors and ligands called the endocannabi noid system. This system is composed of two receptors, CB1(39) and CB2 (47, 66), and endogenous ligands such as anandamide (8). CB1 receptors are concentrated in the brai n and spinal cord, with some found in peripheral tissue. CB1 was first cloned from a rat br ain cDNA library (39). Human CB1 was first cloned in 1991, and encoded a prot ein with 472 amino acids (4). The mouse CB1 sequence has also been cloned (7), and show ed 99% and 97% identity to rat and human CB1, respectively, at the amino acid level (34). The CB2 receptors are largely present in the periphery, especially in immune cells. CB2 was cloned using polymerase chain reaction (PCR) from a human promyelocyti c cell line (HL60) cDNA library (47). CB2 cDNA was reported to encode a protein of only 360 amino acids and showed only a 44% identity with the human CB1 receptor. The mouse (65) and rat (3) CB2 genes have also been cloned and encode proteins of 347 a nd 410 amino acids, respectively. All these cloning studies suggested that cannabinoid r eceptors (CBRs) were di splayed not only in brain, but also in the immune system. CBRs are members of the seven-transmembrane G protein-coupled recepto r superfamily (25). CB2 is coupled predominantly through Gi/o proteins, i.e., nega tively through the G subunit to adenylyl cyclase and positively

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4 through G to MAP kinase. Additionally, at least for CB1, there is some evidence that shows cannabinoid receptors may signal through Gs (55). Endocannabinoids and Synthetic Cannabinoids It has also been shown that the body naturally contains endogenous compounds. The first endogenous ligand described for ca nnabinoid receptors was anandamide (ANA), an arachidonylethanolamine. Anan damide was isolated from pi g brain (8). A more stable anandamide analogue has also been synt hesized, (R)-(+)-Methanandamide, known as a selective agonist for CB1. Other endocannabinoids have be en isolated from tissues such as 2-arachidonylglycerol, or 2-AG (42), and 2-arachidonylglyceryl et her, or 2-AGE (17). The discovery of these endogenous compounds ha s led to the idea th at the cannabinoid receptors are part of an important physiologi cal control system. This discovery has great implications for the potential of developi ng novel drugs that act at the cannabinoid receptors. Furthermore, research on the structureactivity relationship of THC led to the synthesis of non-classical cannabinoid anal ogues. Bicyclic analogs, such as CP55,940 had been synthesized with affinity higher than THC. CP55,940 is a nonselective, high affinity ligand with potent biological eff ects that are mediated through binding both CB1 and CB2 (43). Several synthetic selective agonists and antagonists of the CB1 and CB2 receptors have already been developed. A number of CB2 selective agonists have been synthesized such as JWH015 (22) and HU 308 (18). Highly selec tive antagonists for CB1 (SR 141716A) (11); and CB2 (SR144528) have also b een synthesized (61).

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5 Table 1. Cannabinoid Receptor Ligands. Summary of the binding affinities (Ki values) and selectivity of cannabinoid receptor ligands used in our research studies (35, 38) Selectivity Compound CB1 Ki (nM) CB2 Ki (nM) nonselective 9THC 53.3 75.3 nonselective CP55,940 1.3 1.3 CB1 selective agonist Methanandamide 20 815 CB2 selective agonist JWH015 CB65 383 1000 13.8 3.3 CB1 selective antagonist SR1 (SR 141716A) 11.8 13,200 CB2 selective antagonist SR2 (SR 144528) 437 0.6

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6 B Lymphocytes B cells are an essential component of th e adaptive immune system and play the major role in the humoral immune response. The principal functions of B cells are to make antibodies against antigens, perform th e role of Antigen Presenting Cells (APCs) and eventually develop into memory B cells after activation by antigen (60). Maturation of B cells begins in the b one marrow of mammals wherei n pre B cells develop into immature and mature B cells expressing surface immunoglobulin (s Ig) receptors for antigen. The mature B cell expresses both IgM and IgD forms of the receptor and these cells migrate to peripheral tissues where they are positioned to sense and respond to antigen (52). Once a B cell encounters its cognate antigen and receives additional signals from a T helper cell and other accessory cells, it can further differentiate into plasma cells or memory B cells (60). Purified B cells cultured in vitro need two major signals to survive and proliferate, e.g. stimulation with anti-CD40 and IL -4. Anti-CD40 (membrane contact signal) stimulates B cell activation and proliferati on, while IL-4 (cytokine signal) enhances proliferation as well as antibody class switchi ng to produce IgE rather than IgM. Recent reports showed that stimulation of primary B cells in vitro with IL4 and anti-CD40 can also induce a significant increase in syndecam-1/CD138 positive cells, driving B cell differentiation into highly an tibody secreting plasma cells (6). CD138 (syndecam 1) is a proteoglycan that recognizes extracellular matrix and growth factors, appears during activation and differentiation of B cells, and is specific for the terminally differentiated B cell or plasma cell (71).

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7 Effects of Cannabinoids on the Immune System There is much evidence that cannabi noids modulate immunity, and cannabinoid receptors and endogenous ligands have been reported to be produced by cells of the immune system and to regulate immune cel l function with immune suppression being a dominant response (32, 33). In addition, canna binoids have been reported to polarize toward Th2 (humoral) immunity in a variety of m odels and at least a po rtion of this effect is due to a modulation of T he lper cytokines produced by de ndritic cells, NK cells, and T cells (37). Effects of Cannabinoids on B Lymphocytes THC has been reported to either suppress (27) or enhance (51) B cell functions such as antibody formation; however, these studies were done using mixed immune cell populations and therefore the direct effect of the drug on isolated B cells was not determined. Other studies have shown that B lymphocytes express an abundance of CB2 message relative to other immune cells (5, 36) and cannabinoids have been shown to increase B cell function rather than suppress it. For example, proliferation of B cells was reported to be increased by cannabinoid agonists (5), CB2 receptors were reported to be increased in B cells following IL-4 treat ment (36), serum IgE levels were nonspecifically increased in 50% of mariju ana smokers (59), and Th2-type antibody responses to Legionella pneumophila were increased in TH C-treated and immune stimulated mice (51). It is believed that CB2 plays a critical role in the activation of B

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8 cells and in human tonsillar B cells, the level of CB2 mRNA was increased after activation with anti-CD40 antibody (5). Message was also increased in mouse splenocytes following activation (36). Cannabinoids and Class Switching This research project focuses on the eff ect of cannabinoids on B cell functions, specifically differentiation and antibody class switching in vitro From basic immunology, it is known that both a membrane contact signal and cy tokine signals are necessary to induce B-cell pr oliferation and differentiati on. Therefore, to maintain isolated B cells in vitro, the cells are stimulated with anti-CD40 and IL-4. Anti-CD40 (membrane contact signal) stimulates B ce ll activation and proliferation, while IL-4 (cytokine signal) enhances pr oliferation as well as antibody class switching to IgE. Th2 cytokines such as IL-4 stimulate B cells to undergo immunoglobulin (Ig) heavy chain class switching and thus induce these cells to produce IgE and IgG1 rather than IgM and IgG2a (69). Because we had observed that cannabinoids increased IgG1 and not IgG2a antibodies (51) and because IL-4 activity is reported to promote the expression of CB2 mRNA in B cells (65) we examined the po ssibility that cannabinoids act on B cells directly to induce Ig cl ass switching through a canna binoid receptor mediated mechanism.

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9 Cannabinoids and Toll-Like Receptors (TLRs) Toll-like receptors are a type of Pattern Recognition Receptor (PRR) that plays a key role in the innate immune system a nd also in the adaptive immune response by stimulating B cells. Most recent data show that TLRs control multiple cell functions and activate signals that are critic ally involved in the initiati on of adaptive immune responses (24). For example, signaling through TLR4 pr omotes B cell maturati on (20), class switch recombination in early mouse B cells is me diated by Toll-like receptors (16), and TLR4 surface expression on human B cells is modulat ed by IL-4 (45). Therefore, TLR4 must play an important role on B cell class switching. Furthermore, there is some evidence that the CB2 agonist, JWH-133, suppresses LPS-induced up-regulation of TLR4 and MyD88 (73), but these expe riments were done in bone marrow derived dendritic cells. Curre ntly, there is no literature examining the effect of cannabinoids on TLR expression in B cells. Therefore, the role of cannabinoids on TLR expression and signaling on B cells still needs to be elucidated. Cannabinoid Receptor Mechanisms As previously described, it is well know n that B cells from mouse and human express CB2 mRNA in abundance (5, 36), but th e role of cannabinoid receptors and mechanisms of action on B cell activation are unclear; theref ore, studies using cannabinoid agonists and antagonists, and m easuring cAMP accumulation in B cells will provide a better understanding of the role of these receptors in B cell proliferation and

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10 class switching. As previously described, cannabinoid r eceptors are G protein-couple receptors and upon binding a cannabinoid ligand such as THC, these receptor s associate with members of the Gi/o family of proteins. The Gi/o is a heterotrimeric complex of 3 proteins composed of the G and G/ subunits. Upon activation by ligand binding, the G protein complex is activated releasing the G subunit that binds to and inhibits adenylyl cyclase. The G / dimer is also released and can activate adenylyl cyclase, phospholipase pathways, and the MAPK pathway (2) Additionally, at least for CB1, there is some evidence cannabinoid recepto rs may also activate and signal through Gs proteins (55). The pathways activated by G protein-coupled receptors are powerful signaling pathways which have been implicat ed in many cellular differentiation events including class switch recombin ation and responses to cyt okines (2) and these pathways have been reported to be activated by s timulation through cannabinoid receptors (21). Other studies show that STAT6 up regulates CB2 message expression and plays an important role in IL-4 -mediated B cell act ivation and differentiation (65). Therefore, measuring accumulation of cAMP and activa tion of these signaling pathways after cannabinoid treatment may lead to a better understanding of the mechanisms involved in the cannabinoid effects observed in B cells. Project Significance To date, the role of cannabinoids in regul ating the immune system is still unclear. Therefore, examining the effects of cannabi noids on B lymphocytes will contribute to our

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11 understanding of the advantages and disadvant ages of the medical use of cannabinoid based drugs. Furthermore, cannabinoid receptor studies will provide the basis for future drug development, making a grea t contribution to the field of immune-mediated diseases. The current study examines a model of B cell class switching and the effects cannabinoids have in the regul ation of B cell function. B cell class switching to IgE is a hallmark event in allergies and asthma, and th e spreading epidemic of these diseases has increased the need for more research in this area.

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12 OBJECTIVES It is well understood from previous litera ture and our own preliminary data that purified B cells treated with IL-4 and anti -CD40 proliferate and undergo class switching from IgM to IgE (19). However, the modula tion by cannabinoids and their receptors in this class switching process has not been explor ed. The objective of this project is to get a better understanding of the role of cannabi noids and receptors on B cell function and antibody formation. It is hypothesized that B cell differentiation and class switching can be modulated by cannabinoids and cannabinoid re ceptors. In order to test this hypothesis, the following aims are proposed. Aim 1. To Determine the Extent of Cannabinoid Receptor 2 Expression in B Cell Activation Of all immune cells tested, B cells express the highest level of CB2 mRNA (36). It has been reported that CB2 receptor expression is up regulated during human tonsillar B cell activation through CD40 (5). Othe r studies indicated that the CB2 transcript was 2 fold higher in abundance in murine B cells than in whole splenocyte preparations (36). In summary, data from previous studies have confirmed that the expression of CB2 mRNA is more abundant in B cells. However, there is no conclusive data on CB2 receptor

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13 expression in B cells at the protein level. Therefore, it is nece ssary to analyze the expression of CB2 receptors at the protein level. Ou r own preliminary studies have shown that CB2 protein is more abundant on activated primary B cells cultured for five days with IL-4 and anti-CD40. Primary unstimulated B cells have very low CB2 levels. Furthermore, murine B cell line experiments have shown that CB2 levels are also abundant on a mature B cell line, K4 6u, and a pre-B cell line, 18.81 (unpublished observation). Therefore, it is proposed that CB2 receptors are highly expressed on activated B cells; and CB2 receptor expression will change as the activity of B cell changes. B cells will be activated with di fferent activators such as anti-CD40 and recombinant IL-4, then CB2 mRNA and protein expression w ill be measured at different time points by RT-PCR and flow cytometry, respectively. It is hypothesized that CB2 receptor expression will change as B cell act ivity, such as proliferation and class switching, changes. Aim 2. To Determine the Effects of Canna binoids on B Cell Activation and Class Switching Previous studies have shown that IL-4 and CD40 ligand are sufficient to induce resting murine B cells to di vide and switch antibody isotypes from IgM and IgD to IgG1 and IgE (19). Anti-CD40, PMA, IFN, and marijuana smoking have been shown to increase CB2 expression while LPS, and TGFhave been shown to suppress CB2 expression (35). Cannabinoids, independent of effects on helper T cells, may modulate antibody production by directly stimulating B cell proliferation a nd class switching in

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14 combination with other B cell activating fact ors. Splenic B cells from C57BL/6 CB2 wildtype and knockout mice, will be cultured for five days with IL-4 and anti-CD-40. Different time points and concentrations of ligands will be used in the presence or absence of cannabinoids such as CP55,940, methanandamide, JW015, and CB65. Furthermore, CB1 and CB2 antagonists will be also used to treat B cells in the presence of cannabinoids. The specific purpose of this aim will to determine the effects of cannabinoids, cannabinoid agonist s, or antagonists in this in vitro class switching process. It is proposed that cannabinoids can induce an increase in IgE antibod ies by direct action on B cells. Isotype switched B cells will be distinguished by analyzing IgE, IgG1, IgG2a IgA, IgD, and IgM positive and negative popul ations by flow cytometry. B cell activation will be assessed by analyzing B cell surf ace markers such as CD45, CD19, MHCII, CD80, CD23, and CD138. B cell proliferati on will be measured with CyQUANT™ NF Cell Proliferation Assay Kit, a nd B cell counts with trypan bl ue. It is hypothesized that cannabinoids can regulate B cell proliferation and class switching in a cannabinoid receptor dependent way. Aim 3. To Determine the Molecular Mechanisms Involved in Cannabinoid Regulation of B Cell Antibody Class Switching Different in vitro studies have shown that CD40L and IL-4 are sufficient to stimulate B cells to divide, class switch, a nd secrete antibodies (15, 19). It appears that anti-CD40 can stimulate B cell division while IL-4 enhances division as well as antibody class switching to IgE. Furthermore, canna binoids may modulate this class switching

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15 effect. Data from previous aims show CP55940 treatment significantly increased IgE surface expression and secretion. But, the m echanism is still unclear. Therefore, we propose to find the possible mechanisms involve in cannabinoid regulation of B cell class switching by examining cannabinoid receptors, G protein-coupled mechanisms, and toll like receptors. From previous literature, we know that cannabinoid receptor s suppress adenylyl cyclase and cAMP through the ac tivation of Gi/o protein co upling (55). It is also well known that certain CB2 agonists are known to inhibit forskolin stimulation of cAMP (14). Forskolin is labdane diterpene produ ced by the Indian coleus plant ( Plectranthus barbatus ). It works by binding and activating the enzyme ade nylyl cyclase, increasing intracellular levels of cAMP. Therefore, measuring cAMP levels after forskolin and cannabinoid treatment can be used to dete rmine if cannabinoids are acting through Gi protein-coupled mechanisms. G protein-couple d mechanisms can mediate class switching as has been observed with other agents that increase cAMP (62). To examine the direct effect of CP55940 on B cell cAMP production B cells will be pre-treated with IL-4 and anti-CD40 for 48 hours, harvested, and stimul ated with Forskolin or CP55940 for 15 min. cAMP will be measured by luminescence. We will also determine if increasing intracellular cAMP by forskolin treatment of B cells can decr ease the expression of IgE. It is hypothesized that ca nnabinoids induce B cell class switching through Gi proteincoupled mechanisms involving CB1 and CB2 receptors; in addition, toll like receptors might also be involved in drug effects on antibody production.

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16 MATERIALS AND METHODS Mice C57BL/6 mice, 6-12 weeks of age, were obtained from our CB2 breeding colony housed and cared for at the University of S outh Florida Health Science Center animal facility, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Canna binoid receptor gene defi cient mice (CB2-/-) on the C57BL/6 background were bred by USF animal f acility staff from stocks provided by Dr. Nancy Buckley (California State Polytechnic U.) Cannabinoid Drugs CP55940 [(-)cis -3-[2-Hydroxy-4-(1,1-dimethyl heptyl) phenyl]-trans-4-(3hydroxypropyl) cyclohexanol], (R)-(+)-M ethanandamide [(R)-N-(2-Hydroxy-1methylethyl)-5Z,8Z,11Z,14Z-icosatetraenam ide], JWH 015 [2-Methyl-1-propyl-1Hindol-3-yl)-1-naphthalenylmethano ne], a nd CB65 [N-Cyclohexyl-7-chloro-1-[2-(4morpholinyl)ethyl]quinoli n-4(1H)-one-3-carboxamide] were obtained from Tocris Bioscience, Ellisville, MO. CP55940 is nonselective with Ki value for both CB1 and CB2 of 1.3 nM. Methanandamide is a CB1 selective agonist with Ki values for CB1 and CB2

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17 of 20 nM and 815 nM, respectively. CB2 selective agonists are JWH 015 with Ki values of 383 and 13.8 nM for CB1 and CB2, respectively (35) and CB65 with Ki values of 1000 nM and 3.3 nM for CB1 and CB2, respectively. CP55940 was first diluted in 95% ethanol to a concentration of 100 mM and then in 10% fetal calf serum-RPMI 1640 medium to a working concentration of 100 uM. Methana ndamide was diluted in ethanol to a concentration of 5 mg/ml a nd then in 10% fetal calf serum-RPMI 1640 medium to a working concentration of 140 uM. JWH 015 wa s first diluted in 95% ethanol to a concentration of 10 mM and then in 10% fetal calf serum-RPMI 1640 medium to a working concentration of 100 uM. CB65 wa s first diluted in 95% ethanol to a concentration of 10 mM and then in 10% fetal calf serum-RPMI 1640 medium to a working concentration of 100 uM. SR141716A (CB1 antagonist) and SR144528 (CB2 antagonist) were obtained from the Research Technology Branch of the National Institute on Drug Abuse (Rockville, MD) and were first diluted in 95% ethanol to 20 mg/ml and then in 10% fetal calf se rum-RPMI 1640 medium to a working concentration of 20 ug/ml. Preparation of Purified B lymphocytes Spleens were collected from C57BL/6 male and female mice at 6 to 12 weeks of age, and processed in a StomacherTM 80 lab blender for single cell suspensions. B cells were enriched by magnetic negative selection (EasySepTM from StemCell Technologies). Isolated B cells were cultured in six-well culture plates (C orning Life sciences, Acton, MA) at a concentration of 5.0 x 105cells/ml in RPMI 1640 medium supplemented with 5

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18 uM 2-mercaptoethanol, 2 mM L-glutamine, 1% antibiotic/antimycotic solution (Sigma, St. Louis, MO), 10% heat inactivated feta l calf serum (Hyclone; Logan, UT), 0.1 10 ng/ml recombinant IL-4 (BD Bioscience PharMingen, San Jose, CA, USA) and 500 ng/ml purified hamster anti-mouse CD40 antibody (BD Bioscience PharMingen). The medium and reagents were replenished ev ery 48 hrs during culture. B cells were harvested after five days in culture and th e purity of the cells was determined by flowcytometry staining using fluorochrome-conj ugated monoclonal antibodies (mAbs) to CD19 and CD45R/B220 (BD Bioscience PharMi ngen). The B cell purity was greater than 98% as analyzed by flow Cytome try on a BD Canto II (BD Bioscience). B Cell Proliferation Assay Unstimulated, stimulated untreated (I L-4 + anti-CD40), and treated (ETOH, CP55940, JW015, or Methanandimide) B cells were cultured for five days and harvested at different time points. Ce llular DNA content was measured following the protocol of the Cyquant NF cell proliferation assay kit (Invitrogen/Molecular Probes, Eugene, OR). Cellular DNA content is proportional to cell num ber. Cell number was calculated based on a standard curve. The extent of prolifer ation was determined by comparing cell counts for samples treated with drugs with untreated controls. Cell Viability To assess cell viability, cells were cultur ed as previously described and stained

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19 with 7-amino-actinomycin-D (7-AAD) (BD Ph armingen). Viable cells (7-AAD negative) and dead cells (7-AAD positive) we re quantified by flow cytometry. Viable cell counts of whole splenocytes and B cells isolated from 6-12 weeks old C57BL/6 wildtype and CB2 knockout mice were obtained by the trypan blue staining method. Cannabinoid Treatment of Purified B lymphocytes B cell cultures were stimulated with IL -4 (0.1 ng/ml) and anti-CD40 (500ng/ml), and different experimental groups were set up. Stimulated B cells were treated with the CB1 receptor agonist, methanandamide (0.5 or 1 uM), the CB2 receptor agonists, JWH 015 or CB65 (0.5 or 1 uM), or the non-sele ctive agonist, CP55940 (0.5 or 1 uM), or equivalent concentrations of th e drug diluent, ethanol (ETOH; vehicle control). In studies with antagonists, B cells were pretreated with either the CB1 antagonist, SR141716A (0.1 uM), or the CB2 antagonist, SR144528 (0.1 uM) for 15 minutes prior to CP55940 treatment. B cells were cultured for five days followed by flow cytometry and ELISA analysis. Forskolin and Isobutylmethylxanthine (IBMX) Treatment of Purified B Lymphocytes B cells were stimulated with IL-4 (0.1-5 ng/ml) and anti-CD40 (500ng/ml) and treated with Forskolin or IBMX (Sigma, St Louis, MO), 10 and 100 uM. B cells were cultured for five days followed by flow cy tometry staining for IgE surface expression.

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20 Immunoglobulins, Cell Surface Markers, and Toll like Recep tors Analysis by Flow Cytometry To evaluate the effects of CP 55940, JW015, CB65 and methanandamide on antibody class switching, B cell surface maker expression, and TLR 2 and 4 expression, treated and untreated cells were harvested after five days in culture. Prior to staining, Fc receptors on B cells were blocked with mouse Fc block, CD16/CD32 (BD Bioscience PharMingen). Cells were stained with fluor ochrome-conjugated mAbs (BD Bioscience PharMingen) to surface immunoglobulins such as fluorescein isothiocyanate (FITC)conjugated anti-IgE and allophycocyanin (A PC)-conjugated anti-IgM. B cell surface marker phenotype was assessed by flow-c ytometry staining using fluorochromeconjugated mAbs to CD19, CD45R(B220), CD138, I-Ab (MHCII), CD80(B7-1), CD23, TLR2, TLR4 (BD Bioscience PharMi ngen). Cells were stained at 4 for 30 minutes, then washed in PBS containing 2% fetal calf seru m and fixed in 1% paraformaldehyde. Cells were acquired on a BD Canto II and analyzed by BD DIVA and Flowjo software. Intracellular CB2 Receptor Analysis by Flow Cytometry To assess the levels of CB2 protein in unstimulated and stimulated B cells, the cells were fluorescent stained before (day 0) and after (day 5) stimulation. Cells were then blocked with mouse Fc block (C D16/CD32) and normal donkey serum (NDS) (Chemicon International, Temecula, CA). In order to stain the intracellular CB2 receptors, the cells were fixed and permeabilized with cytofix/cytoperm solution (BD Bioscience)

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21 to allow the antibody to enter the cell and bind the peptide mapping to the C-terminus of the mouse CB2 receptor. Intracel lular and surface CB2 receptors were stained indirectly in the permeabilized cells with goat anti-mouse CB2 as primary antibody and FITCconjugated donkey anti-goat IgG as sec ondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). According to the manufacturer, the anti-CB2 antibody is raised to a 1520 amino acid peptide within the last 50 ami no acids of the C-terminus. Cells were incubated for 30 minutes on ice with both an tibodies, washed, and resuspended in PBS containing 2% fetal calf serum. Cells were acquired on a BD Calibur or BD Canto II and analyzed by Cellquest or Flowjo software. Mass Spectrometry Analysis of CB2 Peptide Peptide was sent to Moffitt Proteomics Facility for Mass Spectrometry (MS) analysis. After reverse phase peptide extr action, MS and MS/MS sp ectra were acquired from a-cyano-4-hydroxycinnamic acid matrix deposits in positive ion mode using a matrix assisted laser desorption ionizati on mass spectrometer (4700, Applied Biosystems, Framingham, MA). Results showed the peptide is 15 amino acids (a.a) long and is composed of residues 320-334 of the CB2 protein. Detection of Secreted IgE in Culture Supernatants by ELISA To assess IgE secretion, B cells were s timulated with 0.1 ng/ml recombinant IL-4 and 500ng/ml anti-CD40, and treated with either the CB1 receptor agonist,

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22 methanandamide (0.5 and 1 uM), or the nonselective agonist, CP55940 (0.5 and 1 uM), or an equivalent concentration of drug d iluent, ethanol (ETOH, vehicle control). Supernatants from B cell cultures were coll ected at five days post-treatment and the levels of secreted IgE in supernatants were measured using BD OptEIA kit (BD Bioscience PharMingen). Nine ty-six-well, enzyme immuno assay plates (Corning Life Science), were coated with 50 ul of anti-mouse IgE (100ng/ml) in 0.1 M NaHCO3, pH 9.5, overnight at 4C. The wells were blocked with 150 ul of 0.5% bovine serum albumin/0.05% Tween 20 in PBS for 1 hr at 37 C. B cell supernatants from cannabinoid treated and untreated cultures or serial dilutions of Ig E standards were added and incubated for 2 hrs at 37C followed by addi tion of biotinylated detection antibody and streptavidin-horseradish per oxidase (1:250 in 50 ul) for 1 hr at 37. The wells were washed between each addition. Tetramethyl benz idine (Sigma) substrate was added to the wells and allowed to develop at room temperature for 5-10 min. The reaction was stopped with 1 N sulfuric acid. Resu lts were read at 450nm on an Emax microplate reader (Molecular Devices; Menlo Park, CA). The concentrations of secreted IgE were calculated from standard curve samples done for each plate. cAMP Detection in Forskolin and Cannabinoid Treated B Cells B cells were pre-treated with IL-4(1 ng/ml) and anti-CD40 (500ng/ml) for 48 hrs., harvested, counted with trypan blue in a he macytometer, and resuspended in induction buffer at a concentration of 1x 106cells /500 ul. 10 ul cell suspensions were dispensed into each well and stimulated with either 10 ul of Forskolin (100 uM), forskolin plus

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23 CP55940, or CP55940 (0.5 and 1 uM) for 15 min. cAMP concentration was measured following the standard protocol from the cAMP-Glo Assay (Promega San Luis, CA ) Luminescence was read with a 96-well plate -reading luminometer. cAMP (nM) levels were calculated from Standard curve. Activation-Induced Cytidi ne Deaminase RT-PCR B cells were isolated as previously descri bed, cultured for up to five days with IL4 (3ng/ml) and anti-CD40 (500 ng/ml), sample s were harvested every 24 hrs. Total RNA was extracted from B cells by standard t echniques using TriReagent (Sigma) and quantitated using RiboGreen RNA quantitation Kit (Molecula r Probes, Eugene, OR). The extracted RNA was treated with DNase us ing the DNA-free kit from Ambion (Austin, TX). 3 ug of total RNA were used for c DNA synthesis using the avian myeloblastosis virus (AMV) reverse transcriptase (Promega ). 2 ul of RT product were used for conventional PCR, which was carried out with the GoTaq DNA polymerase (Promega). The primer pairs used were as follows: mouse AID forward 5 -AGA TAG TGC CAC CTC CTG CTC ACT GG-3 and reverse 5 -GGC TGA GGT TAG GGT TCC ATC TCA G-3 (product size 209 bp) (30). Parallel PCR r eactions were performed with B-actin to normalized cDNA concentrations. All PCR wa s performed in a MyCycler (BioRad) 95 C 2min, 95C 45 s, 55C 2 mi n. 72C 2 min. for 30 cycles. PCR products were separated on a 2% gel and photographed.

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24 Statistical Analysis The standard error of the mean (SEM) was calculated for all experiments. SEM was estimated by taking the sample standard deviation (SD) and divi ding it by the square root of the sample size (N). SEM = SD/ N where SD reflects the variability of individual data points, and the SEM is the variability of means (67). Data were analyzed by two-tailed Student’s t test. A value of p < 0.05 was considered significant.

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25 RESULTS Aim 1. To Determine the Extent of Cannabinoid Receptor 2 Expression in B Cell Activation B Cell Number Because cannabinoid receptors have been s hown to play an important role in B cell biology, primary B cells were used as the main model to study cannabinoid effects and CB2 receptor expression. CB2 knockout (KO) mice are available and they have a phenotype similar to wildtype (WT) mice, but because we were going to be using spleen cells from both strains, we wanted to exam ine the cellularity of spleens from WT and KO mice. Whole splenocytes were isolated from 6-12 weeks-old WT and KO mice and B cells were enriched by negative selection. W hole splenocytes and B cells were stained with trypan blue, and live cel ls were counted with a hemacy tometer. As shown in figure 1, KO mice had a significantly lower numbers of leukocytes and B cells compared to WT mice, but the overall percentage of B ce lls in both groups was similar (approximately 30%).

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26 Figure 1. C57BL/6 WT vs. CB2 KO Cell Number. Graph shows cell co unts/spleen of whole splenocytes and B cells is olated from C57BL/6 WT and CB2 KO mice. 6-8 weeks old male and female mice were used. Data represent the mean of 10 experiments S.E.M. p <0.05, t test for WT vs. KO cell counts. 0 20 40 60 80 100 120 Whole splenocytesB cells # cells /spleen ( x 106 ) wt ko* 0 20 40 60 80 100 120 Whole splenocytesB cells # cells /spleen ( x 106 ) wt ko* *

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27 B Cell Purity and Viability B cell purity and viability were analyzed at different time points in culture. The results in Figure 2A show the scatter charac teristics of primary unstimulated B cells at day 0 and stimulated B cells after five days in culture with IL-4 and anti-CD40. After enrichment, B cells were >98% CD19 positive, IgE negative, and nearly 100% viable. Viability studies as measured using 7-ami no-actinomycin-D (7-AAD) showed that gated CD19 positive B cells were 98% viable at days 0 and 5. B cell characteristics change and the cells get bigger and more gr anular as they are cultured with IL-4 and anti-CD40. At low concentrations of IL-4 (0.1 ng/ml), sw itching to surface IgE expression occur at a relatively low level (3%). As the concentration of IL-4 is increased (10ng/ml), IgE surface expression increased dramatically (97%) as shown in Figure 2B.

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28 2A 2B SSC SSCIL-4 (0.1 ng/ml)CD19IL-4 (10 ng/ml) IgEFSC FSCDay 0 Day 598% 7AADCD19 97% 3% 0% 98% Figure 2. B Cell Purity and Viability. B cells were enriched by negative selection and purity was assessed by flow cytometry staini ng with anti-CD19-PE. Scatter graphs for days 0 and 5 show the populations we ga ted on, which are CD19+ and nearly 100% viable (Fig. 2A). Y-axis represents CD19+ and x-axis represents 7AAD+. IgE surface expression was assessed by flow cytometry stai ning with anti-IgE-FITC (Fig. 2B). There is no IgE surface expression on unstimulated B ce lls, but after five da ys in culture, IgE positivity increased to 3% or 97% depending on the amounts of added IL-4 (0.1 or 10 ng/ml). Y-axis represents CD19+ and x-axis represents IgE+. 50,000 events were analyzed per sample. Data are represen tative of 10 experiments.

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29 B Cell Differentiation into Plasma Cells Recent reports showed that stimulation of primary B cells with IL4 and anti-CD40 induced a significant increase in syndecam-1/C D138 positive cells associated with B cell differentiation into immunoglobulin secreting plasmablasts (6). CD138 (syndecam 1) is a proteoglycan that recognizes extracellular matrix and growth factors, appears during activation and differentiation of B cells, and is specific for the terminally differentiated plasma cell (71). Figure 3A shows sca tter characteristics of primary unstimulated B cells at day 0 and stimulated B cells after 5 days in culture with recombinant IL-4 and antiCD40. The morphological characteris tics change as the cells ar e cultured with IL-4 and anti-CD40 in that unstimulated cells are sm aller and less granular becoming larger and more granular af ter activation. To further analyze cell changes, surf ace staining of the plasma cell marker, CD138, was performed on B cells from C57BL/6 WT and CB2 KO mice after activation with different concentrations of IL-4 (0.1 – 5 ng/ml) and anti-CD40 (500 ng/ml) for 5 days. Unstimulated cells have relatively low levels of CD138 (4%), however, stimulation with varying concentrations of IL-4 in creased CD138 expression from 16 to 47%. Similar results were observed with B cells from CB2 KO mice (data not shown). This increase in CD138 expression along with th e changing morphology of the cells suggests a transition from B cells to plasma cells in the cultures as expected in response to activation with IL-4 and anti-CD40 (Figure 3B).

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30 Figure 3. B Cell Differentiation into Plasma Cells. Top panel (Fig. 3A) shows IL-4 is a critical CD138 activator, as the concentration of Il-4 is incr eased in culture, there is an increase in CD138 surface expression. Unstimulat ed cells have relatively low levels of CD138 (4%). Stimulated cells (Day 5), at low concentrations of IL-4 (0.1 ng/ml), have approximately 16% CD138 expression. As the co ncentration of IL-4 is increased (1, 3, and 5ng/ml), CD138 surface expression is also increased (47%). Bottom panel (Fig. 3B) shows B cell viability is not affected as the concentration of Il-4 is increased. Data is representative of 3 experiments. 3A 3B 0.1 3 5 1 CD138 Counts 16% 33 44% 47 7AAD Counts 11 6% 13 12 IL-4 (ng/ml) FSC SSC Day 0 Day 5

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31 B Cell Phenotype As previously described, primary cultures of mouse B cells can be induced to proliferate and undergo Ig class switching to IgG1 and IgE under the influence of added anti-CD40 antibody and recombinant IL-4 ( 63). To further characterize changes associated with cell stimulati on, cells were analyzed at diffe rent time points in culture by flow cytometry for the surface expressi on of CD19, MHCII, CD23, CD80, and CD45 (B220). Figure 4 shows there is no diffe rence in surface mark er expression (CD19, MHCII, CD80, and CD45) between day 0 a nd 5 except for CD23, which is decreased after stimulation with IL-4 a nd anti-CD40 for five days. The phenotype of unstimulated WT and CB2 KO B cells was also analyzed at different time points in culture. Figure 5 show s there is no difference in surface marker expression (CD19, CD45, CD80, MHCII, CD23) between WT and CB2 KO unstimulated B cells. Their surface marker phenotypes are relatively the same.

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32 CD19 CD45R (B220) MHCIICD80 (B7-1)CD23 0 20 40 60 80 100% gated Day 0 Day 5 CD19CD45R (B220)MHCIICD80 (B7-1)CD23 0 20 40 60 80 100% gated Figure 4. B Cell Surface Markers. Unstimulated (day 0) and stimulated (day 5) B cells were stained with different surface mark er antibodies raised against CD19, CD45, MHCII, CD80, and CD23, and analyzed by fl ow cytometry. Data are expressed as percentage (%) of gated B cells. Gates are shown on the scatter in figure 2A. 10,000 events were analyzed per sample. Data repres ent the mean of 3 experiments S.E.M. p <0.05, t test for day 0 (unstimulated) vs. day 5 (stimulated) B cells. *

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33 Figure 5. B Cell Surface Markers in WT vs. CB2 KO Mice. Unstimulated (day 0) B cells from WT and CB2 KO mice were stained with different surface marker antibodies raised against CD19, CD45, MHCII, CD80, a nd CD23 and analyzed by flow cytometry. Data are expressed as percentage (%) of gate d B cells. Gates are shown on the scatter in figure 2A. 10,000 events were analyzed per sample. 0 20 40 60 80 100 120 controlCD19CD45MHCIICD23% gated KO WT

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34 CB2 Immunoreactive Protein Expression in B Cells It has been reported that CB2 receptor expression is up regulated during human tonsillar B cell activation through CD40 (5). Other stud ies indicated that the CB2 transcript is 2 fold higher in murine B cells than in whole splenocyte preparations (36). In general, data from previous studies ha ve confirmed that the expression of CB2 mRNA is more abundant in B cells than in other immune cell types, but because CB2 protein expression data in B cells is lacking, we deci ded to develop a method to measure this in purified mouse B cell populations. CB2 receptor expression was measured on primary murine B cells and different B cell lines, such as K46u and 18.81. Primary B cells were activated with anti-CD40 and IL-4, and CB2 expression was measured at the mRNA and immunoreactive protein levels by RT-PCR a nd a newly developed flow cytometry technique. Primary B cell results showed immunoreactiv e protein expression was very low in unstimulated cells, however, the expression in creased to 95% positive after stimulation with IL-4 and anti-CD40 for five days (Figur e 6A). There was also a ten-fold difference in mean fluorescence intensity (MFI) pe r cell between unstimulated (day 0) and stimulated cells at day 5 (Figure 6B). The an tibodies used in this study are specific for a CB2 peptide and therefore these results suggest that IL-4 a nd anti-CD40 can dramatically increase the expression of specific immunor eactive protein on B cells. Furthermore, B cell line studies have shown that CB2 levels are also abundant on two murine cell lines; a pre-B cell line, 18.81, and a mature B cell line, K46u (Figure 7).

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35 6A 6B Figure 6. CB2 Immunoreactive Protein Expression in B Cells. Unstimulated and stimulated B cells were stained intracellularly for CB2. Cells were collected at days 0 and 5, blocked with Fc block and NDS, fixed w ith paraformaldehyde, and permeabilized with saponin prior to staining with primary goat anti-mouse CB2 and secondary FITCconjugated donkey-anti-goat IgG. Dash line hi stogram represents unlabeled cells, solid line histogram represents cells stained with secondary antibody only, and filled histogram represents cells stained with anti-CB2 and secondary antibody. Data are expressed as percentage (%) of total cells Lower panel shows mean fluorescence intensity (MFI) of CB2 positive cells of three independent experiments (Fig. 6B). Data represent the mean of 3 experiments S.E.M. p <0.05 comparing Day 5 vs. Day 0 MFI. CB2 % of Max Day 0 Day 5 95% Da y s in culture 0 5 0 1 00 200 300 400 500 600 MF *

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36 18.8I K46u Figure 7. CB2 Receptor Expression in the B Cell Lines, 18.81 and K46u. Shows intracellular CB2 staining in B cell lines labeled with C-terminus anti-CB2. Indirect labeling with 1 goatanti-mouse CB2 and 2 donkey anti-goat-FITC. Samples were analyzed by flow cytometry. Black histogram represents unlabeled cells, red histogram represents cells stain with secondary antibody only, and green histogram represents cells stained with anti-CB2 and secondary antibody. 10,000 events were analyzed per sample. Data are representative of 3 different experiments. CB2

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37 Cross-Reactivity of CB2 Antibody We were somewhat surprised at the st rong immunostaining results obtained above (up to 95%) with the anti-CB2 peptide antibody obtained from Santa Cruz Biotechnology. To examine this further, we ordered the hom ologous specific peptid e (15-20 aa) used to generate the antibody and set up peptide blocking experiments with anti-CB2 antibody and B cells. The results in Figure 8 show th at the peptide has excellent blocking potential showing that the antibody indeed has strong reactivity for the i mmunizing peptide used by Santa Cruz. However, we wanted to see if this short peptide was common to other proteins and thus could pr oduce cross-reacting antibodies. We sequenced the peptide by mass spectrometry and identified it to ha ve the sequence YLQGLGPEGKEEGPR as shown in Figure 9 This sequence was entered into an NCBI blast search resulting in a list of 197 proteins within the mouse genome that had sequence similarity with this peptide. Some of the protei ns that showed a high percenta ge of similarity included the AT-rich interactive domain-containing prot ein 5A (90%), immunoglobulin superfamily, member 8 (75%), and the opioid growth factor receptor (69%). From these results, it is clear that the Santa Cruz antibody could have sp ecificity to antigenic epitopes represented in other mouse proteins.

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38 Figure 8. CB2 Specific Peptide Blocks CB2 Antibody Binding. Graph shows intracellular CB2 staining in primary B cells labele d with Santa Cruz C-terminus anti-CB2 can be completely blocked when using imm unizing peptide. Samples were analyzed by flow cytometry. 10,000 events were analyzed pe r sample. Data represent the mean of 3 experiments S.E.M. p <0.05 comparing blocking peptide bar with CB2 bar. 0 5 10 15 20 25 30 35 40 unl2controlCB2Blocking Peptide% gated *

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39 800 4000m/z 1.7E+4 0 50 100 Intensity C YLQGLGPEGKEEGP R M -33 -SH Disulfidebound Dimer M+12 CB2 320-334 D YLQGLGPEGKEEGPR? The Moffitt Proteomics Facility is Supported by the Department of Defense through Moffitt’s National Functional Genomics Center and by the National CancerInstitute. Figure 9. Mass Spectrometry Analysis of CB2 Peptide. After reverse phase peptide extraction, MS and MS/MS spectra were acqui red from a-cyano-4-hydroxycinnamic acid matrix deposits in positive ion mode using a matrix assisted lase r desorption ionization mass spectrometer (4700, Applied Biosystems, Framingham, MA). The peptide is 15 a.a. long, based on residues 320-334 of the CB2 protein.

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40 CB2 Specific Immunoreactivity Incr eases in Stimulated B Cells We support a colony (founders from Nanc y Buckley) of mice that have the C terminal two-thirds of the CB2 gene deleted. Therefore, these CB2 KO mice do not produce CB2 protein containing the peptide seque nce identified above by MS, and cells from these mice should therefore not show immunoreactivity with the anti-CB2 antibody from Santa Cruz if the antibody only reacts with CB2 protein and not cross-reacts with other proteins. B cells from KO and wild t ype mice were stimulated with IL-4 and antiCD40 for 5 days and the immunoreactivity de termined by flow cytometry. The results show (Figure 10A) that immunor eactivity increases with stimulation over time in cells from both groups of mice. However, the num ber of % positive cells was greater in wild type mice than in KO mice suggesting a certain degree of CB2 specific binding in the wild type mice. To estimate specific immunor eactivity, we subtract ed the % reactivity observed in KO cells from that observed in w ild type cells. Thes e results (Figure 10B) showed a steady increase in positive cells w ith time following stimulation suggesting that CB2 protein increased in B cells as they progressed through differentiation and maturation associated with IL-4 and anti-CD40 treatment.

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41 A B Figure 10. CB2 Specific Immunoreactivity Increases over Time. Top panel (A) shows CB2 levels in B cells from wildtype and knockout mice stimulated with IL-4(3ng/ml) + anti-CD40(500ng/ml). Bottom panel (B) shows CB2 specific fluorescence on wildtype B cells after subtracting nonspecific fluorescen ce. Cells were collected at different time points, blocked with Fc block and normal donkey serum, fixed and permeabilized with BD Cytofix/Cytoperm solution prior to stai ning with primary goat, anti-mouse CB2 and secondary FITC-conjugated, donkey,anti-goat IgG. 10,000 events were analyzed per sample. Data represent the mean of 3 experiments S.E.M.. 0 10 20 024487296 hrs% specific CB2 protein CB2 0 10 20 024487296 hrs% specific CB2 protein CB2 0 10 20 30 40 50 60 wtkowt kowtkowtkowtko 024487296 hrs% g ated 0 10 20 30 40 50 60 wtkowt kowtkowtkowtko 024487296 hrs% g ated

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42 Aim 2. To Determine the Effects of Cannabinoids on B Cell Activation and Antibody Class Switching Effects of Cannabinoids on B Cell Proliferation and Viability Cannabinoids, independent of effects on helper T cells, may modulate antibody production by directly stimulating B cell prolif eration and class switc hing in combination with other B cell activa ting factors. Splenic B cells, from C57BL/6 CB2 wild type and KO, were cultured for five days with IL-4 a nd anti-CD-40 in the pres ence or absence of cannabinoids such as CP55,940, JWH015, CB65, and methanandamide. B cell proliferation was measured with CyQUANT™ NF Cell Proliferation Assay Kit. Before assessing cannabinoid effects on B cell prolifera tion, the basic system of unstimulated and stimulated (IL-4 + anti-CD 40) B cells was analyzed. Figure 11 shows a three-fold difference in proliferation between B cells treated with a low concentration of IL-4 (0.1 ng/ml) and those treated with a hi gher concentration of IL -4 (10ng/ml) after 120 hours in culture. These results directly show that IL-4 induces B cell proliferation, and the level of B cell proliferation is de pendent on the IL-4 concentration. Unstimulated, stimulated untreated (I L-4 + anti-CD40), and stimulated drug treated (ETOH, CP55940, JW015, or methananda mide) B cells were cultured for five days and harvested at different time po ints. Cellular DNA content was measured following the protocol from the Cyquant NF cell proliferation a ssay kit wherein the cellular DNA content is proportional to the cell number. We used the minimum concentration of IL-4 in order to see either and increase or decrease in drug effect. The

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43 results showed that none of the 3 cannabi noid-based drugs significantly affected the cellular proliferation (Figure 12). The sl ope of the time curves and the maximum proliferation responses were the same among all groups suggesting that cannabinoid treatment had no effect on the B cell proliferation response to IL-4. We also wanted to assess the drug effects on cell viability. For this, cells were cultured as previously described and stained with 7-amino-acti nomycin-D (7-AAD). Viable cells (7-AAD negative) and dead cells (7AAD positive) were quantitated by flow cytometry (Figure 13).

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44 0 1 2 3 4 5 6 7 8 9 10 0 hrs18 hrs120 hrscells x 106 0.1 ng/ml 10ng/ml Figure 11. B Cell Proliferation in response to IL-4. Cells were cultured with 0.1 ( ) or 10 ng/ml ( ) of IL-4 plus anti-CD40 (500ng/ml), then proliferation with CyQuant NF proliferation assay (Invitrogen) was performed at 0, 18 and 1 20 hrs. Data represent the mean of 3 experiments S.E.M. p <0.05 comparing cells treated with 0.1 vs. 10 ng/ml of IL-4. *

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45 0 0.5 1 1.5 2 2.5 3 02448120 hrscell # x 106 Control Meth JW015 CP55940 Figure 12. No Effect of Cannabi noids on B Cell Proliferation. B cells were cultured with IL-4 (0.1ng/ml) plus anti-CD40 (500ng/ml ), and treated with vehicle control, methanandamide or CP55940 (0.5 & 1 uM). Pr oliferation was measured with CyQuant NF proliferation assay at 0, 24, 48 and 120 hrs. y axis represents number of cells (x106) and x axis represents time (hrs). Data repr esent the mean of 3 experiments S.E.M..

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46 0 10 20 30 40 50 60 70 80 90 100 110Control0.510.510.51Viable cells % g ated ETOH%METH uMCP55940 uM CD19 7AAD SSCFSCDay 5 Figure 13. No Effect of Canna binoids on B Cell Viability B cells were cultured with 0.1 ng/ml of Il-4 plus anti-CD40 (500ng/ml) a nd various drugs for 5 days, then viability was measured by flow cytometry with 7AAD. Panel A; upper graph shows scatter and gating, bottom graph shows viability stai ning of CD19+ B cells. Panel B shows percentage of viable cells with and without cannabinoid treatment. Data represent the mean of 3 experiments S.E.M.. A B

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47 Effects of Cannabinoids on Surface Marker Expression To evaluate the effects of CP55940, JW015, and methanandamide on B cell differentiation and surface maker expression, puri fied B cells were stimulated for 5 days with IL-4 and anti-CD40 in ei ther the presence or absence of the various cannabinoids. As a measure of differentiation, the B ce ll surface marker phenotype was assessed by flow-cytometry using fluorochrome-conj ugated mAbs to CD19, CD45R(B220), CD138, I-Ab (MHCII), CD80(B7-1), and CD23. Figure 14 shows that the purified B cells were highly positive for CD19, B220, and MHCII. The expression of these markers did not change following stimulation with IL-4 for 5 days nor did treatment with methanandamide or CP55940 alter the expres sion of these markers. However, the expression of CD23 and CD138 did change CD23 decreased following stimulation while CD138 increased (Figure 15) suggesting th at under the influence of IL-4 and antiCD40 stimulation the cells were differentiating fr om B cells to a more plasma cell type of phenotype. CD23 is a low affinity receptor for IgE on B cells while CD138 is a common plasma cell marker in human and mice. Unstim ulated B cells were about 80% positive for CD23, but after 5 days in culture, expression was significantly decreased in the control cultures as well as in cultures treated with methanandamide, JW015, and CB65. However, CP55940 treatment attenuated this drop in CD23 surface expression. CD138 expression increased in the IL-4 stimulated c ontrol cultures and treatment with any of the drugs had no effect on this increase. CD80 is a molecule found on activated B cells and monocytes, which provides a co-stimulatory signal necessary for T cell act ivation and survival. Our data showed,

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48 unstimulated B cells expressed low levels of CD80 and after cell activation and cannabinoid treatment, the CD80 surface expression increased. However, the changes detected were not significant (Figure 15).

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49 Figure 14. Cannabinoids Have no Effect on Surface Expression of CD19, MHCII, and B220. B cells were stimulated with IL-4 (0.1ng/ml) and anti-CD40(500ng/ml) with or without drug treatment. Cells were treated with 0.5 or 1 uM CP55940 or Methanandamide, harvested at day five, a nd stained with different surface marker antibodies raised against CD19, MHCII, and B220. Data are expressed as percentage (%) of gated B cells. Gates are shown on th e scatter in figure 2A. 10,000 events were analyzed per sample. Data represent th e mean of 3 experiments S.E.M.. ETOH %METH uM 0.0050.011 0.5 Controls Day 0 Day 5 CP55940 uM 0.5 1 0 10 20 30 40 50 60 70 80 90 100% gated (CD19+MHCII+B220+)ETOH %METH uM 0.0050.011 0.5 Controls Day 0 Day 5 CP55940 uM 0.5 1 ETOH %METH uM 0.0050.011 0.5 Controls Day 0 Day 5 CP55940 uM 0.5 1 CP55940 uM 0.5 1 0 10 20 30 40 50 60 70 80 90 100% gated (CD19+MHCII+B220+)

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50 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 ETOH %METH uM 0.0050.01 JW015uM 0.5 1 CP55940 uM 0.5 1 1 0.5 Controls Day 0 Day 5 0.5 1 CB65 uMCD23 CD80 0 10 20 30 40 50 60 70 80CD138 Figure 15. Effects of Cannabinoids on Surface Expression of CD23, CD80, and CD138. B cells were stimulated with IL-4 (0.1ng/ml) and anti-CD40 (500ng/ml) with or without drug treatment. Cells were treated with 0.5 or 1 uM Methanandamide, CB65, JW015, or CP55940, harvested at day five, and stained with different surface marker antibodies raised against CD23, CD80, and CD1 38. Data are expressed as percentage (%) of gated B cells. Gates are shown on th e scatter in figure 2A. 10,000 events were analyzed per sample. p <0.05. Significance was only de tected when comparing stimulated (Day 5) controls with untreated (Day 0) controls. Ther e was no significance on the drug treated samples compared to day 5 a nd vehicle controls. Data represent the mean of 3 experiments S.E.M.. *

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51 Effects of Cannabinoids on Class Switching Cannabinoids had no significant effect on B cell proliferation, viability, or surface marker expression. On the other hand, imm unoglobulin staining data showed there was a decrease in IgM surface expression after the ce lls were stimulated with IL-4 and antiCD40. Furthermore, there was a significan t increased in IgE su rface expression after stimulation and CP55940 treatment, which is a hallmark of antibody class switch recombination. To date, no studies have been reported on the eff ects of cannabinoids on B cell immunoglobulin class switching. Therefor e, in the current study we examined the direct effect of the cannabi noid agonists on class switching from IgM to IgE in B cell cultures. B cells were stimulated in culture with IL-4 (0.1 ng/ml) and anti-CD40 (500ng/ml), then treated with 0.5 or 1 uM CP55940 or methanandamide (Figure 16). Cells from the control group were untreated, and vehicle control gr oup were treated with equivalent amounts of ethanol. All cells were harvested at day 5 and two-color stained with anti-CD19-PE and anti-IgE-FITC. CD19+ IgE+ B cells were gated and analyzed by flow cytometry. Only B cells treat ed with CP55940 (binds to both CB1 and CB2) were shown to have increased expression of su rface IgE by day 5; treatment with 0.5 uM CP55940 showed a two-fold increase in su rface IgE, while treatment with 1uM CP55940 showed a three-fold increase in surfac e IgE. Methanandamide (selective for CB1 only) treated cells had very low leve ls of surface IgE similar to unl abeled and vehicle controls and in initial studies had no effect at concentrations ranging from 0.1 to 10 M (data not shown). These results suggested that CB2 ligation may be more important than CB1 in cannabinoid-induced class switchi ng in B cells stimulated with IL-4 and anti-CD40.

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52 Besides enhancing IgE surface expression, CP55940 was also shown to enhance IgE secretion into culture supernatants. B cells were stimulated in culture with IL-4 and anti-CD40 and in presence of two di fferent concentrations of CP55940 or methanandamide as described above. Untreat ed control cells and vehicle treated cells were also studied. All cells were harvested at day 5, and supernatants were collected for IgE analysis by ELISA (Figure 17). Culture s treated with 0.5 uM CP55940 showed a fifteen-fold increase in IgE le vels (~5000 ng/ml) compared to vehicle control. 1 uM CP55940 treated cultures showed a five-fold increased in IgE levels (~2500 ng/ml) compare to vehicle control. Methanandamide had very little effect on IgE secretion similar to untreated and vehi cle controls. Overall, only cultures treated with CP55940 showed enhanced IgE secretion by day 5.

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53 0 10 20 30 40 50 60 70 80% gated Control ETOH % 0.005 0.01 METH uM 0.5 1 CP55940 uM 0.5 1 * Figure 16. CP55940 Enhances Antibody Cl ass Switching from IgM to IgE. B cells were stimulated with IL-4 (0.1ng/ml) and anti-CD40(500ng/ml) with or without drug treatment for 5 days. B cells were trea ted with 0.5 uM or 1 uM CP55940 or methanandamide, cells were harvested at day five, and surface IgE levels were measured by flow cytometry with anti-IgE-FITC. Y-axis shows percent of gated cells, CD19+IgE+ B cells. Gates are shown on the scatter in figure 2A. 10,000 events were analyzed per sample. Data represent the mean of 8 experiments S.E.M.. p <0.05 comparing CP55940 treated cells with vehicle controls.

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54 7000 0 1000 2000 3000 4000 5000 6000IgE (ng/ml) Control ETOH % 0.005 0.01 METH uM 0.5 1 CP55940 uM 0.5 1 Figure 17. CP55940 Enhances IgE Secretion. B cells were stimulated with IL-4 (0.1ng/ml) and anti-CD40 (500ng/ml) with or w ithout drug treatment for 5 days. B cells were treated with 0.5 or 1 uM CP55940 or meth anandamide, supernatants were collected at day 5, and IgE secretion was measured by ELISA. Y-axis represents secreted IgE levels in ng/ml. Data represent the mean of 3 experiments S.E.M.. p <0.05 comparing CP55940 treated cells with vehicle controls.

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55 Aim 3. To Determine the Molecular Mechanisms Involve in Cannabinoid Regulation of B Cell Antibody Class Switching CB2 Antagonist Attenuates CP55940 Effect on Surface IgE Expression The above data showed the nonselective agonist, CP55940, enhanced IgE surface expression and secretion in B cell cu ltures. On the other hand, the CB1 selective agonist, methanandamide, had no effect on B cell cl ass switching. Therefore, we suspected CB2 receptors were involved in the class switchi ng effect observed when B cells were treated with CP55940. In order to confirm CB2 involvement, B cells were treated with CB1 or CB2 antagonists prior to CP55940 treatment. Cells were stimulated as before with IL-4 and anti-CD40 and then pret reated with either the CB1 antagonist, SR141716A, or the CB2 antagonist, SR144528 (0.1 uM), for 30 min prior to CP55940 (0.5 uM) treatment. Controls were treated with either antagonist only. The data showed CB2 receptor involvement in that pretreatment with the CB2 antagonist attenuated the CP55940 effect to a greater extent than pretreatment with the CB1 antagonist (Figure 18). These data suggest a more robust role of CB2 in the regulation of class switching.

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56 0 10 20 30 40 50 60% gated Control CP55940 SR1 + CP55940 SR2 + CP55940 SR1 SR2 Figure 18. Treatment with Antagonists Shows CB2 Involvement. B cells were treated with CB1 (SR1) or CB2 (SR2) antagonist (0.1 M) pr ior to CP55940 (0.5 M) treatment and cultured for 5 days. Surface IgE levels we re analyzed by flow cytometry with antiIgE-FITC. Y-axis shows percent of gated cells, CD19+IgE+ B cells. Gates are shown on the scatter in figure 2A. 10,000 events were analyzed per sample. Data represent the mean of 3 experiments S.E.M.. p <0.05 comparing SR2 antagonist pretreated cells with CP55940 treated.

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57 CP55940 Effect on IgE is Partially Inhibited in CB2 KO B cells The CP55940 effect was attenuated to a greater extent by the CB2 antagonist (SR2), which suggested that CB2 was mediating the class swit ching effect (Figure 18). Another way to test CB2 involvement is to use B cells from CB2 KO mice, treat with cannabinoids, and compare the re sults with those obtained in w ild type B cells. Spleens were collected from C57BL/6 wild type and CB2 knockout mice and B cells were enriched by negative selecti on, and cultured with IL-4 (0.1 ng/ml) and anti-CD40 (500ng/ml), then treated with CP55940 (0.5 and 1 uM). After five days in culture, the cells were stained for surface IgE and analyzed by flow cytometry. The results showed the CP55940 effect on IgE was partially inhibited in CB2 KO B cells (Figure 19).

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58 0 10 20 30 40 50 60 70 80 ControlETOH % 0.0050.01 METH uM 0.5 1 CP55940 uM 0.5 1 % IgE*0.0050.010.5 10.5 1 % IgE Control ETOH % METH uM CP55940 uM 0 10 20 30 40 50 60 70 80KO WT Figure 19. CP55940 Effect on IgE is Partially Inhibited in CB2 KO B cells. B cells from CB2 KO and wild type mice were stimul ated with IL-4 (0.1ng/ml) + antiCD40 (500ng/ml) with or without drug treatmen t for 5 days. Cells were treated with either 0.5 or 1 uM CP55940 or methanandamide harvested at day five, and surface IgE expression measured by flow cy tometry with anti-IgE-FITC. p < 0.05 for CP55940 treated cells versus the respect ive vehicle (EtOH) controls. Da ta represent the mean of 3 experiments S.E.M..

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59 CP55940 Inhibits Intracellular cAMP Pretreatment with CB2 antagonist attenuated the CP55940 effect on IgE surface expression and CP55940 effect on Ig E was partially inhibited in CB2 KO B cells. These results suggested a role for CB2 which is a G protein-coupled receptor. Cannabinoid receptors are coupled predominantly through Gi/o proteins, negatively through the G subunit to adenylyl cyclas e and positively through G / to MAP kinase (55). It is well known that certain CB2 agonists are known to inhibit fors kolin stimulation of cAMP (14). Therefore, measuring cAMP levels in B cel ls after forskolin and cannabinoid treatment can be used to determine if cannabino ids are acting through G protein-coupled mechanisms. To examine the direct e ffect of CP55940 on B cell cAMP production cells were pre-treated with IL-4 and anti-CD40 fo r 48 hours, harvested, and stimulated with forskolin and/or CP55940 for 15 min. cAMP accumulation was measured by luminescence (Figure 20). The results s howed that CP55940 treatment alone had a negative effect on the intracellular level of cAMP. Furthermore, forskolin increased the cAMP level in B cells and this increase wa s attenuated in a concentration-dependent manner by co-treatment with CP55940. These results suggest that CP55940 can signal through a Gi/o protein-coupled receptor and activate the G i subunit to inhibit adenylyl cyclase.

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60 CP55940 uM 0.5 1 Forskolin 100 uM CP55940 uM 0.5 1 Control -20 -10 0 10 20 30 40 50 606cAMP nM Figure 20. CP55940 Inhibits Intracellular cAMP in B cells. Cells were pre-treated with IL-4(1 ng/ml) and anti-CD40 (500ng/ml) fo r 48 hrs, harvested, and stimulated with Forskolin (100 uM) and/or CP55940 (0.5, 1 uM) for 15 min. cAMP was measured by luminescence. Y-axis represents cAMP (nM) le vels as calculated from Standard curve. Data represent the mean of 3 experiments S.E.M..

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61 Forskolin and Isobutylmethylxanthine (IBMX) Inhibit IgE Surface Expression Cannabinoids have been shown to incr ease surface IgE expression and secretion, and receptor involvement was supported by ca nnabinoid inhibition of cAMP levels in forskolin stimulated B cells. Therefore, we tested if the effect on IgE surface enhancement could be affected by agents known to increase cAMP levels such as forskolin and phosphodiesterase inhibitors (IBMX). Forskolin activates the enzyme adenylyl cyclase and increases the intracellu lar levels of cAMP. However, IBMX is a phosphodiesterase inhibitor and acts by blocking the degradation of cAMP (53). In this experiment, B cells were cultured with a high concentration of IL-4 (5 ng/ml) to get an optimal IgE response, then some cells were treated with forskolin, IBMX, or both (10100 uM) at 0 and 48 hrs, then harvested at day five. The results showed that when B cells were treated with forskolin, IBMX, or bot h; IgE surface expression was significantly inhibited compared to the control group (Figure 21).

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62 Figure 21. Forskolin and IBMX Inhibit IgE Surface Expression on B Cells. B cells were stimulated with IL-4 (5 ng/ml) and anti-CD40 (500ng/ml) and cultured for up to 5 days. Forskolin, IBMX, or both were added at day 0. Cells were harvested at day 5 and stained for surface IgE. Y-axis shows percen t of gated cells, CD19+IgE+ B cells. Gates are shown on the scatter in figure 2A. 10,000 events were analyzed per sample. p < 0.05, t test for forskolin, IBMX, or both treated cells versus the control group (black bar). Data represent the mean of 2 experiments. 0 10 20 30 40 50 60 70 control (IL-4 (5 ng/ml) + anti-CD40) IBMX (100uM)Forskolin (100 uM)IBMX + F% gated (IgE)* * 0 10 20 30 40 50 60 70 control (IL-4 (5 ng/ml) + anti-CD40) IBMX (100uM)Forskolin (100 uM)IBMX + F% gated (IgE)* *

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63 Forskolin Inhibits CP55940-Induced IgE Surface Expression on B Cells Our data showed the nonselective ca nnabinoid agonist, CP55, 940, enhanced surface IgE expression and secretion and inhibited forskolin stimulated cAMP production. This data seemed in conflict with previous reports showing that increasing rather than decreasing cAMP in B cells le d to an increase in IgE production (9). Therefore, we tested if the cannabinoid effect on IgE surface enhancement could be affected by an agent such as forskolin that increases cAMP levels. The results showed that when B cells were treated with bo th forskolin and CP55940, IgE surface expression was significantly inhibited compared to the CP55940 treated controls (Figure 22). Thus, it appears that in the presence of cannabinoids, a decrease in cAMP mediates an increase in IgE production by B cells. These data are at variance with other reports using different GPCR ligands showing an opposite associati on between cAMP and IgE production (40). However, one of the relevant points in th eir study is the finding that p38 mitogenactivated protein kinase (M APK) phosphorylation and activat ion resulted in increased production of IgE. Therefore, p38 MAPK coul d also be an alternative mechanism of action for the cannabinoid effect on IgE production.

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64 Figure 22. Forskolin Inhibits CP55940-Induced IgE Surface Expression on B Cells. B cells were stimulated with IL-4 (0.1 ng/ ml) and anti-CD40 (500ng/ml) and culture for up to 5 days. CP55940, Forskolin, or both were a dded at day 0. Cells were harvested at day 5 and stained for surface IgE. Y-axis shows percent of gated cells, CD19+IgE+ B cells. Gates are shown on the scatter in figure 2A. 10,000 events were analyzed per sample. p < 0.05, t test for CP55940 and forskolin treated cells versus the CP55940 treated controls (black bars). Data repres ent the mean of 3 experiments S.E.M.. 0 10 20 30 40 50 60 70% gated (IgE+)CP55940 uM+ Forskolin uM 10 100 Forskolin uM 10 100 Control (0.1 ng/ml IL4) CP55940 uM 0.5 1 0.5 1 0.5 1 ** 0 10 20 30 40 50 60 70% gated (IgE+)CP55940 uM+ Forskolin uM 10 100 Forskolin uM 10 100 Control (0.1 ng/ml IL4) CP55940 uM 0.5 1 0.5 1 0.5 1 0 10 20 30 40 50 60 70% gated (IgE+)CP55940 uM+ Forskolin uM 10 100 Forskolin uM 10 100 Control (0.1 ng/ml IL4) CP55940 uM 0.5 1 0.5 1 0.5 1 CP55940 uM+ Forskolin uM 10 100 Forskolin uM 10 100 Control (0.1 ng/ml IL4) CP55940 uM 0.5 1 0.5 1 0.5 1 ** *

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65 Cannabinoids Enhance Toll-Like Receptor 4 Surface Expression Toll-like receptors such as TLR4 have b een recently shown to mediate B cell activation and antibody production ( 58). From studies such as these, it is now apparent that TLRs play a role in activating adaptive immunity as well as innate immunity. Because our previous studies showed cannabinoids induced IgE production in B cells cultures, we next examined if cannabinoids induced TLR expression on B cells. B cells were stimulated with IL-4(0.1 ng/ml) and an ti-CD40 (500ng/ml), then treated with 0.5 uM or 1 uM CP55940, CB65, JW015, methanandami de, or equivalent amounts of vehicle control, ethanol. All cells were harvested at day 5 and two-color stained with anti-CD19APC and anti-TLR4-PE (Figure 23A). CD19+ TLR4+ B cells were gated and analyzed by flow cytometry. The results showed th at stimulation with IL-4 and anti-CD40 increased TLR4 expression by 5 days in cu lture, and treatment with CP55940, CB65, and methanandamide caused a significant incr ease in surface expression of TLR4 over control. JW015 had no significant effect on surface TLR4 possibly due to its lower potency for cannabinoid receptor stimulation. We also examined cannabinoid effects on TLR2 expression. The results showed, that unlike TLR4, TLR2 expression was decreased following stimulation with IL-4 and anti-CD40 (F igure 23B), and none of the cannabinoids caused a change fr om control in expression of TLR2. These results suggest that cannabinoids increase selectively TLR4 expression and not TLR2 in B cells stimulated with IL-4 and an ti-CD40. Furthermore, the e ffect was mediated by agonists with potency for both CB1 and CB2 suggesting both receptors are involved in the drug effects.

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66 0 5 10 15 20 25 30 35 40 45 50% gated (TLR4 ) ** * *ETOH %JW015 uM 0.0050.01 METH uM 0.5 1 CP55940 uM 0.5 1 1 0.5 Controls Day 0 Day 5 0.5 1 CB65 uM 0 10 20 30 40 50% gated (TLR 2) ETOH %JW015 uM 0.0050.01 METH uM 0.5 1 CP55940 uM 0.5 1 1 0.5 Controls Day 0 Day 5A B Figure 23. Cannabinoids Enhance TollLike Receptor 4 Surface Expression B cells were stimulated with IL-4 (0.1ng/ml) and anti-CD40(500ng/ml) for 5 days. Then, treated with 0.5 uM or 1 uM CP55940, JW015, CB65, or Methanandamide. Cells were harvested at day five, and surface TLR4 levels were measured by flow cytometry with anti-TLR4PE (Figure 21A). Y-axis shows percent of gated cells, CD19+TLR4+ B cells. B cells were also stained with anti-TLR2-FITC (Fi gure 21B). Y-axis shows percent of gated cells, CD19+TLR2+ B cells. Gates are shown on the scatter in figure 2A. 10,000 events were analyzed per sample. Data represent the mean of 4 experiments S.E.M.. p <0.05, t test for cannabinoid treated sample s compared to vehicle controls.

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67 Both CB1 and CB2 are Involved in TLR4 Enhancement The previous results with selective canna binoid receptor agonists suggested that both receptors were involved in the increased expression of TLR4 on B cells. In order to further test this, B cells were treated with CB1 or CB2 antagonist prior to treatment with the nonselective agonist, CP55940. All B cells were stimulated in culture with IL-4(0.1 ng/ml) and anti-CD40 (500ng/ml). Then, B ce lls were treated with either the CB1 antagonist, SR141716A, or the CB2 antagonist, SR144528, (0.1 uM) 15 min prior to CP55940 (0.5 -1 uM) treatment. Controls were treated with either SR1 or SR2. The results showed that the CP55940-induced increase in TLR4 was attenuated by both antagonists suggesting that bot h receptors were involved in the effect (Figure 24).

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68 0 5 10 15 20 25 30 35 40 45 50c o ntr o l sr1 sr2 cp0.5 cp 1 sr1 + cp0 5 sr1 + c p1 s r 2 + cp 0.5 sr2 + c p1% gated (TLR4) Figure 24. TLR4 Enhancement is Mediated by CB1 and CB 2 B cells were treated with CB1 (SR1) or CB2 (SR2) antagonists (0.1 M) prior to CP55940 (0.5 and 1 M) treatment and cultured for 5 days. Surface TLR4 levels were analyzed by flow cytometry with anti-TLR4-PE. Y-axis shows percent of gated cells, CD19+TLR4+ B cells. Gates are shown on the scatter in figure 2A. 10,000 events were analyzed per sample. Data represent the mean of 3 experiments S.E.M..

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69 Forskolin Inhibits CP55940-Induced TLR4 Surface Expression on B Cells Our data showed cannabinoids enhanced surface IgE and TLR4 expression and inhibited forskolin-stimulated cAMP production. Therefore, we tested if the cannabinoid effect on TLR4 surface enhancement could be aff ected by an agent such as forskolin that increases cAMP levels as has been shown above in the case of IgE. Indeed, when B cells were treated with both forskolin and CP55940, TLR4 surface expression was significantly inhibited compared to the CP55940 treated controls (Figure 25). Thus, it appears that in the presence of cannabinoids, a decrease in cAMP mediates an increase in TLR4 production by B cells, similar to the results observed with IgE.

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70 0 10 20 30 40 50 60 ) % gated (TLR4 +)CP55940 uM+ Forskolin uM 10 100 Forskolin uM 10 100 Control (0.1 ng/ml IL4) CP55940 uM 0.5 1 0.5 1 0.5 1 * * Figure 25. Forskolin Inhibits CP55940-indu ced TLR4 Surface Expression on B Cells. B cells were stimulated with IL-4 (0.1 ng/ml) and anti-CD40 (500ng/ml) and culture for up to 5 days. CP55940, Forskolin, or both were added at day 0. Cells were harvested at day 5 and stained for surface TLR4. Y-axis shows percent of gated cells, CD19+TLR4+ B cells. Gates are shown on the s catter in figure 2A. 10,000 events were analyzed per sample. p < 0.05, t test for CP55940 and forskolin treated cells versus the CP55940 treated controls (black bars).

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71 Activation-Induced Cytidine Deaminase (AID ) Gene Expression Increases after B Cell Activation It is well known that CS R requires at least two signals The first is delivered by cytokines, such as IL-4, that induced germline (GL) transcription, and the second signal can be provided by the CD40L. The most important gene induced by these molecules is activation-induced cytidine deaminase (AID)( 30). Our data above showed IL-4 and antiCD40 induced CB2 receptor activation and canna binoids increased IgE surface expression and secretion. However, the genes involved in this system have not been investigated. Since there is plenty of evid ence that activation-induced cytidine deaminase (AID) is required for class switch recombin ation (CSR)(48), we decided to examine AID expression after B cell activation with IL-4 (3ng/ml) and anti-CD40 (500 ng/ml). We cultured the cells for up to five days and ha rvested samples every 24 hrs, performed RNA extraction and RT-PCR. Our results show an increased in AID mRNA after 48hrs in culture as previously reported (30). Although, the AID expression is in creased in the IL-4 and anti-CD40 system, the expression of this gene after cannabinoid treatment is yet to be investigated.

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72 C 0 24 48 72 96 AID -actin hrs Figure 26. AID Expression in Mouse B Cells. Unstimulated (0 hr) and IL-4 (3ng/ml) and anti-CD40 (500 ng/ml)-stimulated cells were harvested at differe nt time points (2496 hrs). Total RNA was extracted and AID transcripts were measured by RT-PCR. Bottom panel shows the -actin controls.

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73 DISCUSSION B Cells are Potential Targets of Cannabinoids Several in vivo and in vitro studies have shown that marijuana, synthetic, and endogenous cannabinoid compounds are immune modulators. They have been shown to modulate immune cells including B and T ly mphocytes, macrophages, dendritic cells, and natural killer cells (32, 33). Cannabinoids su ch as THC have been reported to either suppress (27) or enhance (51) murine B cell functions such as antibody formation. In humans, synthetic cannabinoids such as CP 55940 have been reported to enhance B cell proliferation (5). However, most of th ese studies were done on mixed immune cell populations, therefore, the direct effect of cannabinoids on is olated B cells could not be determined. Our research study is the first to examine cannabinoid effects on purified B cell populations in vitro The main findings of our proj ect are the observations that CB2 immunoreactivity increased over time in stimul ated B cells, cannabinoids enhanced IL-4induced IgE production, cannabinoids enhan ced TLR4 surface expression, and based on our findings we will discuss the possible mech anisms involved in the cannabinoid effect on B cell class switching and TLR4 modulation.

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74 CB2 Specific Immunoreactivity Increases in Stimulated B Cells CB2 receptors are expressed more abundan tly in the immune system than CB1; however, the role of these rece ptors is still not clear in bo th the normal functioning of the immune system and in the immunomodulati on effect of cannabinoid-based drugs (32, 33). The role of these receptors in B cell bi ology has been of particular interest because B cells express the highest level of CB2 mRNA among immune cells (12), have been shown to be activated by cannabinoids (5), and CB2 message is increased in B cells by IL-4, the potent B cell activ ating cytokine (65). Although, several previous studies reported that IL-4 (65) or anti-CD40 stimulated (5, 36) B cells increased CB2 mRNA; our results are the first to show that B cells s timulated with both agen ts, IL-4 and anti-CD40, increased the expression of CB2 immunoreactive protein as measured by flow cytometry (Figure 6). Surprisingly, the CB2 commercially available anti body used detected very high protein levels (>95%), therefore, we ex amined the antibody specificity further by ordering the homologous specific peptide (1520 aa) used to generate the antibody and setting up peptide blocking experiments with anti-CB2 antibody and B cells. As expected, the peptide had excel lent blocking potential show ing that the antibody indeed has strong reactivity for the immunizing peptid e used by Santa Cruz (Figure 8). However, we wanted to see if this short peptide wa s common to other proteins and thus could induce cross-reacting antibodies. We sequen ced the peptide by mass spectrometry and identified it to have the sequence YL QGLGPEGKEEGPR as shown in Figure 9 This sequence was entered into an NCBI blast search resulting in a list of 197 proteins within

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75 the mouse genome that had sequence similarity w ith this peptide. From these results, it is clear that the Santa Cruz antibody could ha ve cross-reactivity to antigenic epitopes represented in other mouse proteins. Furt hermore, results from WT and KO B cells showed that immunoreactivity increases with stimulation over time in cells from both groups of mice (Figure 10A). However, th e number of % positive cells was greater in WT than in KO mice suggesting a certain degree of CB2 specific binding in the WT mice. Therefore, the “specific” immunoreactivity was assessed by subtrac ting the % reactivity observed in KO cells from that observed in WT cells (Figure 10B). Although this specific immunoreactivity conversion makes the perhaps invalid assumption that some of WT reactivity is CB2 specific, the conversion does suppor t a few previous reports in other systems that B cell activation increases CB2 expression. In conclusion, these results s uggest a steady increase in CB2 positive cells with time following stimulation, suggesting that CB2 protein increased in B cells as they progressed through differentiati on and maturation associated with IL-4 and anti-CD40 treatment. Cannabinoids Enhance IL-4-Induced IgE Production We are interested in cannabinoid regu lation of IgE production because of the importance of this immunoglobulin in huma n disease. Cross-linking of IgE bound to Fc receptors on mast cells and basophils is responsible for Type I hypersensitivity (62). Some of the manifestations of IgE-mediated hypersensitivity reactions include hay fever,

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76 asthma, hives, food allergies, and eczema (60) Therefore, examining agents capable of regulating IgE production is critical fo r understanding disease pathogenesis and developing new effective therapies. From the previous literature, it is well unde rstood, that primary cultures of mouse B cells can be induced to pr oliferate and undergo Ig clas s switching to IgG1 and IgE under the influence of added an ti-CD40 antibody and recombinant IL-4 (63). The IL-4 is the main stimulus of class switching because it induces the transcri ption of all germline genes as well as the germline gene leading to production of IgE (70). Our results show that the degree of IgE class sw itching is dependent on the con centration of IL-4 added to the cultures and not on the concentration of anti-CD40 confirming the important role of this cytokine in class switching in highly pur ified B cell populations. It is known that B cells in response to IL-4 and anti-CD40 undergo class switch recombination (CSR) resulting in the expression of the sequence in the C region of the H chain (70). We are the first to examine CP55940 effects on IL-4 and anti-CD40stimulated B cells and the results show that this non-sele ctive cannabinoid agonist enhances the class switching event so that by 5 days in culture the drug significantly increased IgE surface expression and secretion (Figures 16 a nd 17). CP55940 binds equally to both CB1 and CB2; interestingly, results with the CB1 selective agonist, methanandamide, showed no augmentation of class switching. This fi nding coupled with our data showing the CP55940 effect was attenuated to a greater extent by the CB2 antagonist suggests that CB2 has a more prominent role in mediating the class switching effect.

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77 Molecular Mechanisms Involved in the Cannabinoid Effect on IgE Gi/o Protein-Coupled Mechanisms Cannabinoid receptors are G protein-coupled receptors (GPCR) and therefore act through G-coupled mechanisms. CB2 is coupled predominantly through Gi/o proteins, i.e., negatively through the G subunit to adenylyl cycl ase and positively through G to MAP kinase (55). In our results, receptor involvement was supported by a concentration dependent inhibition of cAMP levels by canna binoid treatment in forskolin stimulated B cells (Figure 20). The second messenger cAMP is a modul ator of cellular maturation and differentiation possessing both inhibitory and st imulatory properties. Furthermore, agents that elevate intracellular cAMP levels have been shown to have immunosuppressive and anti-inflammatory properties (28) Cyclic nucleotides appear to play a significant role in the modulation of immediate hypersensitivity reactions, although their exact function is not well understood, it is known th at substances which alter cAMP levels significantly alter allergic symptoms. Thus substances that increase intracellular cAMP seem to relieve allergic symptoms and are used ther apeutically, and agents which decrease cAMP have been shown to aggravate these allergic conditions(13). However, the mode of action and the mechanism of these effects still need to be elucidated. Our data showed that CP55940 enhanced surface IgE expression and secretion and inhibited forskolin stimulated cAMP pr oduction. These data seem ed in conflict with previous reports that increasing rather than decreasing cAMP in B cells led to an increase

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78 in IgE production (10, 62). However, th e studies showing an opposite association between cAMP and IgE production were done using GPCR ligands (13, 40) other than cannabinoids and our results demonstrate for the first time that ca nnabinoid ligands can both decrease cAMP and increase IgE in IL-4 and anti-CD40 stimulated B cells suggesting a more complex role of cyclic nucleotides and other GPCR mechanisms in IgE expression and secretion. To pursue this fu rther, we tested if the cannabinoid effect on IgE surface enhancement could be affected by agents, such as forskolin and phosphodiesterase inhibitors (IBMX), known to increase intracellu lar cAMP levels. Indeed, when B cells were treated with fors kolin and IBMX, or a combination of both, the IL-4 induced IgE surface expression was significantly inhibited compared to the control group (Figure 21). Furthermore, forskolin trea tment also inhibited the cannabinoid effect on IgE surface expression compared to the CP55940 treated controls (Figure 22). These results s uggest that the CP55940 agonist is acting negatively through the G subunit to lower cAMP resulting in the activation of downstream signaling pathways that increase IgE surface expression. It is possible that cAMP and canna binoids are acting through mechanisms involving Ca+2 mobilization. For example, cAMP elevating agents suppressed IL-5 synthesis by down-regulating the Ca2mobilization pathway (28). Calcium mobilization plays an important role in B ce ll activation (72) in that cell activation leads to stimulation of phospholipase C (PLC) with hydrolysis of phosphotidylinosito l 4,5-bisphosphate (PIP2), generating inositol triphosphate (IP3 ) and diacylglycerol (DAG) (29). The IP3 generated mobilizes calcium by binding to the IP3 receptor causing an increase in intracellular free calcium concentration; the DAG generated causes the activation of

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79 protein kinase C (PKC), an enzyme that ca talyzes the phosphoryla tion of several B cell proteins. Elevating cAMP may disrupt Ca+2 mobilization because it has been reported the IP3 receptor regulating Ca+2 stores is inactivated by cA MP-induced protein kinase A (PKA) phosphorylation leading to a reduction in the release of Ca 2+ (29). Besides mechanisms involving cAMP a nd PKA, it is also possible that cannabinoids are working thr ough the modulation of other kinases such as PKC and MAP kinases (Figure 27). For example, evid ence supports the role of PKC in antibody class switching in that PKC KO mice we re not able to class switch to IgG2a/2b antibodies (56). Furthermore, studies have shown in CHO-transfected cells expressing CB2 that cannabinoid agonists activated MAP kina se and this appeared to be dependent on protein kinase C and indepe ndent of inhibiting adenylyl cyclase (46). Cannabinoids, in addition to working through the G subunit and inhibiting adenyl yl cyclase, also work through the G subunit inducing signaling pathways involving MAP kinase, Src, IP3K, and PLC (Figure 27) (2). In this regard, it has been shown that stimulation of CB1 and CB2 receptors leads to phosphorylation a nd activation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK and Jun N-te rminal kinase (JNK) (21). Previous data from our own laboratory support the role of p38 kinase in the cannabinoid-induced modulation of dendritic cell f unction (unpublished). Therefore, examining p38 MAPK, PKA, and CREB are crucial steps for fu ture directions of this project.

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80 GDP GDP GTP G i/o G s Signal Transduction Signal Transduction (+) Adenylatecyclase MAPK IP3K PLC (-) Adenylate cyclase PKC IP3 DAG (+) Adenylate cyclase PKA GTP GDP Ligand Figure 27. G Protein-Coupled Mechanisms. This is a schematic representation of ligand-mediated activation of a G-protein-coupl ed receptor, which results in dissociation of the heterotrimeric G-protein complex a nd activation of downstr eam signaling events. This diagram shows the ability of G family members to alter adenylate cyclase (AC) activity. Gi activation inhibits AC activity, while the Gs activation results in the stimulation of AC. The dimer, can also activate specif ic signaling pathways as well, including AC activity, PLC and MAPK. Adapted from reference (2).

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81 Other Possible Mechanisms We know from previous liter ature and our own data, that B cells in response to IL-4 and anti-CD40 undergo class switch recomb ination (CSR) resulti ng in expression of the H chain of IgE (70). The molecular mechanisms for CSR involve non-homologous recombination in the switching sites of the H chain genes as well as the up-regulation of the enzyme, activation-induced cytidine deamin ase (AID). There is evidence that IL-4, through mechanisms involving STAT6 and the cAMP response element binding (CREB), induces AID expression leading to IgA isotype switching (30). Since IL-4 is involved in CSR to both IgA and IgE, there may be also the possibility that cannabinoids are working through mechanisms involving STAT6, resultin g in IL-4-induced AID expression and isotype switching. We have data showing an increased in AID message after 48hrs of B cell stimulation with IL-4 and anti-CD 40 (Figure 26).Although, AID expression is increased in the IL-4 and anti -CD40 stimulated B cells, the ex pression of this gene after cannabinoid treatment is ye t to be investigated. Cannabinoids Induce TLR4 Surface Expression Besides increasing the pr oduction of IgE in B cells, we also examined cannabinoid effects on B cell maturation and activation. The drug effects on the expression of activation markers and receptors on stimulated B cells were measured by flow cytometry. We found that the e xpression of CD19, MHC class II, B220, CD23, CD80, and CD138 were unaffected by cannabinoid treatment over 5 days in culture. We

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82 also examined the expression of TLRs on activ ated B cells in the presence of various cannabinoids. TLRs are a type of pattern rec ognition receptors (PRR) that play a key role in the function of antigen-presenting cells such as dendritic cells; how ever, their role and function on lymphocytes is less clear. On dendritic cells, TLR4 is a signal-transducing receptor for lipopolysaccharide (LPS) (26, 57), a nd activation of this receptor causes the release of antimicrobial peptides, infl ammatory cytokines and chemokines, and costimulatory molecules that initiate the innate immune response. TLRs also control multiple cell functions and activate signals that are critically involved in the initiation of adaptive immune responses (24) and signa ling through TLR4 promotes B cell maturation (20) and CSR in mouse B cells (16). Furthermore, TLR4 surface expression on human B cells has been shown to be modulated by IL -4 (45). Therefore, TLR4 expression appears to be involved in adaptive immunity and B cell function. There is some evidence that the CB2 agonist, JWH-133, suppresses LPS-induced up-regulation of TLR4 and MyD88 (73), but these experiments were done in bone marrow derived dendritic cell cultures. Curre ntly, there is no literature examining the effect of cannabinoids on TLR expression on B cells. Our study is the first to show that activation of B cells with IL-4 and antiCD40 increases TLR4 and decreases TLR2. We also showed that cannabinoid treatment incr eased further the expression of TLR4 but had no effect on the level of TLR2 (Figure 23). CP55940 increased TLR4 and is a nonselective agonist bind ing equally to both CB1 and CB2; interestingly, results with the CB1 selective agonist, methanandamide, and the CB2 selective agonist, CB65, also showed a significance augmentation of TLR4 surface expr ession. This finding coupled with our data showing the cannabinoid e ffect was attenuated by both CB1 and CB2 antagonists

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83 (Figure 24) suggests that cannabinoids ar e inducing TLR4 surface expression through a mechanism involving both CB1 and CB2. Possible Mechanism of cAMP Regu lation of TLR4 Expression Toll-like receptors such as TLR4 have been recently shown to mediate B cell activation and antibody production (58) and TLR 4 surface expression on B cells is upregulated by IL-4 treatment (45). Our own data supports this upregulation by IL-4 and anti-CD40 and in addition shows the TLR4 expression is further increased by cannabinoids in a CB1 and CB2 dependent mechanism (Figure 23 and 24) but suppressed by Forskolin (Figure 25). There are very few studies reported on the molecular mechanisms regulating TLR4 expression. We speculate that a ro le for NFkB might be involved in our observations and this stems from recent da ta showing that stimuli known to enhance cAMP are capable of suppressing NFB (68). In immune cells IgE switching requires induction of C germline transcripts which is me diated by binding of STAT-6 and NF B to the C promoter with the expr ession of these factors re gulated by IL-4 and CD40, respectively (23, 44). Also, Gprotein-coupled receptors ca n increase NF-kB through the PLC and PKC pathways (50). Since in our syst em we see an increase in IgE and TLR4 in the presence of anti-CD40 and cannabinoids and a decrease of both products by an increase in cAMP, we speculate that NF kB production might be involved in the regulation of TLR4 as well as IgE.

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84 Activates ACForskolin cAMP Downregulates AC/cAMP/PKA Transcriptional/Translational Regulation IgE & TLR 4 G i/o G i/o PKA Increase ? PKC Figure 28. Possible Mechanisms Involved in Re gulating Cannabinoid Effects on IgE and TLR4 Surface Expression. This is the proposed mechanism by which cannabinoids such as CP55940 are acting to upregulate B cell activation. Cannabinoids bind to G protein-coupled receptors, then the G i subunit dissociates from the B and binds Adenylyl cyclase (AC), downre gulating AC, cAMP, and Protei n Kinase A. However, the subunits can also activate PKC, MAPKs, and tr anscription factors, resulting in a series of events that promote transcriptional regula tion ultimately leading to an increase in IgE and TLR4 surface expression. On the other side Forskolin acts by binding directly to AC, increasing cAMP and resulting in a seri es of events that inhibit IgE and TLR4 surface expression. Solid lines represent stimulatory signa ls, dash lines represent inhibition.

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85 SUMMARY Cannabis is one of the oldest psycho tropic drugs known to humanity, and it has been used for centuries as a remedy to alleviate different ailments(1). Recently, cannabinoid-based drugs have been gaining po litical and public support to be used for the treatment of chronic diseases (32). However, to date the potential risks and benefits of marijuana are not well understood; conseque ntly, leading to an increased demand for cannabinoid research. The current study investig ated the role of synt hetic cannabinoids in the immune system and found that these drugs affect the maturation and activation of B lymphocytes in that these cells are induced to express more IgE and TLR4 proteins. Cross-linking of IgE bound to Fc receptors on mast cell and basophils mediates the pathophysiology of Type I allergic diseases (62) Therefore, these stud ies suggest that the endocannabinoid system may be involved in the regulation of the allergic response and point the way to new strategies of controlli ng allergic diseases us ing cannabinoid-based drugs. In summary, our project c ontributed three main findings to the field. First, we showed that CB2 receptors in B cells were increased after activation with IL-4 and antiCD40. Then, we demonstrated that CP 55940, a synthetic cannabinoid, induced IgE surface expression and secretion through mechanisms involving CB2 receptors. Last, we showed several cannabinoids were also capable of inducing TLR4 surface expression

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86 through mechanisms involving cannabinoid rece ptors. Although the detailed mechanisms by which cannabinoids are mediating these responses are not well understood, G-proteincoupled mechanisms appear to play a si gnificant role in the modulation of B cell activation. Our results suggest cannabinoids negatively regulate cAMP in B cells resulting in increased IgE. In addition, an increase in TLR4 on activated B cells is also regulated by cannabinoid receptors and cAMP. Our studies presented here, show that cannabinoids can directly increase the matu ration and differentiation of B cells in a substantive way in relation to mechanisms i nvolving the role of B ce lls in the allergic response. By investigating the effect of cannabinoids on B cell ac tivation, our results contribute to the understanding of the cannabinoid effects in the immune system, and provide new insights for future drug developmen t in the treatment of allergic diseases.

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ABOUT THE AUTHOR Marisela Agudelo was born in 1979 in Maracay, Venezuela. However, she was raised by her mother and grandparents in Medellin, Colombia. In 1996, she graduated from “La Consolacin” Catho lic High School and migrated to the United States where she met her husband. With the encouragement and support of her family, she started her career at Valencia Community College in Kissi mmee, Florida. She then transferred to the University of South Florida and complete d her Bachelor’s Degree in Microbiology in 2003. The same year, she started the Doctor of Philosophy in th e Multidisciplinary Biomedical Science (MBS) Program at USF College of Medicine. Marisela met her mentor Dr. Thomas Klein in 2001 during a resear ch internship in his lab. Two years later, in 2003, she joined the lab and has been worki ng on this research project ever since. The data in this research were published in the Journal of NeuroImmune Pharmacology in 2008.