Pathophysiology of interleukin-two induced neurologic toxicity

Interleukin-2 (IL-2) induces neuropsychiatric toxicity in patients, which is often dose-limiting and currently not well understood due to lack of suitable experimental methods. We therefore evaluated a number of experimental techniques to quantify changes in the brain vasculatures in a well-characterized IL-2 treatment murine model. Direct measurement of small molecule accumulation, wet versus dry brain weight comparison, and proton density MRI lacked sensitivity and reliability to detect small changes in the brain water content. Successful methods included dynamic contrast enhanced (DCE) MRI and immunohistochemistry using specific endothelial markers to identify vasodilation in carefully matched regions of the mouse brain. Both methods demonstrated significant vasodilation of the brain blood vessels following IL-2 treatment. DCE MRI further indicated that IL-2 increased brain blood vessel permeability to the contrast agent. Using these techniques, we evaluated the mechanism involved in IL-2- induced neuropsychiatric toxicity. We initially established that mice exhibited behavioral changes following IL-2 treatment, indicating that IL-2 affected the brain in the murine model as in patients. The spontaneous movement, rearing, and grooming significantly decreased by day 2 following IL-2 treatment in the C3H/HeN mice, while the motor strength and coordination of these mice were affected by day 4. Vasodilation and microvascular permeability evaluated by immunohistochemistry and DCE MRI were not iv observed until day 4, suggesting that IL-2-induced behavioral changes occurred before detectable vasodilation and microvascular permeability. Further experiments established that IL-2 mediates its cardiovascular toxicity via its receptor on leukocytes, instead of a direct action on endothelial cells. This finding supports the concept that the dose-limiting toxicities associated with IL-2 therapy are mediated by the secondary inflammatory cytokine storm. We therefore evaluated the role of nitric oxide (NO) produced by endothelial NO synthase (eNOS) in vascular changes in the brain following IL-2 treatment. The data indicated that activation of eNOS-mediated changes in vascular permeability and vasodilation correlate with motor function changes induced by IL-2, while changes in spontaneous behavior following IL-2 treatment are not mediated by NO.

PATHOPHYSIOLOGY OF INTERLEUKIN-TWO INDUCED NEUROLOGIC TOXICITY by Yetty Yennawati Irwan A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Pathology Department of Pathology The University of Utah December 2010 Copyright  Yetty Yennawati Irwan 2010 All Rights Reserved Th e Uni v e r s i t y o f Ut a h Gr a dua t e S cho o l STATEMENT OF DISSERTATION APPROVAL The dissertation of Yetty Yennawati Irwan has been approved by the following supervisory committee members: Wolfram E. Samlowski , Chair 9/29/2009 Date Approved John H. Weis , Member 9/29/2009 Date Approved Lorise C. Gahring , Member 9/29/2009 Date Approved Glen R. Hanson , Member 9/29/2009 Date Approved Matthias Schabel , Member 9/29/2009 Date Approved and by Peter E. Jensen , Chair of the Department of Pathology and by Charles A. Wight, Dean of The Graduate School. ABSTRACT Interleukin-2 (IL-2) induces neuropsychiatric toxicity in patients, which is often dose-limiting and currently not well understood due to lack of suitable experimental methods. We therefore evaluated a number of experimental techniques to quantify changes in the brain vasculatures in a well-characterized IL-2 treatment murine model. Direct measurement of small molecule accumulation, wet versus dry brain weight comparison, and proton density MRI lacked sensitivity and reliability to detect small changes in the brain water content. Successful methods included dynamic contrast enhanced (DCE) MRI and immunohistochemistry using specific endothelial markers to identify vasodilation in carefully matched regions of the mouse brain. Both methods demonstrated significant vasodilation of the brain blood vessels following IL-2 treatment. DCE MRI further indicated that IL-2 increased brain blood vessel permeability to the contrast agent. Using these techniques, we evaluated the mechanism involved in IL-2- induced neuropsychiatric toxicity. We initially established that mice exhibited behavioral changes following IL-2 treatment, indicating that IL-2 affected the brain in the murine model as in patients. The spontaneous movement, rearing, and grooming significantly decreased by day 2 following IL-2 treatment in the C3H/HeN mice, while the motor strength and coordination of these mice were affected by day 4. Vasodilation and microvascular permeability evaluated by immunohistochemistry and DCE MRI were not iv observed until day 4, suggesting that IL-2-induced behavioral changes occurred before detectable vasodilation and microvascular permeability. Further experiments established that IL-2 mediates its cardiovascular toxicity via its receptor on leukocytes, instead of a direct action on endothelial cells. This finding supports the concept that the dose-limiting toxicities associated with IL-2 therapy are mediated by the secondary inflammatory cytokine storm. We therefore evaluated the role of nitric oxide (NO) produced by endothelial NO synthase (eNOS) in vascular changes in the brain following IL-2 treatment. The data indicated that activation of eNOS-mediated changes in vascular permeability and vasodilation correlate with motor function changes induced by IL-2, while changes in spontaneous behavior following IL-2 treatment are not mediated by NO. To my father who always believed in me TABLE OF CONTENTS ABSTRACT ................................................................................................................ iii LIST OF FIGURES .................................................................................................. viii LIST OF ABBREVIATIONS ...................................................................................... x ACKNOWLEDGMENTS ........................................................................................ xiii CHAPTER: 1. INTRODUCTION ................................................................................................. 1 Interleukin-2 ..................................................................................................... 2 IL-2 Receptor ................................................................................................... 5 Signaling Pathways and Biological Activities of IL-2 .................................... 7 Preclinical Studies and Clinical Uses of IL-2 .................................................. 9 Dose-limiting Cardiovascular Toxicities of IL-2 Therapy ............................ 10 Nitric Oxide ................................................................................................... 11 Nitric Oxide Synthase .................................................................................... 15 Dose-limiting Neuropsychiatric Toxicity of IL-2 Therapy ........................... 21 Sepsis ............................................................................................................. 24 References ...................................................................................................... 24 2. QUANTITATIVE ANALYSIS OF CYTOKINE-INDUCED VASCULAR TOXICITY AND VASCULAR LEAK IN THE MOUSE BRAIN .................... 42 Abstract .......................................................................................................... 43 Introduction .................................................................................................... 44 Materials and methods ................................................................................... 47 Results ............................................................................................................ 54 Discussion ...................................................................................................... 75 Acknowledgments.......................................................................................... 82 References ...................................................................................................... 82 3. NEUROLOGIC TOXICITY CORRELATES WITH ENDOTHELIAL NITRIC OXIDE SYNTHASE INDUCED CEREBRAL VASODILATION vii AND MICROVASCULAR PERMEABILITY FOLLOWING MURINE INTERLEUKIN-2 TREATMENT ...................................................................... 87 Abstract .......................................................................................................... 88 Introduction .................................................................................................... 88 Materials and Methods ................................................................................... 90 Results ............................................................................................................ 93 Discussion .................................................................................................... 107 Acknowledgments........................................................................................ 115 References .................................................................................................... 115 4. DISCUSSION .................................................................................................... 121 References .................................................................................................... 130 LIST OF FIGURES Figure Page 1.1 The crystal structure of IL-2 generated using RasMol software........................4 1.2 The structure of nitric oxide synthase (NOS) dimer ........................................17 2.1 Cross section drawing of the mouse mid-brain section and the representative sections from the control and IL-2 treated mice .......................51 2.2 Direct comparison of wet versus dry brain weight from IL-2 treated mice .....56 2.3 Evaluation of IL-2 induced vascular leak in the brain using [125I]-labeled albumin ............................................................................................................59 2.4 Evaluation of changes in brain water content using tritiated water .................61 2.5 Evaluation of blood vessel permeability using sodium fluorescein in the mouse pleural fluid, lungs, and brain ...............................................................65 2.6 Immunostaining of brain sections to evaluate endothelial cell morphology ...68 2.7 Evaluation of proton density in the brains of IL-2 treated mice using a 7T MRI system ......................................................................................................71 2.8 Evaluation of IL-2 induced changes in the brain blood vessel permeability by dynamic contrast enhanced (DCE) MRI ................................74 3.1 IL-2 induced behavioral changes in mice .................................................. 96-97 3.2 Vasodilation of brain blood vessels in IL-2 treated mice ..............................100 3.3 Effects of IL-2 on brain plasma volume and microvascular permeability in mice evaluated by DCE MRI .....................................................................103 3.4 IL-2 induced vasodilation and microvascular permeability in the brain are abrogated in eNOS-/- mice ...................................................................... 105-106 ix 3.5 IL-2 induced behavioral changes were attenuated in eNOS-/- mice ....... 109-110 4.1 Assessment of whether IL-2 induces hypotension via IL-2 receptor expression on endothelial cells or leukocytes ................................................126 4.2 DCE MRI following 3 days of IL-2 treatment to evaluate early changes in vasodilation and microvascular permeability induced by IL-2 in the brain ..129 LIST OF ABBREVIATIONS IL Interleukin NK Natural killer IL-2R IL-2 receptor IL-2Rα IL-2R alpha chain IL-2Rβ IL-2R beta chain γc Gamma chain JAK Janus kinases STAT Signal transducers and activators of transcription LAK Lymphokine-activated killer TNF Tumor necrosis factor IFN Interferon GM-CSF Granulocyte-macrophage colony-stimulating factor VLS Vascular leak syndrome NO Nitric oxide cGMP Cyclic guanosine monophosphate sGC Soluble guanylate cyclase iNOS Inducible nitric oxide synthase CNS Central nervous system xi NOS Nitric oxide synthase nNOS Neuronal NOS eNOS Endothelial NOS mtNOS Mitochondrial NOS NADPH Nicotinamide adenine dinucleotide phosphate FAD Flavin adenine dinucleotide FMN Flavin mononucleotide BH4 Tetrahydrobiopterin O2 Oxygen CaM Calmodulin Ca2+ Calcium Ser Serine Thr Threonine O2 - Superoxide ONOO- Peroxynitrite MRI Magnetic resonance imaging BBB Blood-brain barrier DCE Dynamic contrast enhanced ICU Intensive care unit NaFl Sodium fluorescein Iso-B4 Isolectin GS-IB4 vWF von Willebrand Factor ROI Region of interest xii SI Signal intensity PD Proton density fPV Fractional plasma volume KPS Endothelial transfer coefficient PS Permeability surface area product SEM Standard error of the mean WT Wild-type BM Bone marrow KO Knockout ACKNOWLEDGMENTS First and foremost, I would like to thank Dr. Wolfram Samlowski, my PI, for his mentorship. I am grateful for the opportunity to do this research in his lab. It has been a very challenging project and a tough journey, but we made it. I thank him for all his support and advice, and for reviewing all my papers. His work ethic is definitely an inspiration to me. I want to thank John McGregor, our lab manager, for all his consultations whether in research or in life. I thank him for his support throughout my many difficult times. His jokes always make me laugh and he taught me so many American metaphors. I also thank him for ordering all my research reagents and necessities, for helping me in some experiments, and for proofreading all my writings. I could not have asked for a better lab manager than him. Next I want to thank Shweta Tharkar, our current research assistant in the lab, for all her help. She is very organized and it is good to have her in the lab. I also would like to thank the previous members of the lab, Courtney McKinney and Muralidhar Kondapaneni. Courtney was our previous lab technician. She is an expert in immunohistochemistry and taught me the technique. Murali was a previous graduate student in our lab. He laid down the ground work for nitric oxide's contribution in IL-2 induced toxicities. Both Courtney and Murali were good coworkers and friends to me. I would like to thank my supervisory committee members - Dr. Glen Hanson, John Weis, Lorise Gahring, and Matthias Schabel - for their constructive criticisms and xiv experimental ideas. I thank Dr. Gach, who operates the MRI scanner at the Nevada Cancer Institute and Gopalkrishna Veni, who used to work for him. Gopal helped me analyzing the proton density MRI images and later helped me getting my DCE MRI images from the scanner. I also thank Yi Feng for his technical advice in DCE MRI and his MATLAB codes to analyze the DCE MRI data. I would like to thank Dr. James Symanowski, the Head of Biostatistics at the Nevada Cancer Institute, for his help in statistical analysis of the immunohistochemistry and behavioral data. I thank Dr. Victor Laubach for his generous donation of iNOS knockout mice and Novartis for its donation of IL-2 to our lab. Last but not least, I would like to thank my family and friends for their support throughout my graduate school journey. I would not have made it without them. My father, who passed away 5 years ago, remains my constant motivation to get this PhD degree. I cannot be more thankful to my mother, who always makes the long trip from Indonesia to the US at least once a year to keep me company, cook for me, and do all the housework so I can focus with the lab work. I also thank my American families: Dad (Harry), Mom (Cathy), Molly, Lee, and Sandy Stoltzfus. They were my host families when I arrived in the US 12 years ago, but they always treat me like their own. Without them, I may not have stayed in the US and obtained this higher education. Finally, I would like to thank God for all His blessings, especially a beautiful daughter, who kept me company during the final stage of achieving this degree and became an extra motivation for me. I love you very much, Fei Fei. CHAPTER 1 INTRODUCTION 2 Interleukin-2 Interleukin-2 (IL-2) was first identified in 1976 as the active component of a lymphocyte conditioned medium that supported the long-term growth of T lymphocytes in vitro (1) and hence named T-cell growth factor (TCGF). In 1981, it was characterized as a variably glycosylated 15,500 Dalton protein (2). IL-2 was then purified to homogeneity (3) and its cDNA was cloned in 1983 (4). The gene that expresses IL-2 spans four exons and is located on chromosome 4q26-q27 in human or chromosome 3 in mouse (5). The human cDNA encodes a polypeptide composed of 153 amino acids, with the first 20 amino acids serving as a signal sequence and are cleaved prior to secretion. The murine amino acid sequence is about 60% homology to the human sequence. Both are secreted as single polypeptides with molecular weights of 15-17 kDa (6). The size and charge variability of IL-2 depends on the degrees of glycosylation, which have no effect on activity (6). Therefore, recombinant IL-2 is equally effective in stimulating T-cells proliferation as glycosylated IL-2. Recombinant human IL-2 has been found to be effective in mice. The crystal structure of IL-2 was determined in 1991 (7), which showed that IL-2 folds into four α (alpha) helical domains (named A, B, C, and D) connected by 3 loops (A-B, B-C, and C-D loops) and has a single disulfide bridge (Fig. 1.1). The first and last two α helices are each connected by a long crossover loop, resulting in an "up-up-down-down" topological structure because the first two α helices (the A and B helices) appear to orient in an upward direction while the last two α helices (C and D) can be oriented in a downward direction as viewed from the NH2- to COOH-terminal. IL-2 also has two more small α helices, named A' and B'. 3 Figure 1.1 The crystal structure of IL-2 generated using RasMol software. 4 5 In the immune system, a small amount of IL-2 is mainly secreted by antigen-activated T-helper (Th1) lymphocytes. Additionally, dendritic cells, monocytes, natural killer (NK) cells, and activated CD8+ T-cells can also produce IL-2. In the normal brain, IL-2 expression is extremely low and regionally restricted. IL-2 in the brain is produced by the glial cells, which include microglial cells, oligodendrocytes, astrocytes, and ependymal cells. IL-2 Receptor IL-2 was the first cytokine identified to mediate its effects via a cell surface receptor that has similar characteristics to those of classic hormone receptors, including stereospecificity, saturability, and high affinity (8). IL-2 binds to a well-characterized heterotrimeric receptor on its target cells (9). The IL-2 receptor (IL-2R) is composed of three noncovalently linked distinct chains: 1) the alpha chain (IL-2Rα, CD25), 2) beta chain (IL-2Rβ, CD122), and 3) a common cytokine receptor gamma chain (γc, CD132) (10-18). IL-2Rα is essential for specific binding of IL-2. By itself, IL-2Rα binds to IL-2 with a low affinity (binding affinity Kd ≈ 10 nM) and does not play a role in signal transduction (19). IL-2Rβ alone also has a very low affinity (Kd ≈ 100 nM) whereas isolated γc has no detectable binding affinity to IL-2, but the complex of IL-2Rβ and γc binds to IL-2 with an intermediate affinity (Kd ≈ 1 nM) (20). This two-part receptor complex is found on macrophages, NK cells, and resting T cells (9). Upon IL-2 binding, IL-2Rβ and γc complex is sufficient and necessary for effective signal transduction via the heterodimerization of their cytoplasmic domains (21-22). A complex of all three IL- 2R subunits (IL-2Rα, IL-2Rβ, and the γc) binds to IL-2 with high affinity (Kd ≈ 10 pM) 6 and is the receptor form found on activated T cells (18) that mediates most biological activities of IL-2 in vivo (9). There is homology between the mouse IL-2R and human IL- 2R. In the mouse brain, IL-2 receptors have been found on microglial cells and on myelin (23-24), mainly in the hippocampus, frontal cortex, and striatum regions (25). The recently elucidated structure of the IL-2 and IL-2R quaternary complex revealed many details of the interactions between IL-2 and its various receptor complexes. The structure of IL-2Rα and its mode of interaction with IL-2 differ from the typical cytokine receptor (26). IL-2Rα folds into two "sushi-like" domains, which form five-stranded β-sheet sandwiches (27). A long connecting peptide of the IL-2Rα, between its globular head and the transmembrane segment, allows its binding domain to extend away from the cell surface and reach the dorsally located binding site on IL-2 (28), capturing the IL-2 molecule and setting it against the βγ complex. IL-2Rβ and γc, on the other hand, contain a "WSXWS" motif in the C-terminal domain and two disulfide bonds in the N-terminal domain that are characteristics of the class I cytokine receptor superfamily (29). They consist of N- and C-terminal fibronectin-III domains, which are characterized by a β-sandwich sheet containing seven antiparallel strands arranged in a three-on-four topology, and their bases converge to form a Y shape where IL-2 binds to (28). The formation of the IL-2 and IL-2R quaternary complex is mediated by four binding sites: IL-2/IL-2Rα, IL-2/IL-2Rβ, IL-2/γc, and IL-2Rβ/γc (28). IL-2/IL-2Rα interface is the largest of the three interfaces with a hydrophobic center, a polar periphery featuring five ion pairs, and seven hydrogen bonds between IL-2 and IL-2Rα (27). Interestingly, IL-2Rα does not associate with IL-2Rβ or γc and the three contact sites on IL-2 normally do not overlap with each other, except for a small region of helix A that 7 wedges tightly between IL-2Rβ and γc to form a three-way junction for a composite binding site for the final γc recruitment (27). There are only minor conformational changes observed in the IL-2 structure upon forming a quaternary complex with IL-2R, the most significant is the arrangement of the BC loop due to crystal-packing interactions (27). Whereas IL-2Rα chain (CD25) is specific to the IL-2 receptor, IL-2Rβ is also a subunit of the IL-15 receptor while the γc is shared by IL-4, IL-7, IL-9, IL-15, and IL-21 receptors (30). Hence the redundancy of IL-2 functions with those of other cytokines sharing a common γc in their receptors. IL-2 exerts its diverse activities by binding to different receptor complexes of IL-2R depending on which components of the receptor are expressed on the cell surface. A crucial number of IL-2 receptors must be activated before a single T cell will make the irreversible, all-or-none commitment to pass through the G1 restriction point to undergo proliferation (31-32). A number of other cells (i.e.,monocytes, fibroblasts, keratinocytes, and perhaps endothelial cells) express IL-2Rβ and γc on their cell surface, but it is not clear whether IL-2 binds to these receptors and plays a physiological role in these cells. Signaling Pathways and Biological Activities of IL-2 IL-2 binding to IL-2R activates numerous intracellular tyrosine kinases, leading to a rapid increase in tyrosine phosphorylation of many cellular proteins and subsequent increase in expression of several nuclear proto-oncogenes critical for proliferation (33). The cytoplasmic domains of IL-2Rβ and γc associate with Janus kinases JAK1 and JAK3, respectively (34-35). IL-2 binding to IL-2R activates these Janus kinases, leading 8 to phosphorylation and concurrent induction of STAT (signal transducers and activators of transcription) proteins (36). Although several STAT-like DNA binding activities seem to be involved in the IL-2 signaling pathway (37-38), activation of STAT5 protein in particular has been functionally linked to the IL-2Rβ subunit via a redundant tyrosine-containing motif found in several cytokine receptors (39-41). Upon phosphorylation, STAT5 proteins dissociate from the IL-2Rβ, dimerize, and translocate to the nucleus where they promote the transcription of IL-2 specific genes. Additionally, IL-2 binding to IL-2R also triggers other signaling pathways, such as phosphorylation of the Src-family protein tyrosine kinases Lck (leukocyte-specific protein tyrosine kinase) and Syk (spleen tyrosine kinase), expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), stimulation of the phosphatidylinositol-3-kinase-AKT pathway, and stimulation of the Ras-Raf-mitogen-activated-protein-kinase pathway, leading to the activation of Fos- and Jun-containing transcription factor complexes (42). IL-2 plays a key role in cell-mediated immunity (43). At low concentrations (10- 100 IU/ml), IL-2 is a potent autocrine T-cell growth factor and is the key cytokine responsible for rapid activation, clonal expansion, and differentiation of antigen-activated T-cells (44). It also plays a role in the proliferation and survival of mature B-cells, and in enhancing the phagocytic capability, oxidative burst, and microbicidal activities of monocytes (i.e., macrophages and neutrophils). Moreover, IL-2 promotes the proliferation of NK cells and activates their cytotoxic function. High concentrations of IL-2 (> 600 IU/ml) rapidly activate a population of NK cell-derived cytotoxic lymphocytes named lymphokine-activated killer (LAK) cells (45). These cells are termed "nonspecific" killer cells because their cytotoxicity does not require antigen presentation 9 in the context of self-MHC on target cells (46). Thus LAK cells demonstrate killing against almost all cultured and freshly isolated malignant cells (47-48), including multidrug-resistant cancer cells (49). Activation of lymphocytes by IL-2 also strongly induces production of inflammatory cytokines. These secondarily released cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, tumor necrosis factor (TNF), interferon (IFN)-γ, lymphotoxin (LT), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (44). Lastly, both IL-2 and IL-2R knockout mice have been found to suffer from lymphoproliferation followed by lethal autoimmunity, indicating that IL-2 also has a crucial role in the termination of T-cell responses and maintenance of self-tolerance (50). IL-2 regulates the T-cell tolerance via a group of cells called T regulatory cells (Treg) and this function is the only known nonredundant activity of IL-2. Preclinical Studies and Clinical Uses of IL-2 Activation of cytolytic T-cells and LAK cells cytotoxicity against tumor cells in vitro by IL-2 led to the evaluation of this cytokine as an anti-cancer drug. In preclinical studies, concurrent administrations of IL-2 and LAK cells on tumor-bearing mice resulted in the regressions of liver and lung metastases (51-52). These tumor regressions were associated with prolonged survival of the experimental mice (53-54), and correlated with both increasing doses of LAK cells (up to 108 cells/mouse) and IL-2 (up to 105 IU every 8 hours) (51, 55-56). Higher doses of IL-2 were lethal to experimental mice due to the development of cardiovascular toxicities. In clinical studies, cancer therapy using a high-dose i.v bolus of IL-2 induced 10- 15% partial and 5-7% complete responses in patients with metastatic renal cell carcinoma 10 and malignant melanoma (57-58). About 60% of the complete responses proved durable with up to 20 years of follow-up (59). IL-2 therapy was consequently approved by the FDA for treatment of metastatic renal cell carcinoma and melanoma in 1992 and 1998, respectively. To date, IL-2 remains the only clinically available agent that can induce durable complete remissions of metastatic melanoma and renal cell carcinoma in patients despite the dramatic increase in treatment options in the past 4 years. Other agents, such as V600E B-raf specific inhibitor for treatment of melanoma (60) or inhibitors of mTOR (mammalian target of rapamycin) and VEGF (vascular endothelial growth factor) pathways for treatment of renal cancer (61), predominantly induce tumor growth arrest, requiring ongoing administration of these expensive drugs to maintain clinical responses. Attempts to improve IL-2 based immunotherapy include concomitant adoptive transfer of in vitro activated LAK cells (62-64) or tumor specific CD8+ T-cells (termed tumor infiltrating lymphocytes or TILs) or vaccines directed against the cancer cells. These modifications were performed to enhance the immune activities towards the tumor, but have not been proven superior to the treatment with IL-2 alone (57, 65). Dose-limiting Cardiovascular Toxicities of IL-2 Therapy The effectiveness as well as a broader utilization of IL-2 therapy as a cancer treatment is limited by its severe dose-limiting toxicities, which include cardiovascular and neuropsychiatric toxicities (64, 66-67). The two most prominent cardiovascular toxicities of IL-2 administration are vascular leak syndrome (VLS) and hypotension. IL-2 induced VLS is characterized by generalized extravasations of intravascular fluid into interstitial tissues. It is associated with marked fluid retention (weight gain), noncardiac 11 pulmonary edema, and reversible renal dysfunction (68). In conjunction with VLS and intravascular volume depletion, IL-2 also induces hypotension, due to a marked decrease in systemic vascular resistance and hypoperfusion of vital organs, such as the heart and kidneys (69). These IL-2 induced cardiovascular toxicities are thought to be indirectly mediated by the inflammatory cytokines released upon IL-2 administration, termed a "cytokine storm" (70). These secondarily released cytokines, particularly IL-5, IL-6, IFNγ, GM-CSF, and TNFα (71-74), reach high levels in patient serum. High dose of steroids can block IL-2 induced cardiovascular toxicity, but also completely abrogate therapeutic responses, indicating that these secondarily released cytokines may also contribute to the IL-2 treatment response (75). These secondary inflammatory cytokines, mainly IFNγ and TNF, can induce the production of nitric oxide (NO) through activation of the inducible L-arginine:NO synthesis pathway (76-80). The levels of nitrate (metabolites of NO) in the serum and urine of patients have been shown to be elevated 6-10-fold on days 5-7 after a 5-day treatment course of IL-2 (81). Studies in our lab have shown that the mechanism of IL-2 induced VLS is via induction of NO synthesis by endothelial NO synthase (82) whereas the hypotension appears to be due to catecholamine oxidation by superoxide and/or peroxynitrite (83). Nitric Oxide In 1980, a small molecule released by the endothelial cells that diffuses to and relaxes the adjacent muscle was identified (84) and named endothelial derived relaxing factor (EDRF). This vasodilator was later demonstrated to be NO (85-87). There are 12 various stimuli that can act on the endothelial cells to cause relaxation of vascular smooth muscle and therefore vasodilation. A few stimuli, such as adenosine, histamine (via H2 receptors), and atrial natriuretic peptide, cause vasodilation independent of the endothelium whereas acetylcholine, substance P, vasoactive intestinal peptide, histamine, bradykinin, and some other vasodilators act via endothelium by causing the release of NO (88). Many additional endothelium-derived vasodilator and vasoconstrictor players have also been identified (i.e., prostaglandin H2, endothelin-1, and the endothelium-derived hyperpolarizing factor), but none of these molecules play such a crucial role in the regulation of vascular tone and homeostasis as NO. The fluid shear stress (shear stretch) exerted on the endothelium by the flowing blood is the main stimulus for a continuous synthesis of NO in vivo (89-90). Changes in shear stress are detected by the stretch receptors on endothelial cells, which are connected to the endothelial cytoskeleton by cell membrane adaptor proteins (i.e., integrins). Activation of the endothelial cytoskeleton results in the release of NO from endothelial cells that diffuses to the vascular smooth muscle cells and induces vasodilation and increased blood flow. Nitric oxide, with a chemical formula of NO, is a small gas molecule under ambient conditions. It is extremely labile, with a half-life of only about 3-5 seconds, after which it is oxidized into nitrites and nitrates by water or oxygen (91). NO is also highly lipophilic and readily diffusible, and therefore can diffuse and traverse multiple cell membranes to reach its numerous targets, making it an ideal signaling messenger. The known repertoire of NO functions in physiologic and pathological processes has steadily increased in recent years along with a better understanding of its diverse biochemical targets (91-92). NO interacts with various types of protein bound metal centers and has a 13 susceptibility to interact with cysteine thiol groups and other nucleophilic amino acids, such as tyrosine residues in a process called nitrosation. It also has several molecular targets, which upon interaction can modulate gene expression, mRNA stability and translation, as well as intracellular signaling (93). NO relaxes smooth muscle by stimulating the synthesis of cGMP (cyclic guanosine monophosphate) (94). It augments cGMP levels by interacting with the heme-containing soluble guanylate cyclase (sGC) (95-96), displacing the iron from the plane of the heme molecule (97). This interaction leads to sGC structural change and activation, resulting in increased intracellular cGMP production (98). Subsequent events include the activation of calcium activated potassium channels (99), activation of cGMP-dependent protein kinases (100), and decreases in cytosolic calcium (Ca2+) concentration in cardiac and endothelial smooth muscle cells via alterations in calcium transporter function (101- 103). The final result of these events is decreased phosphorylation of myosin light chain, resulting in smooth muscle relaxation (104). The mechanism of action of most pharmacologically useful vasodilators involves either direct release of NO or indirect metabolism of nitrates to NO (105). Nitroglycerin and the organic nitrates, which are potent treatments of cardiac angina, act by stimulating guanylate cyclase after they are converted to NO, resulting in blood vessel relaxation (106-107). In contrast, when various derivatives of arginine that inhibit NO synthase are administered to humans (108-109) or experimental animals (110-112), they result in a rapid rise in blood pressure, suggesting that NO plays a direct role in the regulation of blood pressure. NO is also involved in vascular remodeling and angiogenesis, and has been implicated in atherosclerosis, a pathological damage of endothelium resulting in 14 stroke. NO can inhibit blood clotting by preventing platelet aggregation (113-115) and adhesion (116-117). Moreover, NO is thought to play a key role in diffusion across endothelium as it has been shown to regulate focal adhesion integrity via FAK (focal adhesion kinase) phosphorylation in endothelial cells, which is important in the regulation of tight junction and of macromolecule transport across endothelium (118). NO has also been suggested to interact with hemoglobin (Hb) when Hb is in the oxygen partial pressure (pO2)-dependent allosteric transition, be carried away by the red blood cells, and then released in the relatively ischemic tissues (119). NO subsequently induces vasodilation in oxygen-poor loci, diverting blood flow and thus increasing oxygen delivery to where it is most needed (120). Abnormalities in this process are associated with sickle cell anemia (121). In the immune system, NO plays a key role in mediating the bactericidal and tumoricidal actions of macrophages (79, 122-124). Inflammatory stimuli, such as endotoxin, stimulate the production of very large amounts of NO by the inducible NO synthase (iNOS), enabling macrophages to kill tumor cells and bacteria. Besides killing bacteria, NO also can inhibit viral replication (125-126). In the central nervous system (CNS), beginning with influential works of Garthwaite (127-128) and Snyder (129-131), NO has been identified as an important messenger molecule (132-136). The brain is capable of synthesizing NO (137-139). NO has been proposed to mediate neuronal plasticity, which underlies both development and information storage in the brain, by potentiating neurotransmitter release (91). NO signaling in the CNS appears to be essential for two forms of neuronal plasticity: 1) long-term depression (LTD) in the cerebellum (138) and 2) long-term potentiation (LTP) in 15 the hippocampus (140). Via its regulation of synaptic plasticity and neurotransmitter release, NO contributes to brain development, memory formation, and behavior (91). Excess levels of NO are neurotoxic and have been implicated in stroke and other neurodegenerative diseases in vivo (141). Nitric Oxide Synthase The specific action of NO depends on its enzymatic source, namely the nitric oxide synthase (NOS) enzyme family. Because NO cannot be released, stored, or inactivated by conventional regulatory mechanisms, NOS is one of the most highly regulated enzymes in biology. There are four major isoforms of NOS named after the tissue, cell source, and the order of which the isoform was characterized: 1) neuronal NOS (nNOS or NOS-1) (130, 142-143), 2) inducible NOS (iNOS or NOS-2) (144-147), 3) endothelial NOS (eNOS or NOS-3) (148), and, more recently, 4) mitochondrial NOS (mtNOS) (149-152). All NOS isoforms are self-sufficient enzymes with two main functional domains fused into a single polypeptide (153). The C-terminal reductase domain contains the binding sites for NADPH (nicotinamide adenine dinucleotide phosphate), FAD (flavin adenine dinucleotide), and FMN (flavin mononucleotide). The N-terminal catalytic or oxygenase domain binds the redox cofactor, tetrahydrobiopterin (BH4), the heme prosthetic group, and the substrates L-arginine and oxygen (O2). This architecture is similar to that of cytochrome P-450BM-3 (154). Two NOS peptides dimerized in a head-to- tail manner (Fig. 1.2) to ensure proper transfer of electrons from the reductase domain of one monomer to the oxygenase domain of the second monomer (155). 16 Figure 1.2 The structure of nitric oxide synthase (NOS) dimer. Arrows indicate the reaction mechanism of NO synthesis by the enzyme. 17 18 NOS oxidizes the guanidine group of L-arginine, in the presence of NADPH and O2, and in a process that consumes five electrons to produce NO with stoichiometric formation of L-citrulline. Thus, L-Nω-substituted arginine analogs, such as L-Nω-methyl-arginine (L-NMA) and L-Nω-nitro-arginine (L-NNA), serve as NOS inhibitors (156). The catalysis process from arginine to NO takes two independent monooxygenase reaction steps. The first step is a two-electron oxidation of L-arginine to L-Nω-hydroxyarginine (LHA), utilizing 1 equivalents of NADPH and 1 equivalents O2, and releasing one molecule of water (157-158). The reducing equivalents from NADPH are transferred through FAD to FMN and finally to the heme (159). This hydroxylation reaction is accelerated by BH4, requires calmodulin (CaM) and Ca2+ as activators, and is blocked by carbon monoxide (CO) (157, 160-161). CaM is believed to facilitate the flow of electrons from the reductase domain to the monooxygenase domain and also from FAD to FMN (162). In the second step, one electron (or 0.5 equivalents of NADPH) is essential to form a second equivalent of reduced oxygen molecule (163), which then attacks the guanidine carbon of LHA to form a tetrahedral intermediate. This tetrahedral intermediate collapses to form the carbonyl group of L-citrulline with simultaneous expulsion of NO. The complete stoichiometry of NO synthesis by NOS therefore utilizes 1 mole of L-arginine, 1.5 moles of NADPH, and 2 moles of O2 and produces 2 moles of water, 1 mole of L-citrulline, and1 mole of NO (164). The involvement of BH4 and CaM is unique to NOS. The CaM/Ca2+ binding domain lies in the center of the enzyme. nNOS, eNOS, and mtNOS are expressed constitutively in mammals and their activities are regulated by CaM binding in a Ca2+ concentration dependent manner (165-166). Conversely, iNOS has CaM bound 19 permanently as an additional subunit and thus is not controlled by CaM binding nor it is Ca2+ concentration dependent, but is under transcriptional regulation instead (167). The mammalian NOS enzymes are products of distinct genes located on different chromosomes but they exhibit about 50 to 60% sequence homology with each other (164). nNOS is 155 kDa and found in peripheral and central neurons, noncholinergic and nonadrenergic neurons, endometrium, pancreatic islets, skeletal and smooth muscle, neutrophils and epithelium (92, 168). It has four splice variants and accounts for the majority of physiologic processes attributed to NO in the nervous system (169). iNOS is a 125 kDa protein expressed in macrophages, NK cells, neutrophils, smooth muscle, liver, heart, endothelium and other cell types (168). It is crucial for immune function and is inducible in response to glucocorticoids, cytokines, which include IL-1, TNF-α, IFNγ, and lipopolysaccharide (LPS), a bacterial cell-wall component that elicits symptoms of sepsis (170). eNOS is 125 kDa (168) and is the main isoform found in the endothelium. It is also found in smooth muscle, heart, brain, and other sites (168). mtNOS is 144-kDa and associates with the mitochondrial inner membrane (171). It produces NO to regulate mitochondrial respiration (172). Unlike nNOS and iNOS, which are mainly cytosolic, eNOS is predominantly localized to the plasma membrane (148). eNOS bioactivity and mobilization to specific organelles are highly regulated by multiple posttranslational modifications, such as acylation and phosphorylation. eNOS is irreversibly myristoylated at its N-terminal glycine (173). Myristoylation targets the enzyme to the cell membrane, where it is doubly palmitoylated at the N-terminal cysteine residues 15 and 26, a modification that further anchors eNOS to the plasmalemmal caveolae of endothelial cells (174). The N- 20 myristoylation is sufficient and necessary for eNOS membrane association (175-176), palmitoylation (177-178), compartmentalization to the Golgi complex of cells, and most importantly, efficient NO synthesis (179). Palmitoylation is required for localization of eNOS in caveolae (180-181) and regulation of the frequency and magnitude of NO release in vivo in response to stimuli (182). In contrast to the myristoylation, palmitoylation and the consequential caveolar targeting are reversible and controllable as the thiopalmitoyl bonds are labile, hence creating an additional level of dynamic regulation of eNOS activity (183). Another dynamic control of eNOS involves various pathways of phosphorylation and dephosphorylation. eNOS phosphorylation is usually associated with its agonist-triggered translocation to the cytosol (184). eNOS has multiple phosphorylation sites, including Ser (Serine) 635 and Ser 1177, which are stimulatory, and Thr (Threonine) 495 and Ser 116, which are inhibitory (164). (Note that the numbering system refers to phosphorylation sites of human eNOS isoform.) The protein kinase Akt (protein kinase B) is a key enzyme that phosphorylates eNOS at Ser 1177 in response to agonists, resulting in eNOS released into the cytosol and activation at basal levels (185). Kinase Akt is activated by phosphoinositide-3-kinase (PI3K) (186), which in turn is activated by many eNOS agonists (i.e., vascular endothelial growth factor). eNOS Ser 635 represents a second stimulatory phosphorylation in respond to basal stimuli, such as shear stress, and agonists downstream of protein kinase A (187). In contrast, phosphorylation of eNOS at Thr 945 inhibits enzyme activity by preventing CaM binding (188). When eNOS is uncoupled from essential cofactors or is in the oxidized state, it produces superoxide (O2 -) instead of NO (189). This situation has been demonstrated in 21 diseases, such as atherosclerosis, hypertension, hypercholesterolemia, and diabetes (189- 191). O2 - and NO react rapidly, generating peroxynitrite (ONOO-), a potent oxidizing agent (192-194). This reaction further decreases NO availability while promoting lipid and protein oxidation (195). ONOO- itself plays a crucial role in eNOS uncoupling (196) by oxidizing the essential cofactor BH4 to the inactive pterin, 7,8-dihydrobiopterin (BH2) and removing zinc from the zinc-thiolate cluster (197). Dose-limiting Neuropsychiatric Toxicity of IL-2 Therapy When clinical cardiovascular toxicities of IL-2 are attenuated with pressor agents and fluid administration, the effective dose escalation of IL-2 therapy is further limited by the onset of neuropsychiatric toxicity. In data reported in 1987, a large percentage of patients were found to develop striking neuropsychiatric changes after the onset of IL-2 treatment, with 30-50% of patients experienced cognitive and behavioral changes (67). All of these patients returned to their baseline cognitive scores at follow-up. Based on our clinical experiences, perhaps as high as 40% of patients receiving IL-2 therapy have subtle neuropsychiatric changes, such as mental slowing or confusion, and approximately 10-20% of patients develop more severe manifestations, such as agitation, hallucinations, or delusions. IL-2 induced neuropsychiatric toxicity is the least predictable and treatable compared to the cardiovascular toxicities. Management is generally by holding further doses of IL-2 and allowing patients to recover spontaneously. In 1989, a study was conducted to look at the effects of IL-2 in the brain of patients with extracranial cancer without evidence of intracranial metastases using T2- weighted MRI before and after IL-2 therapy (198). The results suggested a massive 22 increase of cerebral water content after IL-2 therapy in both the gray matter (12.6±7.3%) and white matter (17±6.2%). However, the range of these measurements actually varied from 3-48% and only 3 out of these 7 patients were mildly lethargic, raising questions concerning the accuracy of these data. The magnitude of these apparent cerebral water content increases are far greater than the magnitude of VLS measured in any other organ or even compared to the overall weight changes in the intact IL-2 treated animals or patients (approximately 6-8%), and thus are difficult to believe. Previous studies in animal models showed increased permeability of horseradish peroxidase into the brain of cats and rats after a single bolus intravenous injection of recombinant IL-2, suggesting that IL-2 disrupted the blood-brain barrier (BBB) and cerebrovascular morphological integrity (199-200). Another study in a rat gliosarcoma model suggested that IL-2 increased the permeability of carbon-14-labeled aminoisobutyric acid from the BBB into the brain of tumor-bearing rat brains, but not in normal brains (201). Since tumors are known to have abnormal blood vessel permeability and accordingly could have contributed to the increased brain edema following IL-2 treatment, the significance of this finding is uncertain. The pathophysiology of IL-2 induced neuropsychiatric toxicity therefore remains poorly understood to date. Further efforts to study this toxicity have been limited by the availability of suitable experimental techniques and the technical challenges of performing dynamic contrast enhanced (DCE) MRI (magnetic resonance imaging) studies in critically ill patients, who normally are hospitalized in the ICU (intensive care unit). It would be quite difficult to perform DCE MRI studies once these patients display neuropsychiatric toxicity (delirium) as they are frequently receiving intravenous pressor 23 agents, intubated, and would not be stable enough to undergo lengthy MRI scans. Additionally, the gadolinium-based contrast agent used for DCE MRI may have an adverse effect for IL-2 treated patients, who usually develop a certain degree of acute renal dysfunction, as this agent has been shown to be associated with the development of nephrogenic fibrosing dermopathy (NFD) and nephrogenic systemic fibrosis (NSF) in patients with kidney dysfunction (202-204). It is likely that the "cytokine storm" released following IL-2 therapy induces changes in brain microvasculature, such as vascular leak and brain edema, which correlate to changes in the CNS function and thus trigger neuropsychiatric toxicity. Therefore, development and evaluation of a number of potential experimental methods to quantify changes on the brain blood vessels in a well-characterized IL-2 treatment murine model (83, 205) are required to delineate IL-2 induced neuropsychiatric toxicity. Once successful assays have been identified, the mechanisms of this toxicity can be determined. The role of NO and superoxide, the major players in systemic IL-2 induced VLS and hypotension, respectively, in IL-2 induced CNS changes is currently unknown. Detailed understanding of the mechanisms by which IL-2 induces neuropsychiatric toxicity will be useful in identifying possible pharmacologic inhibitors of this process to be used in patients, which may enable dose escalation of IL-2 treatment to achieve the effective high dose. In addition, this knowledge may also be broadly applicable to other conditions that give rise to an inflammatory "cytokine storm," such as clinical administration of cytokines IL-1, TNF, and IFNγ, as well as endotoxin and bacterial sepsis syndromes. 24 Sepsis A potential implication of this study in other area of Biology is sepsis. Sepsis is a clinical syndrome characterized by a systemic response to an infection. There are roughly 750,000 cases of sepsis annually in the United States (206-207). 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Martin S, Maruta K, Burkart V, Gillis S, Kolb H. 1988. IL-1 and IFN-gamma increase vascular permeability. Immunology 64: 301-5. 212. Maruo N, Morita I, Shirao M, Murota S. 1992. IL-6 increases endothelial permeability in vitro. Endocrinology 131: 710-4. 213. Nooteboom A, Van Der Linden CJ, Hendriks T. 2002. Tumor necrosis factor-alpha and interleukin-1beta mediate endothelial permeability induced by lipopolysaccharide-stimulated whole blood. Crit. Care Med. 30: 2063-8. CHAPTER 2 QUANTITATIVE ANALYSIS OF CYTOKINE-INDUCED VASCULAR TOXICITY AND VASCULAR LEAK IN THE MOUSE BRAIN 43 Abstract A storm of inflammatory cytokines is released during treatment with pro-inflammatory cytokines, such as interleukin-2 (IL-2), closely approximating changes initially observed during sepsis. These signals induce profound changes in neurologic function and cognition. Little is known about the mechanisms involved. We evaluated a number of experimental methods to quantify changes in brain blood vessel integrity in a well-characterized IL-2 treatment mouse model. Measurement of wet versus dry weight and direct measurement of small molecule accumulation (e.g. [3H]-H2O, sodium fluorescein) were not sensitive or reliable enough to detect small changes in mouse brain vascular permeability. Estimation of brain water content using proton density magnetic resonance imaging (MRI) measurements using a 7 T mouse MRI system was sensitive to 1-2% changes in brain water content, but was difficult to reproduce in replicate experiments. Successful techniques included use of immunohistochemistry using specific endothelial markers to identify vasodilation in carefully matched regions of brain parenchyma and dynamic contrast enhanced (DCE) MRI. Both techniques indicated that IL-2 treatment induced vasodilation of the brain blood vessels. DCE MRI further showed a 2-fold increase in the brain blood vessel permeability to gadolinium in IL-2 treated mice compared to controls. Both immunohistochemistry and DCE MRI data suggested that IL-2 induced toxicity in the brain results from vasodilation of the brain blood vessels and increased microvascular permeability, resulting in perivascular edema. These experimental techniques provide us with the tools to further characterize the mechanism responsible for cytokine-induced neuropsychiatric toxicity. 44 Introduction Cytokines are important signaling proteins secreted by cells to regulate the immune system. Interleukin-2 (IL-2) is a 15 kDa cytokine which activates lymphocytes via its well-characterized heterotrimeric receptor (1-2). IL-2 plays an important role in the development of cell-mediated immunity (3). In the presence of properly processed and presented antigenic peptides, low concentrations (10-100 IU/ml) of IL-2 are essential for the activation of cytolytic lymphocytes and their clonal expansion (4). Additionally, when murine or human lymphocytes are exposed to high concentrations of IL-2 (> 600 IU/ml) over 3-4 days either in vitro or in vivo, IL-2 rapidly activates a population of cytotoxic lymphocytes called lymphokine-activated killer (LAK) cells (5). LAK cells are termed "non-specific" killer cells as their cytotoxicity does not require antigen presentation in the context of self-MHC on target cells nor is it tumor-specific. LAK cells demonstrate cytotoxicity against almost all freshly isolated and cultured malignant cells (6-7), including multidrug-resistant tumor cells (8). Activation of lymphocyte cytotoxicity against tumor cells in vitro by IL-2 led to evaluation of this cytokine as an anti-cancer therapy. In preclinical studies, concomitant IL-2 and LAK cells administrations on tumor-bearing mice resulted in regression of lung and liver metastases (9-10). Tumor regression in experimental mice was associated with prolonged survival (11-12). In clinical studies, high-dose i.v bolus IL-2 treatment induced 5-7% complete and 10-15% partial responses in patients with malignant melanoma and metastatic renal cell carcinoma (13-14). Approximately 60% of complete responses to high-dose IL-2 proved durable, with up to 20 years of follow-up (15). 45 Currently, IL-2 therapy is FDA approved for treatment of metastatic renal cell (RC) carcinoma and melanoma. IL-2 remains the only agent that can induce durable complete remissions of metastatic RC in approximately 5% of patients. Despite the dramatic increase in treatment options for metastatic RC in the past 4 years, including agents that inhibit vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) pathways (16), these agents predominantly induce disease arrest, requiring ongoing administration of expensive drugs to maintain a response. At the present time, IL-2 also remains the only clinically available remission-inducing agent for treatment of metastatic melanoma. IL-2 therapy strongly induces synthesis of inflammatory cytokines termed a "cytokine storm" (17). These secondarily released cytokines, which include IFN-γ, tumor necrosis factor (TNFα, TNFβ), IL-1α, IL-1β, IL-5, and IL-6 (18-21) reach high levels in patient serum and are believed to trigger severe toxicities during IL-2 treatment. This pattern of cytokine secretion has strong similarities to the "cytokine storm" elicited by bacterial endotoxin (22-24). These secondarily released cytokines may also contribute to the therapeutic response to IL-2, as high dose steroids block IL-2 toxicity but also abrogate the therapeutic effectiveness of this agent (25). The major dose-limiting toxicities of IL-2 are vascular leak syndrome (VLS) and hypotension. IL-2 induced VLS is generalized and dose dependent, characterized by loss of intravascular fluid into interstitial tissues. VLS is associated with marked fluid retention (weight gain), reversible renal dysfunction, and noncardiac pulmonary edema (26). IL-2 induced hypotension is due to a marked decrease in systemic vascular resistance, resulting in hypoperfusion of vital organs, such as heart and kidneys (27), in 46 conjunction with VLS and intravascular volume depletion. It has also been recognized that reversible neuropsychiatric toxicity represents the third IL-2 dose-limiting toxicity. Denicoff et al. showed that there were striking neuropsychiatric changes in the majority of patients after the onset of IL-2 treatment, with 30-50% of patients experienced severe behavioral and cognitive changes (28). All patients returned to their baseline cognitive scores at follow-up (28). It should be noted that the pattern of hypotension, VLS, and delirium following IL-2 therapy in patients appears very similar to the clinical presentation of patients who have severe bacterial infection and septic shock (27). The major apparent difference is the complete resolution of renal function changes following completion of IL-2 therapy, unlike the high incidence of acute tubular necrosis during septic shock. It has become increasingly clear that even if hypotension and VLS can be attenuated, dose limitations related neuropsychiatric toxicity prevent effective IL-2 dose escalation. To date, the effects of inflammatory cytokines that trigger neuropsychiatric toxicity in the brain are not well understood. Moreover, technical challenges exist to study these effects after 4-5 days of IL-2 treatment (similar to treatment sequence used in human patients). Studies in patients and animal models have suggested that brain edema could play a role (29-32). However, Alexander et al. suggested that IL-2 induced brain edema occurred predominantly in tumor-bearing rat brain but not in normal brain (30). Since tumors are known to have abnormal blood vessel permeability, it is strongly believed that this could result in increased brain edema following IL-2 treatment. We therefore evaluated a number of potential approaches to more directly evaluate cytokine 47 effects on the non-tumor bearing central nervous system (CNS) using IL-2 treatment as a model in a well-characterized murine system (33-34). Materials and Methods Mice. Pathogen-free C3H/HeN mice were obtained from Charles River Laboratories. Mice were maintained under guidelines established by the Institutional Animal Care and Use Committee (IACUC). The IACUC also approved all the animal experimental protocols. Mice were age- and sex-matched in each experiment. IL-2 treatment. IL-2 treated mice received 150,000 IU of IL-2 (Aldesleukin Proleukin®; Novartis, Emeryville, CA) in 0.2 ml PBS (phosphate buffered saline) intraperitoneally (i.p.), b.i.d. (twice a day) for 5 days (10 doses total) in all experiments, except in experiments using MRI. For MRI studies, mice received 8 doses of 150,000 IU of IL-2 in 0.2 ml PBS i.p., b.i.d. (4 days). Control mice received no treatment, except in wet versus dry brain weight experiments, where control mice were treated with an equivalent volume of PBS (without IL-2). Comparison of wet versus dry brain weight. Brains were harvested from each IL-2 treated and control mouse after 5 days of IL-2 and PBS treatment, respectively. Each brain was weighed before and after drying by Heto vacuum centrifuge (ATR, Laurel, Maryland) overnight in 60°C. The water content of the brain was calculated as: x 100. wet weight wet weight - dry weight %tissue water = [125I]-labeled albumin extravasation in the mouse brain. Approximately 100,000 counts per minute (cpm) of [125I]-labeled albumin (specific activity 1-5 μCi/μg) in 100 μl PBS was injected intravenously (i.v.) into the tail veins of mice 2 h after the last 48 dose of IL-2. Mice were sacrificed 6 h after the last dose of IL-2 and the brains excised. Each brain was analyzed for albumin accumulation. Data was calculated as follow: . total cpm recovered from the mouse brain cpm x 100% % cpm = Tritiated water ([3H]-H2O) extravasation as a potential marker of IL-2 induced VLS. IL-2 treated or control mice were injected with approximately 300,000 cpm of [3H]-H2O in 100 μl PBS i.v. into the tail vein 2 h after the last dose of IL-2 or no treatment (control). Mice were then sacrificed at 4, 12, or 18 h after [3H]-H2O injection. The brain was harvested from each mouse, weighed, and homogenized in 1 ml PBS using Tissumizer (Tekmar, Cincinnati, OH). The homogenate (100 μl) was added into 2 ml scintillation fluid and quantified in a scintillation counter. To correct for tissue quenching, 100 μl brain homogenate from a normal mouse was directly mixed with 300,000 cpm [3H]-H2O in 100 μl PBS and 2 ml scintillation fluid, and quantified in a scintillation counter. The cpm recovered was expressed as percentage of 300,000 cpm and was used to correct the apparent cpm from the IL-2 treated and control mouse brain homogenates. The [3H]-H2O extravasation in each brain homogenate of IL-2 and control mice was expressed as cpm per gram tissue as follow: . % cpm recovered (quenching) x tissue weight (g) homogenate cpm x 100% cpm/gram tissue = Sodium fluorescein extravasation in mouse pleural fluid, lungs, and brain as an indicator of VLS. IL-2 treated or control mice were injected with 1.5 mg fluorescein sodium salt (NaFl; Sigma-Aldrich, St. Louis, MO) in 100 μl PBS i.v. via tail vein, 4 h after the last dose of IL-2. The mice were sacrificed 10 min after NaFl injection. The pleural fluid from IL-2 treated mice was collected and the volume was measured. Since 49 control mice did not have detectable pleural fluid, the thoracic cavity was irrigated with 500 μl PBS, which was collected. The lungs and brain were also harvested and were homogenized in 1 ml PBS each, using Tissumizer (Tekmar, Cincinnati, OH). After centrifugation for 5 min, 100 μl of each homogenate was added in triplicate into a 96- well plate. Also in triplicate, 100 μl of the pleural fluid from each mouse was transferred into another 96-well plate. Plates were read using a Synergy 2 microplate reader (BioTek Instruments, Winooski, Vermont) at 519 nm emission and 495 nm excitation wavelengths. For the pleural fluid, the fluorescence was quantified using a standard curve and multiplied by the total volume recovered to calculate the total amount of NaFl (in μg) that leaked into the pleural fluid. In lung or brain homogenates, the fluorescence was corrected for tissue quenching and quantified using a standard curve. Data were expressed as μg NaFl/organ based on the total volume of homogenate. Evaluation of brain blood vessel morphology using immunohistochemistry. Brains were excised from IL-2 treated or control mice. Each brain was embedded in Tissue-Tek O.C.T. Compound (Ted Pella, Redding, CA) and frozen at -20 °C overnight before sectioning. Matched sections (6 μm) of the mid-brain (Fig. 2.1) were dual-stained with Isolectin GS-IB4 (Iso-B4) from Griffonia simplicifolia, conjugated to Alexa Fluor® 568 (Molecular Probes, Eugene, OR) and rabbit anti-human von Willebrand Factor (vWF; Sigma-Aldrich, St. Louis, MO), followed by a secondary Dichlorotriazinyl-aminofluorescein (DTAF)-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Digital images of 20 specific regions of interest (4 different areas in the left cortex, right cortex, left striatum, right striatum, and the cerebral fissure; Fig. 2.1) in the stained brain sections were acquired using Olympus BX51 50 Figure 2.1 Cross section drawing of the mouse mid-brain section, which includes the cortex and striatum anatomic regions, derived from Dorr et al. (35). Inset figure represents the position at which the tissue slice was taken (Bregma 0.62-0.38 mm). The drawing shows the name of major cerebral vasculature structures found in the specific section. azPA = azygos pericallosal artery. Boxes show the locations of 19-20 digital images obtained from each section for vessel analysis. On the right are representative sections from the control and IL-2 treated mice, which were matched as closely as possible to the drawing on the left, stained with hematoxylin and eosin. These representative sections are located right by the sections used in the immunohistochemistry experiment. 51 52 fluorescent microscope mounted with Olympus DP70 camera. Using ImageJ software (National Institutes of Health, NIH), the blood vessels (dual stained tubular structures) in each digital image were manually outlined and analyzed for the total number and average size of vessels. Evaluation of brain water content using MRI proton density measurements. MRI was performed using a Bruker 7T/20 scanner, a 3.5 cm ID (inner diameter) quadrature transceiver coil, and a 7 cm ID unshielded gradient. Mice were anesthetized by i.p. injection of 80 mg/kg ketamine (Vetalar®; Aveco, Fort Dodge, IA) and maintained inside the magnet using isoflurane (Attane™; Minrad, Bethlehem, PA) inhalational anesthesia as required. Mouse temperature inside the magnet was maintained using a circulating warm water system. Each mouse was scanned both before and after IL-2 treatment. Proton density MRI was sequenced using a Fast Spin Echo sequence with a repetition time (TR) of 24 s, an echo time (TE) of 7.5 ms, and an echo train length of 4. Slice thickness was 1 mm, field of view was 2x2 cm2, and matrix size was 256x192 pixels. The slice thickness and matrix size were optimized to detect proton density changes in the mouse brain of > 1%. The total scan time was 14 min and 24 s. Regions of interest (ROIs) were drawn manually on matched pre- and post-IL-2 mouse's brain DICOM (Digital Imaging and Communications in Medicine) images using OsiriX imaging software (http://www.osirix-viewer.com/). The signal intensity (SI) of each brain slice was normalized using a set of phantoms (60-100% H2O/D2O in 10% increment) and expressed as the percentage of proton density (%PD). We averaged the %PD over 8 MRI brain image slices for each mouse and compared the average %PD before and after IL-2 treatment. 53 Dynamic contrast enhanced (DCE) MRI. The MRI system and anesthesia were described above. Each mouse was scanned before and after IL-2 treatment, serving as its own control. Prior to imaging, the tail vein of each mouse was catheterized using a 30- gauge needle connected to an approximately 1 m long, 0.28 mm ID tube pre-filled with heparinized PBS (approximately 100 μl). DCE-MRI was performed using 2D-FLASH (Fast Low-Angle Shot): TR = 93.5 ms, TE = 3.1 ms, flip angel = 30 º, FOV = 2 x 2 cm, matrix = 128 x 128, slice thickness = 1 mm, number of slices = 11, single scan time = 9 s, number of repetitions = 54. After the first 5 repetitions, 0.1 mmol/kg contrast agent (gadobenate dimeglumine; MultiHance®, Bracco) was administered by bolus injection via the catheter. Customized MATLAB programs (The MathWorks, Natick, MA) were used to calculate the SI of the whole brain ROI in matched mid-brain sections of pre- and post-IL-2 DICOM images. The signal intensities of the left and right maxillary veins were averaged to derive the contrast agent concentrations in the blood pool. The average SI of ROI before contrast agent injection was used as the baseline and subtracted from all SI after contrast agent injection to calculate the increase in SI ( SI). SI was assumed to be proportional to the change in contrast agent concentration when a low concentration of contrast agent was used (36). A two-compartment model was originally developed by Shames et al. (36) to characterize tumor vasculature and adapted by Feng et al. (37) to calculate the tumor fractional plasma volume (fPV), endothelium transfer coefficient (KPS), and permeability surface area product (PS). This method was similarly used to characterize the brain microvasculature. Statistical analysis. All data are expressed as mean ± standard error of the mean (SEM). Statistical analyses on MRI data were performed by paired Student's 2-tailed t- 54 test using GraphPad PRISM software (GraphPad Software, La Jolla, CA). The immunohistochemistry data were analyzed using 2-factor analysis of variance (ANOVA) with a blocking factor for region of the brain. All the other data were analyzed using 2- sample Student's 2-tailed t-test. A p-value ≤ 0.05 was considered significant. Results Comparison of wet versus dry brain weight in IL-2 treated mice. Difference in wet versus dry tissue weight is assumed to be approximately the volume of interstitial fluid in the tissue. It was not certain whether this technique was sensitive enough to detect the small fluid shifts that were likely in the mouse brain related to cytokine treatment. We excised brains from IL-2 treated or control mice (treated with an equivalent volume of PBS) after 5 days of treatment and weighed each brain before and after drying by speed vacuum. The average brain water content of IL-2 treated mice was 43.05±1.84% of the wet weight while the control mice had a mean brain water content of 42.6±1.524% (p = 0.85; Fig. 2.2). It became clear from these results that this method lacked sensitivity to identify relatively small changes in the brain water content of mice related to IL-2 treatment. [125I]-labeled albumin extravasation in the mouse brain. Radioactive labeled [125I]-bovine serum albumin has often been used to study IL-2 induced VLS in mice tissues (38). However, it had not been quantified as a VLS marker in the mouse brain. To test this, we injected IL-2 treated or control mice with [125I]-labeled albumin bolus via tail vein. After 4 h, mice were sacrificed and the brains excised. The amount of [125I]-labeled albumin recovered in the brain was quantified and expressed as a percentage of the total 55 Figure 2.2 Direct comparison of wet versus dry brain weight from IL-2 treated mice. Brains were excised from IL-2 treated and control mice (n = 9). Each mouse brain was weighed before and after drying in a vacuum centrifuge overnight in 60 °C. The wet and dry weight difference was expressed as a percentage of wet weight. Error bars represent SEM. The result was not significant (NS), p > 0.05. 56 57 amount of [125I]-labeled albumin recovered from the mouse. Analysis of [125I]-labeled albumin accumulation in the brain showed no significant increase following IL-2 treatment (p = 0.17; Fig. 2.3). The amount of [125I]-labeled albumin recovered from the brain of IL-2 treated mice was 0.2172±0.04% while the control mice had 0.1364±0.04%. The small percentage of [125I]-labeled albumin recovered from the mouse brain demonstrated that this method also lacked the sensitivity to measure a small increase in [125I]-labeled albumin extravasation in the mouse brain induced by IL-2. Tritiated water ([3H]-H2O) extravasation as a potential marker of IL-2 induced VLS. We hypothesized that vascular leak in the brain should correlate with the ability of water molecules (H2O) to cross the BBB. To test [3H]-H2O as a potential marker of IL-2 induced fluid shifts in the mouse brain, IL-2 treated or control mice were injected with [3H]-H2O bolus via tail vein. At specified times, mice were sacrificed and the brain excised. Each brain was weighed and homogenized. The homogenate was quantified in a scintillation counter, and corrected for quenching of counts by tissue homogenate. At 4 and 12 h after the last IL-2 dose, significant [3H]-H2O retention in the brain of IL-2 treated mice could not be detected compared to the control mice (Fig. 2.4). At 4 h after the last IL-2 dose, there were 787.3±36.48 cpm [3H]-H2O/g brain in the IL-2 treated mice compared to 742.7±17.39 cpm [3H]-H2O/g brain in the control mice (p = 0.332). By 12 h, there were 713±37.29 cpm [3H]-H2O/g brain in the IL-2 treated mice and 653.5±24.28 cpm [3H]-H2O/g brain in the control mice (p = 0.2024). There was a trend toward increased [3H]-H2O retention in the brain of IL-2 treated mice at 18 h (810.8±70.82 cpm [3H]-H2O/g brain) compared to the control mice (552±24.03 cpm [3H]- H2O/g brain), p = 0.003 (Fig. 2.4). Despite use of syngeneic mice and a precisely timed 58 Figure 2.3 Evaluation of IL-2 induced vascular leak in the brain using [125I]-labeled albumin. Mice (n = 6) were injected with approximately 100,000 cpm of [125I]-labeled albumin in 100 μl PBS via tail vein 2 h after the last dose of IL-2 or no treatment (control). After 4 h, mice were sacrificed, the brains excised and analyzed for [125I]-labeled albumin accumulation. The results were expressed as percentage of radiolabeled albumin recovered from the brain with the total amount recovered from the animal. 59 60 Figure 2.4 Evaluation of changes in brain water content using tritiated water ([3H]-H2O). Two hours after the last IL-2 dose or no treatment (control), mice (n = 3 at 4 h, n = 8 at 12 and 18 h) were injected with ~300,000 cpm [3H]-H2O in 100 μl PBS via tail vein. The brain was harvested from each cohort of control and IL-2-treated mice at the specified time (4 h, 12 h, or 18 h) after [3H]-H2O injection. Brains were homogenized and 100 μl was counted in 2 ml scintillation fluid on a scintillation counter. The results were corrected for tissue quenching and expressed as cpm/gram tissue. * indicates p = 0.003. 61 62 treatment schedule, this technique exhibited quite a bit of variability between experiments. This led us to conclude that measurement of [3H]-H2O retention was not a reliable means of measuring IL-2 induced fluid shifts in the mouse brain. Measurement of Sodium Fluorescein (NaFl) extravasation in the mouse pleural fluid, lungs, and brain. NaFl is a small fluorescent molecule (MW = 376.27). This probe may provide increased sensitivity for detection of BBB disruption induced by IL-2 and decrease tissue quenching and volume of distribution problems related to the use of [3H]-H2O. We evaluated NaFl as an indicator for IL-2 induced VLS, based on its prior use as a marker for fluorescence imaging of BBB disruption in rats (39-40). Control and IL-2 treated mice were injected with 1.5 mg NaFl in 100 μl PBS via tail vein and sacrificed after 10 min. Pleural fluid was collected directly from the thoracic cavity of IL- 2 treated mice whereas the thoracic cavity of the control mice was irrigated with 500 μl PBS, which was then collected. The entire recovered volume was quantified. Fluorescence was measured by spectrofluorometer. Using a standard curve, the concentration of NaFl recovered from each mouse was quantified. By multiplying the concentration of NaFl by the total volume of the fluid recovered, we calculated the total amount of NaFl leak. IL-2 treatment increased NaFl extravasation into the pleural space by 9-fold (9.59±0.82 μg total NaFl) compared to the control mice (1.09±0.13 μg total NaFl), p < 0.0001 (Fig. 2.5A). In order to evaluate the usefulness of this technique for solid organs, the lungs and brain from these mice were excised. Each tissue was homogenized in 1 ml PBS, and the amount of NaFl in the homogenate quantified. Following correction of tissue-specific fluorescence quenching, we found 10.34±0.82 μg total NaFl in the lungs of IL-2 treated 63 mice. There was no detectable difference compared to NaFl content in lungs of the control mice (8.88±1.21 μg total NaFl, p = 0.35; Fig. 2.5B). There was also no detectable difference in the amount of total NaFl in the brains of IL-2 treated mice (1.06±0.05 μg total NaFl) compared to the control (1.14±0.06 μg total NaFl), p = 0.34 (Fig. 2.5C). This method proved useful in demonstrating small molecule leak resulting in pleural effusions, but not into solid organs of IL-2 treated mice. Perfusion of mice with saline to decrease intravascular NaFl retention did not seem to influence results in the brain (data not shown). Evaluation of brain blood vessel morphology using immunohistochemistry. It is well known that nitric oxide (NO) produced by endothelial cells or from pharmacologic donors induces vasodilation (41-43). We therefore evaluated whether vasodilation could be identified in brain blood vessels via immunohistochemistry. We prepared 6 μm sections from carefully defined anatomic region of the mouse mid-brain (Fig. 2.1) chosen to reflect highly vascular areas based on a study by Dorr et al. (35). Areas selected included the cerebral fissure, the striatum and the cortex in the highly vascularized anatomic region shown (Fig. 2.1). We tested a number of putative endothelial cell-specific antibodies, CD34, CD144 (VE-cadherin), and anti-von Willebrand Factor (vWF). All of them showed speckled endothelial cell staining (data not shown). As vWF is specifically produced in blood vessels, this marker was selected. However, the granular nature of the stain made it difficult to outline vessels to measure the area. Isolectin GS-IB4 (Iso-B4) demonstrates strong staining of perivascular cells. Unfortunately, Iso-B4 is also known to stain brain microglial cells. By using dual staining of vWF and Iso-B4, we were able to identify the circumference of blood vessels in the 64 Figure 2.5 Evaluation of blood vessel permeability using sodium fluorescein (NaFl) in the mouse A) pleural fluid, B) lungs, and C) brain. Mice (n = 5) were injected with 1.5 mg NaFl in 100 μl PBS via tail vein 4 h after the last IL-2 dose or no treatment (control). After 10 min, mice were sacrificed. The pleural fluid was collected and the brains and lungs were excised from each mouse. The lungs and brain were homogenized in 1 ml PBS and 100 μl of each homogenate or 100 μl of the collected pleural fluid was quantified by spectrofluorometer. The total amount (μg) of NaFl in each sample was calculated by using a standard curve, corrected for tissue quenching, and then multiplied by the total volume of organ homogenate (1 ml) or the measured volume of the collected pleural fluid. 65 66 mouse brain section. We obtained twenty 20x magnification digital images from precise anatomic locations of the brain section (4 of each left cortex area, right cortex, left striatum, right striatum, and the cerebral fissure) using fluorescence microscopy and analyzed each image for the numbers and size of blood vessels. The analysis showed that the average size of vessels in the brain was almost 2-fold larger in the IL-2 treated mice (37750.53±5598 pixel2) compared to the control mice (20652.85±5598 pixel2), p = 0.0389 (Fig. 2.6A), while the number of vessels in the brain of IL-2 treated mice (8.082±1.83) were not significantly different compared to the control mice (7.343±1.83), p = 0.7765 (Fig. 2.6B). The number of blood vessels was not expected to change during 5 days of IL-2 treatment. These results suggest that IL-2 strongly induced vasodilation of the brain blood vessels. Evaluation of brain water content using MRI proton density. Intrinsic MR parameters, such as longitudinal (T1) and transverse (T2) relaxation times, are sensitive to changes in the proton density and therefore the water content of tissues. Therefore, MRI proton density (PD) provides a potential noninvasive technique to quantify changes in the brain water content induced by cytokine treatment. A prior study in IL-2 treated patients using a T2-weighted MRI had suggested that particularly large changes in brain MR signal intensity were induced by IL-2 treatment (29). However, this study showed an extremely wide percentage range (3-48%) of the changes in the cerebral water content, raising questions as to the accuracy and reproducibility of this determination. We developed a high resolution murine imaging technique to critically assess MRI quantified PD changes in the brains of IL-2 treated mice. Imaging phantoms were prepared employing various ratios of H2O/D2O (60-100% in 10% increment), serving as 67 Figure 2.6 Immunostaining of brain sections to evaluate endothelial cell morphology. Brains were excised from IL-2 treated and control mice (n = 3). Sections (6 μm-thick) were carefully prepared to identify specific anatomic regions of the mid-brain. Each frozen section was dual-stained with Iso-B4 and anti-vWF to identify blood vessels and exclude microglial cells. The blood vessels were manually outlined on 20 digitized fluorescence images of each stained section and analyzed for A) the average size of vessels and B) total number of vessels using ImageJ software. The values were compared between pre- and post-IL2. 68 69 references. We scanned anesthetized mice both before and after IL-2 treatment using MRI PD sequence using a 7T Bruker MRI system with a mouse-specific coil. We established that this sequence was sensitive enough to detect an approximate 1% change in PD in the mouse brain. PD was calculated using a ratio of the signal intensity (SI) from the manually drawn ROI (region of interest) around the whole mouse brain with that of the phantom SI. We averaged PD over 8 matched brain slices of pre- and post-IL-2 DICOM images for each mouse. The results of this experiment (Fig. 2.7) established that there was no significant difference (p = 0.7892) in MRI proton density of IL-2 treated mice (73.31±0.7) compared to before receiving IL-2 treatment (73.65±0.7). We also found that results obtained with this technique varied markedly between experiments. We concluded that the MRI PD sequence was too inconsistent to provide useful information concerning the small, most likely perivascular changes in mouse brain water content related to IL-2 administration. Dynamic contrast-enhanced (DCE) MRI. DCE MRI is a quantitative, noninvasive technique to measure vascular permeability using a gadolinium-based contrast agent. DCE MRI has often been used clinically to assess tumor angiogenesis based on tumor vascular permeability. This technique provides physiologically meaningful quantitative assessment of vascular parameters, such as fractional plasma volume (fPV), endothelial transfer coefficient (KPS), and permeability surface area product (PS) (44). fPV describes the plasma volume of the blood vessels in that tissue. KPS reflects the permeability of the contrast agent from the blood vessels to the extravascular and extracellular space (EES) of the tissue. PS describes the surface area of the vasculature that is permeable to the contrast agent. We developed a protocol for DCE 70 Figure 2.7 Evaluation of proton density in the brains of IL-2 treated mice using a 7T MRI system. Mice (n = 5) were scanned both before and after 4 days of IL-2 treatment (150,000 U i.p, b.i.d.) using a MRI PD sequence as described in the Materials and Methods. Regions of interest were manually drawn on matched sections of mouse brain using OsiriX software and expressed as % proton density (PD) based on the phantoms (created using 60-100% H2O/D2O in 10% increment). The %PD was averaged over 8 slices for each mouse and compared between pre- and post-IL-2 treatment scans derived from the same mouse. 71 72 MRI to evaluate the brain microvasculature of IL-2 treated mice. Anesthetized mice were scanned before and after IL-2 treatment using 2D-FLASH MRI sequence. This MRI sequence provides good spatial resolution and has the advantage of allowing rapid scanning. We calculated SI from a manually drawn ROI on a selected mid-brain section of each mouse, matched between pre- and post-IL-2 images, using customized MATLAB programs. The contrast agent concentration in the blood pool was established by averaging the SI from the left and right maxillary veins. We used a two-compartment model consisting of the brain plasma compartment and the EES to calculate fPV, KPS, and PS as described previously by Feng et al. (37). DCE MRI data established a 2-fold increase of fPV in the mice brain after receiving IL-2 treatment (p = 0.0285; Fig. 2.8A). The fPV in the post-IL-2 mice brain was 3.048±0.56 whereas in the pre-IL-2 mice brain was 1.554±0.21. This result suggested vasodilation of the brain blood vessels following IL-2 treatment, independently verifying the results of brain immunohistochemical staining. DCE MRI also showed that IL-2 significantly increased brain microvascular permeability to the contrast agent by 2-fold (p = 0.0079; Fig. 2.8B). KPS value in the mice brain after receiving IL-2 treatment was 0.4305±0.05 ml/min/100 cm3 whereas prior to IL-2 treatment was 0.2039±0.04 ml/min/100 cm3. Finally, the PS was 1.275±0.22 ml/min/100 cm3 in the mice after receiving IL-2 while in the mice prior to the IL-2 treatment was 0.2953±0.05 ml/min/100 cm3 (Fig. 2.8C). These data suggested a significant (p = 0.0083) 4-fold increase in the blood vessel surface area that was "leaky" to the contrast agent after IL-2 treatment. These changes in brain vascular parameters identified using DCE MRI established that IL-2 induces vasodilation of the brain blood vessels and increases perivascular permeability of these vessels. 73 Figure 2.8 Evaluation of IL-2 induced changes in the brain blood vessel permeability by dynamic contrast enhanced (DCE) MRI. Mice (n = 5) were scanned both before and after 4 days of IL-2 treatment (150,000 U i.p, b.i.d.) using 2D-FLASH MRI sequence as described in the Materials and Methods. After baseline imaging, 0.1 mmol/kg contrast agent was administered by bolus injection via tail vein catheterization. Customized MATLAB programs were used to calculate signal intensities from manually drawn ROI on a selected 1-mm mid-brain section of each mouse. We averaged the left and right maxillary veins to derive the contrast agent concentration in the blood pool and a two-compartment model was used to calculate A) fractional plasma volume (fPV), B) endothelium transfer coefficient (KPS), and C) permeability surface area product (PS). IL-2 treatment significantly increased fPV and KPS by 2-fold, and PS by 4-fold. 74 75 Discussion Sepsis causes an estimated 215,000 deaths (9.3% of all deaths) in the US and considerable morbidity, mortality, health care utilization, and cost (45). Patients of early stages of sepsis develop similar symptoms to the side effects of IL-2 therapy, particularly hypotension, vascular leak, and neuropsychiatric toxicity (i.e., delirium). However, as sepsis progresses, patients experience irreversible organ failure, and eventually death. IL- 2 dose limiting toxicities are reversible and therefore may provide useful to model the early cardiovascular and neuropsychiatric effects of sepsis. A poorly characterized percentage of patients receiving IL-2 therapy become mentally slowed or confused (perhaps as high as 40% in our experience) and approximately 10-20% of patients become agitated, sometimes with hallucinations or delusions. The pathophysiology of IL-2 induced neuropsychiatric toxicity remains poorly understood. It is likely that the "cytokine storm" released during IL-2 treatment and sepsis induces changes in brain microvasculature, such as vascular leak and brain edema, which relate to changes in the CNS function and thus cause neuropsychiatric toxicity. We therefore evaluated a number of potential experimental methods to quantify the effect of cytokine-induced changes on brain blood vessels, utilizing a well-characterized IL-2 treatment murine model. We have performed extensive studies of different doses of IL-2 in mice. The dose of IL-2 chosen for the current experiments (150-180,000 IU twice daily) is based on dose and time response studies previously performed in our lab to evaluate changes in systemic vascular permeability and hypotension in our murine model, and is approximately the LD