ENDOGENOUS EXPRESSION OF BRAIN INTERLEUKIN-2 AND THE...

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ENDOGENOUS EXPRESSION OF BRAIN INTERLEUKIN-2 AND THE LINK TO ALTERATIONS IN CHOLINE ACETYLTRANSFERASE EXPRESSION IN

INTERLEUKIN-2 KNOCKOUT MICE

By

DANIELLE MARIE MEOLA

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2012

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© 2012 Danielle Marie Meola

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To my dog Prime, whose lifelong battle with illiteracy inspires me to strive for greatness despite the odds

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ACKNOWLEDGMENTS

First and foremost a special thanks must be extended to my mentor Dr. John

Petitto whose modest personality, sense of humor, and empathetic nature have made

him a pleasure to work for. His style of mentoring has provided me with a true

assessment of what it takes to generate your own ideas, and the hard part, see them

through. It is only the large stick by the door he intermittently threatens me with that

would clue you into his well understood frustration. I would also like to express my

gratitude toward the members of my supervisory committee. In addition to the time they

have volunteered to oversee my progress, each member has imparted wisdom and

attitudes that undoubtedly have influenced my own. Dr. Mark Lewis, who also mentored

me during my undergraduate years, has a kindness and generosity with his time that

has always made me feel welcome and encouraged. One of the best decisions I made

early on in my studies was joining Dr. Jake Streit’s journal club. Though I may not

remember the science we read, the underlying message stuck with me… be skeptical in

your reading, and there’s nothing wrong with going against dogma especially when the

evidence behind it is lacking…just don’t expect an easy paycheck. As for Dr. Michael

King, in addition to the many hours I spent working in his lab, I truly appreciate the

exchange of ideas and the help I’ve received from him regarding the direction of my

work. I am also indebted to him for expanding my taste in music- this guy’s just cooler

than an ice cube. While I have not had the opportunity to get to know Dr. Mark Atkinson

well, I am no less appreciative of his willingness to serve on my committee, and have

benefited from his insightful questions during our meetings. Besides my committee, I

must thank some folks who have helped me in a great number of ways. Dr. Huang Zhi,

an important contributor to my work and delightful person to have around the lab, has

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been paramount to my success. Also, my dear friend Dr. Amber Muehlmann, who has

provided me with guidance, a shoulder to lean on, and comic relief only a friend that has

gone before you can offer. I’d like to thank Bonnie McLaurin for teaching me about

animal procedures, how to navigate the IACUC, and for being that warm presence when

I just needed a hug. Lastly, I must thank my family for their patience, encouragement,

love, and monetary contributions that have made a higher education possible for me.

Most of all, I thank my fiancé Alan for believing in me 8 years ago when he selflessly

packed up his life and followed me to Gainesville even though he’s a diehard

Tennessee fan and Gator hater. I love you sweetheart.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF FIGURES .......................................................................................................... 8

ABSTRACT ................................................................................................................... 10

CHAPTER

1 BACKGROUND AND SIGNIFICANCE ................................................................... 12

Cytokine-Brain Interactions ..................................................................................... 12 Interleukin-2: a Pleiotropic Cytokine ....................................................................... 13

Evidence of IL-2’s Action’s in the CNS.................................................................... 14 Detection of IL-2 in the Brain .................................................................................. 16 Alteration of Septohippocampal System in IL-2 Knock-Out Mice ............................ 16

Morphology and Behavior................................................................................. 16 Cytokine Profile ................................................................................................ 17

Neurotrophic Environment ................................................................................ 18 T Lymphocyte Trafficking to Brains of IL-2KO Mice ................................................ 18 Objectives of This Dissertation Research ............................................................... 19

2 PHENOTYPIC LOSS OF SEPTOHIPPOCAMPAL CHOLINERGIC NEURONS: RELATION TO BRAIN VERSUS PERIPHERAL IL-2 DEFICIENCY ....................... 22

Introduction ............................................................................................................. 22 Materials and Methods............................................................................................ 24

Animals............................................................................................................. 24 Immunohistochemistry...................................................................................... 25 Cytokine Analysis ............................................................................................. 26

Neurotrophin Analysis ...................................................................................... 27 Cell Quantification ............................................................................................ 28

Results .................................................................................................................... 29

Discussion .............................................................................................................. 30

3 EXPRESSION OF GFP IN B6.CG-TG (IL2-EGFP) 17EXR TRANSGENIC MICE: EVIDENCE FOR THE NEURONAL EXPRESSION OF INTERLEUKIN-2 IN DISCRETE REGIONS OF MURINE BRAIN ....................................................... 35

Introduction ............................................................................................................. 35 Materials and Methods............................................................................................ 37

Animals............................................................................................................. 37 Breeding and Genotyping ................................................................................. 37

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Tissue Preparation ........................................................................................... 37

Immunohistochemistry...................................................................................... 38 Results .................................................................................................................... 38

Discussion .............................................................................................................. 39

4 EXPRESSION OF IL-2 IN RESPONSE TO SYSTEMIC LPS CHALLENGE AND FACIAL NERVE AXOTOMY IN B6.CG- TG (IL2-EGFP) 17EVR TRANSGENIC MICE ....................................................................................................................... 47

Introduction ............................................................................................................. 47

Materials and Methods............................................................................................ 48 Animals............................................................................................................. 48 Facial Nerve Axotomy ...................................................................................... 48

Lipopolysaccharide ........................................................................................... 48 Tissue Preparation ........................................................................................... 48 Immunohistochemistry...................................................................................... 49

Results .................................................................................................................... 50 Discussion .............................................................................................................. 50

5 CONCLUSION ........................................................................................................ 52

Summary of the Overall Findings ............................................................................ 52 Implications ............................................................................................................. 54

Caveats and Future Directions ............................................................................... 56 Concluding Remarks............................................................................................... 57

REFERENCES .............................................................................................................. 58

BIOGRAPHICAL SKETCH ............................................................................................ 67

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LIST OF FIGURES

Figure page 2-1 Quantification of ChAT+ cells in the mouse medial septum of subject groups

at 8 weeks of age. Bars represent the mean ± S.E.M. for IL-2WT(C57), IL-2KO/RAG-2KO(KO/KO), and IL-2KO mice. N=5 mice/group. *p< .05. ............... 34

2-2 Comparison of NGF (left) and BDNF (right) protein levels in the medial septum of IL-2KO mice (n=7) vs. IL-2WT littermates (n=6). Bars represent mean ± S.E.M. .................................................................................................... 34

3-1 GFP positive cells in the lateral septum (top) and in the spleen (bottom). Bottom panel illustrates specificity of GFP antibody. Reactivity of primary antibody was quenched with 10µl free recombinant GFP protein prior to use in staining protocol. ............................................................................................. 43

3-2 Representative micropictographs showing expression of GFP in the medial septum (top) and red nucleus (bottom). Arrows annotate cellular GFP expression co-localized with pan-neuronal cell marker NeuN. ........................... 44

3-3 GFP positive cells in the lateral septum (left) and fastigial nucleus and interposed nucleus of the cerebellum (right). Shown here at 10X. ..................... 45

4-1 No evidence of IL-2 expression in injured facial motor nucleus (area indicated by rectangular border). Representative section stained with anti-GFP primary antibody 7 days after periphery nerve axotomy. Note contrast with positively stained cells in reticular nucleus above. .............................................. 51

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LIST OF TABLES

Table page 3-1 List of nuclei positive for IL-2 transgene through rostral-caudal extent of brain

and brainstem. (+) symbol designates relative intensity of GFP staining as visualized by fluorescence immunohistochemistry ............................................. 46

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

ENDOGENOUS EXPRESSION OF BRAIN INTERLEUKIN-2 AND THE LINK TO

ALTERATIONS IN CHOLINE ACETYLTRANSFERASE EXPRESSION IN INTERLEUKIN-2 KNOCKOUT MICE

By

Danielle Marie Meola

December 2012

Chair: John Michael Petitto Major: Medical Sciences

In the peripheral immune system, Interleukin-2 (IL-2) is essential for immune

homeostasis, normal T regulatory cell function, and self-tolerance. IL-2 knockout (IL-

2KO) mice develop spontaneous autoimmunity characterized by increased T cell

trafficking to multiple organs. IL-2 is also expressed in the brain. We previously

described the apparent loss of cholinergic cell bodies in the medial septum of IL-2KO

mice. Here we investigated if loss of brain-derived IL-2, or autoimmunity stemming from

loss of peripheral IL-2, is responsible for the alteration in choline acetyltransferase

(ChAT) expression in the medial septum of IL-2KO mice. To accomplish this objective,

we compared ChAT-positive neurons between IL-2 wild-type (IL-2WT) mice, IL-2KO,

and congenic IL-2/recombinase activating gene-2 KO (IL-2KO/RAG-2KO) mice that lack

IL-2 but fail to develop autoimmunity. IL-2KO and IL-2KO/RAG-2KO mice had

significantly lower numbers of ChAT-positive neurons than IL-2WT mice. This did not

coincide with an overall loss of cells in the medial septum suggesting that loss of brain

IL-2 results in a change in cholinergic phenotype unrelated to cell death. No differences

were noted in the endogenous expression of cytokines and chemokines tested in the

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medial septum. Evaluation of brain derived neurotrophic factor (BDNF) and nerve

growth factor (NGF) levels between IL-2WT and IL-2KO mice in medial septal

homogenates revealed that IL-2KO mice have markedly higher levels of NGF in the

medial septum compared to IL-2WT mice. Our findings suggest that brain-derived IL-2

plays an essential role in the maintenance of septohippocampal projection neurons in

vivo.

In the second part of this dissertation project, to determine the origin of brain-

derived IL-2, we used a novel transgenic mouse model that expresses green

fluorescent protein (GFP) in cells that normally express IL-2. We found that a number

of discrete brain regions express GFP and the expression is selective for neurons.

There are several reports that IL-2 is expressed by rat microglia, however, we found

microglia activated in vivo by peripheral nerve injury or challenged by intraperitoneal

injection of lipopolysaccharide, do not express GFP in our model.

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CHAPTER 1 BACKGROUND AND SIGNIFICANCE

Cytokine-Brain Interactions

The central nervous system (CNS) and peripheral immune system were once

considered separate, compartmentalized systems necessitated by the brain’s

vulnerability to inflammatory processes and marked by the brain’s reduced ability to

respond to immune challenge and reject transplants (Barker and Widner, 2004). It is

now understood that the two systems work in concert to achieve many homeostatic

mechanisms both related to immune protection and normal physiological activities

including neuroinflammation and autoimmunity, viral infection, hypothalamic-pituitary

axis (HPA) regulation, induction of fever, sleep, analgesia, feeding behavior, and

cognition (Ader et al., 2001; Dunn, 2002; Wilson et al., 2002).

The cytokines produced by immune cells, and involved in the intercommunication

of the two systems, are ushered through the blood-brain-barrier (BBB) to initiate their

effects on the CNS by way of various transporters (Banks et al., 2002), the “leaky”

circumventricular organs (CVO), and, as is the case with IL-2, non-saturable

mechanisms that have yet to be determined (Waguespack et al., 1994). Peripheral

leukocytes, in particular activated T cells that enter the brain during certain conditions

(e.g., EAE, facial nerve axotomy, IL-2 deficiency), can also release cytokines in the

CNS (Hickey et al., 1991). In addition to peripheral cytokines finding their way to central

targets, immune competent cells in the brain such as microglia and perivascular

macrophages, that respond to immune challenge in the brain, produce and secrete

many of the same cytokines and may play an important role in both the innate and

adaptive arms of the immune response (Petitto et al., 2001).

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While much attention has been given to immune cytokines expressed by

immune-type cells, neurons have also been shown to express cytokines and cytokine

receptors typically associated with immune functions (Sorkin et al., 1997). The role of

immune cytokines expressed by neurons, and the functional significance of cytokine

receptors on select populations of neurons, is a relatively new area of study and a

promising avenue for discovery regarding the influence of brain-derived cytokines on

normal brain function, and immune-derived cytokines in injury and disease, via

converging signaling pathways. The focus of this dissertation is on Interleukin-2 (IL-2),

which can be produced in the periphery and in the CNS.

Interleukin-2: a Pleiotropic Cytokine

IL-2, as its name suggests, is best known for its actions in the peripheral immune

system where it is commonly secreted by leukocytes to signal activation and

differentiation to a number of cell types most notably, T cells, B cells, and macrophages

(Waldmann, 2002). IL-2 belongs to the four -helix bundle family of cytokines and

signals via a common gamma (c) subunit shared by multiple cytokines including IL-4,

IL-7, IL-9, and IL-15 (Sugamura et al., 1996); a subunit only shared with IL-15 (Giri et

al., 1995); and, in one conformation, an subunit, which confers high affinity binding

(Leonard et al., 1984).

The creation of a transgenic knockout mouse model has provided further

information on the inherent function of IL-2 in the immune system and its role in self-

tolerance (Schmitt et al., 1994). IL-2 knockout (KO) mice develop autoimmune

symptoms including ulcerative colitis, although the manifestation of the phenotype (e.g.

advanced hemolytic anemia) is dependent on the genetic background of the knockout

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mice (Horak, 1995, 1996). The autoimmune phenotype that develops when the IL-2

gene is deleted is T cell dependent and marked by infiltration of auto-reactive T cells to

several tissues and organ systems (Ma et al., 1995). Further investigation of these

knockout mice has revealed that the apparent mechanism of autoimmunity is driven by

the limited development and ability of regulatory T cells (CD4+CD25+ T reg cells) to

promote self-tolerance and suppress T cell responses in vivo (Nelson, 2004). Though

extensive research has characterized IL-2 in the peripheral immune system, increasing

evidence indicates that IL-2 may play a role in normal brain functioning and may

potentially be involved in the pathogenesis of a number of neuropsychiatric and

neurodegenerative diseases where alterations in IL-2 function and IL-2 gene

polymorphisms have been reported (e.g., Alzheimer’s, multiple sclerosis, schizophrenia,

and Tourette syndrome) (Mahendran et al., 2004; Beloosesky et al., 2002; Morer et al.,

2010; Cavanilla et al., 2010).

Evidence of IL-2’s Action’s in the CNS

The effect of IL-2 on cognition and mood in humans were among the earliest

findings that suggested that this cytokine might have neurobiological actions. In early

clinical studies of the cognitive side effects of IL-2 therapy, 50% (i.e., 22 patients out of

44) of the subjects monitored developed cognitive changes, with 15 of them

necessitating acute intervention (Denicoff et al., 1987). In other studies, IL-2 therapy

was found to impair spatial memory and performance in planning tasks (Capuron et al.,

2001a), and induce depressive symptoms as early as two days into therapy (Capuron et

al., 2000). In general, the most notable side effects occurred with higher doses and/or

longer treatment intervals. In addition to cognitive and emotional changes, other

neurological side effects may include drowsiness, aphasia, blurred or double vision, and

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loss of taste (published in the Proleukin package insert - Cetus Oncology—US, Rev

5/92).

The impact of IL-2 on learning and memory has been investigated in

hippocampal slice culture and was found to modulate aspects of both short-term (STP)

and long-term potentiation (LTP) (Tancredi et al., 1990), and has been shown to interact

with NMDA receptors and influence peak amplitudes in a concentration-dependent

manner (Bender et al., 1996).

IL-2 can also modulate the release of some neurotransmitters such as dopamine

(Alonso et al., 1993; Petitto et al., 1997), and acetylcholine (Hanisch et al., 1993; Seto

et al., 1997). The most potent effects have been on acetylcholine (Ach) release from

septohippocampal cholinergic neurons in culture (Hanisch et al., 1993; Seto et al.,

1997). Exogenous IL-2 has also been shown to have trophic effects on fetal septal and

hippocampal cells marked by increased viability and dendritic sprouting (Sarder et al.,

1993; Sarder et al., 1996). Our lab has previously detected IL-2 receptors in the

habenula, medial septum, hippocampal formation, and associated limbic regions in

mouse, and suspect that these effects are a result of direct signaling through IL-2

receptors expressed by these neurons (Petitto and Huang, 2001).

Exogenously administered IL-2 also has multiple effects on the hypothalamic-

pituitary axis (HPA). Effects on pituitary cells include stimulation of cortisol production

and adrenal corticotropin releasing hormone (Hanisch et al., 1994), and increased

pituitary cell responsiveness to corticotropin-releasing hormone (Witzke et al., 2003).

IL-2 has also been shown to regulate the production and secretion of peptides from

hypothalamus (Karanth et al., 1993; Lapchak and Araujo, 1993; Pardy et al., 1993).

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Detection of IL-2 in the Brain

Detection of IL-2 expression in the CNS has been challenging due to the

instability of its mRNA and short half-life of the protein (Chappell et al., 1998). There

has been some success in detecting IL- 2 mRNA in homogenized samples of murine

hippocampus, amygdala, and pre-frontal cortex by reverse transcriptase PCR (Banks et

al., 1991). IL-2 protein has also been detected in the hippocampus, amygdala, pre-

frontal cortex, cerebral cortex, striatum and pituitary gland by enzyme-linked

immunoabsorbent assays (Lee et al., 2008). These studies make evident the normal

expression of IL-2 in the brain, however, they do not provide any information about

cellular origin. In-situ IL-2 immunoreactivity has been mapped to discrete areas of

perfused rat forebrain including the septohippocampal system, hippocampus, and

related limbic regions (Lapchak et al.1991; Villemain, 1991; Lapchak, 1993), however,

the techniques used in these studies (e.g. antiserum directed against recombinant

human IL-2) resulted in poor resolution and IL-2 reactivity appeared scattered and

nonspecific. Overall, studies aiming to describe the origin of IL-2 in the brain have been

inconclusive and have yielded conflicting results that range from widespread expression

that appears non-specific to expression that is very limited or undetectable.

Alteration of Septohippocampal System in IL-2 Knock-Out Mice

Morphology and Behavior

To assess the importance of brain-derived IL-2 on neuronal function in vivo, our

lab has investigated the morphological status of IL-2 related structures in an IL-2KO

mouse model on the C57BL/6 background. IL-2KO mice had a 26% reduction in medial

septal cholinergic neurons as assessed by choline acetyltransferase (ChAT) staining.

Loss of IL-2 selectively affected medial septal cholinergic neurons, as there were no

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differences in GABAergic cells originating from the medial septum and no changes in

the cholinergic neurons in the striatum (Beck et al., 2002). In the hippocampus, IL-2KO

mice had fewer granule cells (Beck et al., 2005a) and reduced infrapyramidal (IP)

mossy fiber length (Petitto et al., 1999); a measure that has been positively correlated

with spatial learning ability (Schopke et al., 1991; Schwegler and Crusio, 1995;

Schwegler et al., 1988).

We previously observed that IL-2KO mice had markedly impaired spatial learning

and memory in the Morris water-maze. Examination of other domains of behavioral

performance showed that IL-2KO mice did not differ from WT controls in measures of

fearfulness or locomotor activity in the elevated plus-maze, or in reflexive startle

responses to auditory stimuli, although, sensory motor gating assessed by pre-pulse

inhibition of acoustic startle (PPI) was increased significantly (Petitto et al., 1999).

Cytokine Profile

IL-2 gene deletion alters the neuroimmunological status of the mouse

hippocampus. Our lab previously measured cytokines in the hippocampus and serum

of IL-2KO mice by multiplex microsphere cytokine analysis (Beck et al., 2005b).

Compared to IL-2WT mice, in the hippocampus of IL-2KO mice we detected an

increase in known T cell chemoattractants interleukin-15 (IL-15), monocyte chemotactic

protein-1 (MCP-1) and Interferon gamma-induced protein 10 (IP-10) (Beck et al.,

2005b). The unique cytokine profiles detected in the serum versus hippocampal tissue

indicated that IL-2 deficiency alters the neuroimmunological status of the hippocampus

by influencing the endogenous production of immune cytokines, rather than by

peripheral cytokines traversing the BBB.

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Neurotrophic Environment

Compared to wild-type mice, IL-2KO mice have drastically reduced

concentrations of brain-derived neurotrophic factor (BDNF) and a reciprocal increase of

nerve growth factor (NGF) in the hippocampus (Beck et al., 2005a). This significant

(~50%) alteration in neurotrophin levels may explain some of the neuropathologies we

have observed in the septohippocampal system of our model.

T Lymphocyte Trafficking to Brains of IL-2KO Mice

Although not presented in its entirety here, prior to the studies that make up the

later chapters of this dissertation, we investigated the trafficking of IL-2WT and IL-2KO

T cells to the septum, hippocampus, and cerebellum of transgenic mice.

In this study, we used an experimental approach that used a combination of

inter-breeding IL-2KO, IL-2WT, and RAG-2KO mice, to produce immunodeficient mice

that either have functional brain IL-2 (IL-2WT/RAG2KO) or lack brain IL-2 (IL-

2KO/RAG2KO). These animals were then reconstituted with either (normal) IL-2WT or

(autoimmune) IL-2KO splenocytes to evaluate whether IL-2 deficiency in the brain, or

rather autoimmunity found in IL-2KO mice is responsible for the upregulation of T cell

trafficking in the brains of IL-2KO mice (Huang., 2009). In IL-2KO/RAG2KO mice that

were reconstituted with a wild-type immune system, the number of T cells found in all

regions quantified (hippocampus, septum, and cerebellum) was doubled compared to

IL-2WT/RAG2KO mice reconstituted with a wild-type immune system. In IL-

2WT/RAG2KO mice that did not lack brain IL-2 and reconstituted with autoreactive T

cells from IL-2 KO mice, there was a comparable increase of T cells in the hippocampus

and septum (Huang et al., 2011). These findings demonstrate that brain IL-2 deficiency,

and possibly subsequent changes in the CNS (e.g. cytokines, chemokines, and

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neurotrophic factors), may lead to the development of T cell mediated inflammatory

processes in the brain. Conversely, as previously shown in our lab using the facial

nerve axotomy model of peripheral nerve injury, T cells that enter the CNS may play a

role in maintaining the viability of neurons. In these studies we demonstrated that in WT

mice, re-injury of the axotomized facial motor nerve results in the reversal of neuronal

atrophy and loss of cholinergic phenotype typically observed after initial injury.

RAG2KO mice, that lack a functional immune system, do not exhibit these effects (Ha et

al., 2007). Therefore, given the known pathology of the medial septum and

hippocampus of IL-2KO mice, wild-type T cells trafficking to the CNS of IL-

2KO/RAG2KO mice may indicate an underlying supportive function of T cells in

response to the neuropathology caused by loss of endogenous IL-2 signaling in the

brain.

Objectives of This Dissertation Research

In the following chapter, we sought to disentangle the contributions of IL-2

deficiency in the brain versus in the peripheral immune system to alterations in

cholinergic expression in the medial septum. While IL-2KO mice of the C57BL/6 strain

are relatively autoimmune resistant, they do develop pathology in the bowel and exhibit

splenomegaly that coincides with an increase of T cells in the CNS (Huang et al., 2009;

Huang et al., 2011). In the IL-2KO model, we cannot be certain whether the changes in

the medial septum are due to related “autoimmunity” in the brain, or rather a

dysregulation of the cholinergic septum due to loss of endogenous IL-2. To make this

distinction, we again bred IL-2KO/RAG2KO mice and compared the number of ChAT

positive cells in the medial septum with those of IL-2KO and IL-2WT mice. This strategy

allowed us to remove the confounding effects of autoreactive T cells as well as draw

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conclusions about the efficacy of T cells (if playing a supportive role) to rescue medial

septal neurons from effects of IL-2 deficiency. In addition to answering this important

question about the nature of our model, we evaluated the expression profile of multiple

cytokines and chemokines in IL-2KO mice to reveal any inflammatory signaling in the

medial septum and to better understand the mechanism by which T cells are drawn to

the CNS in response to loss of IL-2. Lastly, we tested whether the changes in

neurotrophins we previously detected in the hippocampus was consistent with levels in

the medial septum.

Despite interest for many years about IL-2 as a possible neurotrophic factor or

neuromodulator in the septohippocampal system, reliable tools have been lacking to

study the cell types and circuitry involved in IL-2’s actions. In Chapter 3 we describe the

expression profile of an IL2-GFP transgene reporter mouse (IL-2p8-GFP) that could

provide a powerful tool to advance this field. Unlike the autoradiography studies done in

rats, because GFP has a significantly longer half-life than IL-2 and is not secreted from

its cell of origin, we can provide a clear account of IL-2 expression without the problems

of cross-reactivity to other cytokines or background staining of secreted IL-2 that may

explain the unspecific binding and wide range of detection abilities characteristic of

existing methodologies. Here we use fluorescent immunohistochemistry co-labeling

techniques to determine which cell types (i.e. neurons or glia), and which select nuclei

throughout the brain and brainstem, express the IL2-GFP transgene.

In the final chapter, we investigated whether activation of microglia in vivo would

activate the expression of the IL2-GFP transgene in our model. While the expression of

IL-2 from microglia has been reported in rat (Girard et al., 2008), to our knowledge there

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is no evidence of IL-2 expression from murine microglia. Mirroring work done in rats

shown to activate microglia in vivo, we administered intraperitoneal injections of

lipopolysaccharide to IL-2p8-GFP mice to evaluate the expression of IL-2 in the brain

under these conditions. In a second experiment, we tested the expression of IL-2 from

activated microglia in the facial nerve axotomy model. Axotomy of the peripheral nerve

causes a localized proliferation and activation of microglia in the facial motor nucleus.

This paradigm allowed us to investigate the microglial expression of IL-2 under

alternative immune activating conditions and at the same time evaluate whether injury

induces neuronal expression of IL-2 in the facial nucleus.

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CHAPTER 2 PHENOTYPIC LOSS OF SEPTOHIPPOCAMPAL CHOLINERGIC NEURONS:

RELATION TO BRAIN VERSUS PERIPHERAL IL-2 DEFICIENCY

Introduction

Interleukin-2 (IL-2) has been implicated in the pathogenesis of several major

neurological and neuropsychiatric disorders including multiple sclerosis, Alzheimer’s

disease and schizophrenia (Hanisch & Quirion, 1995; Merrill, 1990). The indispensable

role of IL-2 for normal immune system functioning was discovered when IL-2 knockout

(IL-2KO) mice demonstrated that IL-2 deficiency results in increased T cell trafficking

and autoimmunity to multiple organ systems (Horak, 1995; Kundig et al., 1993; Schorle,

Holtschke, Hunig, Schimpl, & Horak, 1991), and by research showing that IL-2 is

essential for immune homeostasis, normal T regulatory cell function, and self-tolerance

(Nelson, 2004; Turka & Walsh, 2008). IL-2 is also expressed by brain cells. IL-2

receptors are enriched in the septohippocampal system where the cytokine has been

shown to have trophic effects on fetal septal and hippocampal neurons, and have potent

effects on acetylcholine release from septohippocampal cholinergic neurons (Awatsuji,

Furukawa, Nakajima, Furukawa, & Hayashi, 1993; Hanisch, Seto, & Quirion, 1993;

Petitto & Huang, 2001; Sarder, Saito, & Abe, 1993; Seto, Kar, & Quirion, 1997). In

addition to IL-2’s actions in the immune and central nervous systems, we have found

that loss of brain IL-2 gene expression results in dysregulation of the brain’s

endogenous neuroimmunological milieu (e.g., alterations in the normal balance of

cytokines and chemokines), and that such effects may be involved in initiating

processes that lead to central nervous system (CNS) autoimmunity (Beck et al., 2005b;

Huang et al., 2009; Huang et al., 2011).

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We found previously that compared to wild-type (WT) littermates, adult IL-2

deficient mice had a marked reduction of choline acetyltransferase (ChAT) positive

medial septum/diagonal band of Broca (MS/vDB) cell bodies (Beck, King, Huang, &

Petitto, 2002). This loss of ChAT-positive neurons was selective for medial septum, as

the cholinergic phenotype of WT and IL-2KO mice did not differ in the number of ChAT-

positive neurons in the striatum, and GABAergic neurons in the MS/vDB did not differ

between IL-2WT and IL-2KO mice (Beck et al., 2002). Central versus peripheral

immunological contributions on brain development and neuropathology are not well

understood. Neuroimmunology studies revealed that T lymphocytes can have important

effects on CNS neurons, and normal peripheral T cell function has been found to be

essential for the preservation of the phenotype of injured motoneurons (Ha, Huang, &

Petitto, 2007; Jones, Serpe, Byram, Deboy, & Sanders, 2005; Schwartz & Moalem,

2001). We previously found in IL-2KO mice that there is a marked infiltration of T cells to

the brain that mirrors, in relative magnitude, the progression of autoimmunity in the

periphery (Huang et al., 2009). In the present study, we sought to test the hypothesis

that the loss of quantifiable medial septal cholinergic neurons in IL-2KO mice is due to

the loss of cholinergic phenotype rather than neuronal cell loss, and that the loss of

phenotype is due to loss of brain-derived IL-2 rather than changes in neuroimmune

status or T cell infiltration. In Experiment 1, we sought to determine if the loss ChAT-

positive neurons in the medial septum was due to loss of central (brain-derived) IL-2,

peripheral IL-2 (autoimmunity), or a combination of both factors. To accomplish this

objective, in Experiment 1 we compared ChAT-positive neurons between IL-2WT mice,

IL-2KO and congenic IL-2KO/RAG-2KO mice bred in our lab. These double knockout

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IL-2KO/RAG-2KO mice have peripheral immunodeficiency resulting from the absence of

mature T and B cells associated with the loss of both RAG-2 gene alleles, and also

have both IL-2 gene alleles deleted. In Experiment 2, we determined if the loss of the

IL-2 gene resulted in changes in the endogenous expression of cytokines and

chemokines in the medial septum. In Experiment 3, we quantified total neurons in the

medial septum to test our working hypothesis that the marked reduction of ChAT-

positive neurons in the medial septum of IL-2KO mice is due to the loss of the

cholinergic phenotype, rather than neuronal cell loss. Exploring a potential mechanism

for downregulation of cholinergic phenotype (Lazo et al., 2010; Van der Zee & Hagg,

2002; Ward & Hagg, 2000), in Experiment 3 we also quantified BDNF and NGF in the

medial septum to assess how levels of these neurotrophic factors correlate with

changes in ChAT-positive neurons in the medial septum of IL-2KO mice.

Materials and Methods

Animals

Mice used in these experiments were cared for in accordance with the NIH Guide

for the Care and Use of Laboratory Animals and housed under specific pathogen-free

conditions. All animals used in these experiments were 8–12 weeks of age, and were

matched for age and balanced for sex. IL-2KO mice were bred in our colony using IL-2

heterozygote by IL-2 heterozygote crosses as described previously (Huang et al.,

2009). The IL-2KO mice, obtained originally from the NIH repository at Jackson

Laboratories, were derived from ten generations of backcrossing onto the C57BL/6

background. IL-2KO/RAG-2KO mice were bred in our colony using recombinase

activating gene 2 knockout (RAG-2KO) mice that were originally obtained from Taconic

farms. The RAG-2 protein is necessary for the recombination of T cell receptors and

25

immunoglobulins, therefore, RAG-2KO mice fail to develop a mature and functional T

and B cells. The breeding of these congenic mice was performed as described

previously by our lab, where IL-2KO mice where bred with RAG-2KO mice, producing

mice with both IL-2 and RAG-2 alleles deleted - referred to here as IL-2KO/RAG-2KO

(Huang et al., 2011). All mice used in study were on C57BL/6 background. Genotypes

of mice were determined by PCR as described previously (Huang et al.,

2009). Statistical analyses for these studies were performed using analysis of variance

(ANOVA), and post-hoc comparisons were performed using Fisher’s post-hoc analysis.

Immunohistochemistry

Mice were anesthetized by a 0.5mg/mL ketamine cocktail in a 3:3:1 ratio

(ketamine/xylazine/acepromazine) and were perfused with 4% buffered formaldehyde.

Brains were dissected, post-fixed 2 hrs, and cryoprotected in 30% sucrose overnight.

Tissue was snap frozen in isopentane and stored at −80°C. Coronal sections were cut

through the brain and brainstem at a thickness of 15 or 40μm. 15µm sections were

collected on Superfrost/Plus slides (Fisher Scientific) and stored at −80°C until staining

could be performed. 40µm sections were collected in 0.1 M phosphate buffered saline

(PBS) and immediately used in staining protocol. Tissue sections were incubated in

normal goat serum (Vector; 1:30 in PBS) for 1 hour at room temperature followed by

overnight incubation at 4°C with the primary antibodies rabbit anti-ChAT (Chemicon;

1:2000 in PBS with 0.3% Triton-X-100 and 1% normal goat serum (NGS)), or rabbit

anti-beta-III tubulin (Chemicon; 1:1000 in PBS with 0.3% Triton-X-100 and 1% NGS,

200 μL/well). Sections were washed and incubated overnight in the secondary antibody,

biotinylated goat anti-rabbit IgG (Sigma B-7389; 1:1000 dilution in PBS with 0.3%

Triton-X-100 and 1% NGS). The sections were then washed and incubated in

26

ExtrAvidin (Sigma E-2886; 1:1000 in PBS) for 2 h. The sections were developed in 0.5

mg/ml 3,3′-diaminobenzidine (DAB), 0.2 mg/ml urea H2O2 for approximately 5 min and

were placed on slides, dehydrated in graded ethanol washes, cleared in two changes of

xylenes, and coverslipped.

Cytokine Analysis

Septal homogenates were analyzed from IL-2KO and WT mice to compare

cytokine levels in the septum as described previously (Beck et al., 2005b). Briefly, mice

were anesthetized with an injection cocktail of 3:3:1 ketamine (100 mg/mL)/xylazine (20

mg/mL)/acepromazine (10 mg/mL) at a dose of 0.015 mL injection cocktail/g body

weight. The animals were then saline perfused. The brains were removed, snap frozen,

and then allowed to equilibrate to −20 °C. The brains were sectioned on a cryostat at

−20 °C at 400 μm thickness and the septum was dissected from sections with a 0.75

mm micropunch on a −20 °C freezing platform. The dissected tissue was weighed on a

microgram scale and then transferred to 25 μL of homogenizing solution (500 mM

NaH2PO4/Na2HPO4 buffer and 0.2% TX-100 in H2O with anti-protease complete TM

cocktail (Boehringer) per mg of wet weight tissue). The tissue was sonicated in the

homogenizing solution for 30 s on ice and centrifuged at 16,000 × g for 15 min at 4 °C.

The supernatant was collected and stored at −20 °C for Luminex analysis. Multiplex

microsphere cytokine analysis was performed to measure a number of cytokines in the

septum of IL-2KO and WT mice using Lincoplex mouse cytokine (Linco, Research, Inc)

and Luminex 100 LabMAP system (Upstate Biotechnology) kits. Assays were

performed according to the manufacturer's instructions, and cytokine concentrations

were calculated using the Softmax program and the linear range on the standard curve

27

(3.2–10,000 pg/mL). Altogether, we attempted to detect a total of 22 different cytokines

and chemokines.

Neurotrophin Analysis

Enzyme-linked immunosorbent assay (ELISA) measurement of NGF and BDNF

was performed as described previously (Beck et al., 2005a). Levels of NGF and BDNF

were analyzed in the homogenates from medial septum using a commercially available

Emax immunoassay system according to the manufacturer's instructions (Promega).

Briefly, the 96-well plates were coated with 1:6250 anti-NGF polyclonal antibodies in

carbonate coating buffer (0.025 M sodium bicarbonate, 0.025 M sodium carbonate, pH

9.7) and incubated overnight at 4 °C. The plates were washed with TBST wash buffer

(20 mM Tris–HCL pH 7.6, 150 mM NaCl, 0.05% (v/v) Tween 20) and blocked with 1×

Block and Sample buffer (provided with kit) for 1 h. The plates were washed again with

TBST, and a set of standard curves were generated in duplicate by performing 1:2

dilutions of a known 500 pg/mL standard in a range from 500 to 7.6 pg/mL followed by a

“blank” well of 0 pg/mL. All added samples and standards were allowed to incubate at

25 °C for 6 h. The plates were washed thoroughly with TBST, and a 1:4000 anti-NGF

monoclonal antibody was added and incubated overnight at 4 °C. The plates were again

washed with TBST, and a 1:100 anti-rat IgG polyclonal antibody conjugated to HRP was

added for 2.5 h at room temperature. The plates were washed, and TMB One Solution

was added for color development for 10 min. The reaction was stopped with the addition

of equal volume of 1 N HCl, and the absorbance was read at 450 nm within 30 min of

the color development reaction. The data were reported as pg of protein per mg wet

weight tissue.

28

Cell Quantification

For quantification of stained neuronal somata of the medial septum cells were

counted using the software MCID 5.1 and the three-dimensional counting box (optical

dissector) method described by Williams and Rakic (Williams & Rakic, 1988) as

described previously by our lab (Beck et al., 2002). All stereology was performed using

a CCD High Resolution Sony camera and a Zeiss Axioplan 2 microscope with a

motorized x-y stage made by Imaging Research, Inc. The latter is capable of making

movements as fine as 0.1 μm. Every third section through the anterior-posterior extent

of the septal region was sampled. The regions to be counted were outlined at 10x

magnification and the size of the counting boxes were generated to be approximately

5% of the most rostral, and therefore, smallest, area of the medial septum (defined by

the section where the corpus collosum first joins in the midline). The size of the outlined

count regions, but not the counting box, varied depending on where the individual

section was taken from the rostral to caudal extent of the medial septum. The defined

counting box was approximately 2-2.5% of the outlined count area of the largest single

section of the medial septum. Quantification of ChAT positive neurons was performed

on 20 µm sections stored at −80°C that were used to assess T cells and microglia from

other brain regions for other ongoing studies in our lab, and the neuronal assessments

were done as described previously in our lab for comparing relative difference between

groups (Ha et al., 2006). Planimetric counting methods were used due to section

thickness. A total 30 sections throughout the entire medial septum per animal were

collected. Eight sections per animal (approximately 1/4 of the entire medial septum),

were used to quantify the number of cholinergic neurons. Sections were chosen

throughout the medial septum at a fixed interval with every fourth section selected for

29

quantification. ChAT-immunostained histological sections were processed for cell

number quantification within the region of interest encompassing the individual left and

right medial septal nuclei defined by a triangular shape that extended, dorso-ventrally,

from the apex of the medial septum to an imaginary line connecting the lower limits of

the anterior commissures on each hemisphere and, medio-laterally, from the midline to

the outer limits of the medial septal area (Lopez-Coviella et al., 2011). Briefly, color

images were taken with a SPOT digital camera at 10X magnification. ImageJ (National

Institutes of Health) was used to view the images and perform the planimetric cell

counting.

Results

In Experiment 1, we compared ChAT-positive medial septal neurons between

IL-2WT, IL-2KO and congenic IL-2KO/RAG-2KO mice. As seen in Figure 1, there was a

significant main effect of subject group [F(2,10)=38.9, p< .05]. Post-hoc analyses

confirmed that IL-2KO and IL-2KO/RAG-2KO mice did not differ from one another,

however, both of these subject groups had significantly lower ChAT-positive neuron

numbers than IL-2WT mice (p<.05).

In Experiment 2, to determine if the loss of IL-2 resulted in changes in the

endogenous expression of cytokines and chemokines in the medial septum, we

compared levels of 22 different cytokines and chemokines between IL-2WT (n=8) and

IL-2KO (n=7) mice. In medial septal homogenates, there were detectable levels of IL-6,

IL-1, IL-17, IL-7, IL-9, IL-12, IL-15, interferon-gamma inducible protein of 10 kD (IP-10),

and monocyte chemoattractant protein-1 (MCP-1). Thus, only those cytokines and

chemokines detected were subjected to statistical analyses. The remainder of the

cytokines and chemokines tested could not be detected, these were: IFN-γ; TNF-α; IL-

30

1α, IL-1β; IL-2, IL-4, IL-5, IL-10, IL-13, kerotinocyte-derived chemokines (KC),

granulocyte-stimulating factor (G-CSF), macrophage inflammatory protein-1 alpha (MIP-

1), and RANTES. Among the cytokines and chemokines that were measurable, there

were no differences between IL-2WT and IL-2KO mice.

In Experiment 3, we quantified total neurons in the medial septum by counting

cells positive for the pan-neuronal marker beta-III tubulin between IL-2WT (n=5) and IL-

2KO (n=5) mice, to test the hypothesis that the marked reduction of ChAT-positive

neurons in the medial septum of IL-2KO mice is due to the loss of the cholinergic

phenotype. We found that there were no differences in beta-III tubulin stained neurons

between the IL-2WT and IL-2KO mice (data not shown). We also compared BDNF and

NGF levels between IL-2WT and IL-2KO mice in medial septal homogenates. Figure 2

shows the results of the comparison of these neurotrophins between the groups. As

seen in Figure 2, IL-2KO mice had markedly higher levels of NGF in the medial septum

compared to IL-2WT mice [F(1,11)=13.3, p<.005]. For BDNF, however, there were no

differences between these subject groups.

Discussion

Consistent with our hypothesis, the results of these quantitative experiments

show that the reduction in stained neurons in the medial septum is not due to cell loss,

but to a change in cholinergic phenotype. Since IL-2KO mice do not produce brain IL-2,

but have peripheral T cell dysregulation and autoimmunity from the loss of IL-2 in the

peripheral immune system, we needed to determine if the reduction in medial septal

ChAT-positive neurons was the result of the peripheral immune alterations associated

with the loss of peripheral IL-2. Using IL-2KO/RAG-2KO mice that lack a functional

immune system, we established that the loss of ChAT-positive cells in the medial

31

septum is due to loss of brain-derived IL-2 rather than the peripheral immune

dysregulation present in IL-2KO mice (Huang et al., 2009). Quantitative comparisons of

total cells in the medial septum of IL-2KO and IL-2WT mice revealed that the reduction

in ChAT-positive cells in the medial septum is indicative of a change in cholinergic

phenotype rather than cell death. Loss of the cholinergic phenotype by medial septal

neurons has been recognized since the seminal study by Hagg and colleagues (Hagg,

Manthorpe, Vahlsing, & Varon, 1988), who demonstrated that loss of medial septal

cholinergic phenotype occurs following axotomy, and is reversible with

intracerebroventricular infusion of NGF.

Surprisingly, although there were no differences in detectable cytokines and

chemokines in the medial septum, we found previously that IL-2KO mice had alterations

in the hippocampus (Beck et al., 2005a). Thus, it appears that loss of IL-2 modifies the

neuroimmunological environment differently in different regions of the brain. The

neurotrophic factor that is specifically responsible for maintaining cholinergic phenotype,

NGF, was elevated while BDNF levels were not abnormal in the absence of IL-2. IL-2

has been found to modify the expression of neurotrophin receptors in lymphocytes

(Besser & Wank, 1999), however, the effects of IL-2 on NGF in the brain is unknown.

Septal cholinergic neurons account for most of the cholinergic innervation of the

hippocampus and play a key role in the regulation of hippocampal synaptic activity

(Lazo et al., 2010). Since neuronal expression of neurotrophins is controlled by some

neurotransmitters and there is a topographical correlation between neurotrophin

expression and cholinergic terminal distribution from the cholinergic basal forebrain (Yu,

Pizzo, Hutton, & Perez-Polo, 1995), it has been investigated whether cholinergic

32

afferents regulate neurotrophin gene expression in the hippocampus. When cholinergic

neurons were selectively and completely destroyed by intraventricular injection of 192

IgG-saporin, resulting in a cholinergic deafferentation of the hippocampus, there were

no significant changes in NGF or BDNF mRNA levels from 1 week to 5 months after the

lesion (Yu et al., 1995). Similarly, in a developmental study using hippocampal slice

culture, changes in neurotrophin expression in the excised hippocampus over time

reflected the changes that occur in vivo (Forster, Otten, & Frotscher, 1993). These

results suggest that cholinergic afferents may not play a significant role in maintaining

basal levels of neurotrophin gene expression in the hippocampus, and that perhaps loss

of IL-2, rather than the consequence of changes in cholinergic functionality, may be

responsible for changes in neurotrophic environment. This dysregulation of

hippocampal and medial septal neurotrophins may be, in part, responsible for the failure

of cholinergic neuronal maintenance seen in the Ms/vDB of IL-2KO mice. Further

studies are needed to determine the purported point of convergence of the IL-2 and

neurotrophin signaling pathways.

In summary, the reduction of cholinergic cells in the medial septum of IL-2KO

mice is due to loss of brain-derived IL-2 rather than neuroimmunological processes

initiated by the peripheral T cell dysregulation and autoimmunity that develops in these

mice. The loss of ChAT staining in the medial septum of IL-2KO mice did not coincide

with loss of total neurons, suggesting that the failure to visualize these cells by ChAT

immunohistochemistry is due to a down regulation of the protein and a consequent

change in cholinergic phenotype. Lastly, we detected an increase in NGF in the medial

septum that mirrored the increase we previously reported in the hippocampus (Beck et

33

al., 2005a). This dysregulation of the neurotrophin environment in the

septohippocampal pathway, in response to loss of brain-derived IL-2, is a likely

candidate in the etiology of the observed changes in phenotype, or conversely, may

play some compensatory function in providing cholinergic support. If the latter is true,

and IL-2 deficiency has direct consequences on cholinergic function, we are compelled

to hypothesize that endogenously produced brain IL-2 has a biologically significant role

in maintaining cholinergic circuitry in the septohippocampal system.

34

Figure 2-1. Quantification of ChAT+ cells in the mouse medial septum of subject groups

at 8 weeks of age. Bars represent the mean ± S.E.M. for IL-2WT(C57), IL-2KO/RAG-2KO(KO/KO), and IL-2KO mice. N=5 mice/group. *p< .05.

Figure 2-2. Comparison of BDNF (left) and NGF (right) protein levels in the medial septum of IL-2KO mice (n=7) vs. IL-2WT littermates (n=6). Bars represent mean ± S.E.M.

35

CHAPTER 3 EXPRESSION OF GFP IN B6.CG-TG (IL2-EGFP) 17EXR TRANSGENIC MICE:

EVIDENCE FOR THE NEURONAL EXPRESSION OF INTERLEUKIN-2 IN DISCRETE REGIONS OF MURINE BRAIN

Introduction

Interleukin-2 (IL-2) is widely regarded as a pro-inflammatory cytokine responsible

for regulating homeostasis, activation, and clonal expansion of T cells in the peripheral

immune system. There are several lines of evidence that suggest IL-2 may also function

as a neuromodulator. Most of our understanding about the role IL-2 may play in the

brain comes from studies that demonstrate how exogenously administered IL-2 to

neurons in culture has dynamic effects on several key functions such as long-term and

short-term potentiation in the hippocampus (Tancredi et al., 1990; Bender et al., 1996),

neurotransmitter release from cholinergic and dopaminergic neurons (Alonso et al.,

1993; Petitto et al., 1997; Hanisch et al., 1993; Seto et al., 1997), and has been shown

to have neurotrophic effects on septal and hippocampal primary cell cultures (Sarder et

al., 1993; Sarder et al., 1996). Despite interest for many years about IL-2 as a possible

neurotrophic factor or neuromodulator, reliable tools have been lacking to study the cell

types and circuitry involved in IL-2’s actions. In-situ IL-2 immunoreactivity has been

mapped to discrete areas of perfused rat forebrain including the septohippocampal

system, hippocampus, and related limbic regions (Lapchak et al.1991; Villemain, 1991;

Lapchak, 1993), however, the techniques used in these studies (e.g. antiserum directed

against recombinant human IL-2) resulted in poor resolution and IL-2 reactivity

appeared scattered and nonspecific. Overall, studies aiming to describe the origin of IL-

2 in the brain have been inconclusive and have yielded conflicting results that range

36

from widespread expression that appears non-specific to expression that is very limited

or undetectable.

B6.Cg-Tg(Il2-EGFP)17Evr (IL2p8-GFP) transgenic mice, generated by targeting

a new upstream regulatory region of the IL-2 gene, reliably express green fluorescent

protein (GFP) in immune cells known to produce IL-2 (Eizenberg et al., 1995). The

expression of GFP in the brains of these animals has not been documented.

Here we report on the expression of GFP from the brains of IL2p8-GFP mice, a

novel approach that could provide a powerful tool to advance this field. Unlike the

autoradiography studies done in rats, because GFP has a significantly longer half-life

than IL-2 and is not secreted from its cell of origin, we can provide a clear account of IL-

2 expression without the problems of cross-reactivity to other cytokines or background

staining of secreted IL-2 that may explain the nonspecific binding and wide range of

detection abilities characteristic of existing methodologies. Here we use fluorescent

immunohistochemistry co-labeling techniques to determine which cell types (i.e.

neurons or glia), and which select nuclei throughout the rostral-caudal extent of the

brain and brainstem, express the IL2-GFP transgene.

We observed GFP expression in the immune cells of the spleen and thymus, and

in neuronal populations of the septal nuclei, thalamus, hypothalamus, striatum, cortex,

and brainstem. Our findings suggest that IL-2 is produced by neurons in discrete

regions of the mouse brain and supports the hypothesis that IL-2 is used in normal brain

signaling. IL-2 has been implicated in a number of psychiatric and neurodegenerative

disorders where aberrant levels of central and peripheral IL-2 have been reported

(Mahendran et al., 2004; Beloosesky et al., 2002; Cavanilla et al., 2010). The mounting

37

evidence supporting a role of IL-2 in normal brain functioning should lead to a new

perspective in evaluating IL-2 levels in assessing and studying neurological disease.


Animals

All mice in this study were cared for in compliance with the NIH Guide for the

Care and Use of Laboratory Animals. Mice were housed in microisolater cages under

specific pathogen free conditions.

Breeding and Genotyping

Female B6.Cg-Tg (Il2-EGFP) 17Evr (IL2p8-GFP) mice were obtained from the

Mutant Mouse Regional Resource Center and bred with C57BL/6 mice obtained from

Jackson Laboratories. Transgene positive offspring were identified by PCR analysis of

tail DNA. PCR primers in the IL-2 proximal promoter (IL2-1F: 5′-

CATCCTTAGATGCAACCCTTCC-3′) and the GFP coding sequence (GFP-1R: 5′-

GCTGAACTTGTGGCCGTTTAC-3′) were used, amplifying a 830-bp product in

transgene-positive mice. PCR conditions were as follows: 94°C, 5 min, then 35 cycles of

93°C, 30 s; 62°C, 15 s; 72°C, 45 s, followed by a final 5 min at 72°C, using an i cycler

(BioRad).

Tissue Preparation

Mice were anesthetized by a 0.5mg/mL ketamine cocktail in a 3:3:1 ratio

(ketamine/xylazine/acepromazine) and were perfused with 4% paraformaldehyde (PF).

Brains were dissected, post-fixed in 4% PF, and dehydrated in 30% sucrose overnight.


throughout the brain and brainstem at a thickness of 30μm. Sections were collected on

38

Superfrost/Plus slides (Fisher Scientific) and stored at −80°C until staining could be

performed.


Tissue sections were air-dried and were incubated in normal goat serum (Vector;

1:30 in PBS) for 1 hour at room temperature followed by overnight incubation at 4°C

with the primary antibodies mouse anti-NeuN (MAB377; 1:250; Millipore), and rabbit

anti-GFP (A-11122; 1:5000; Life Technologies). Phosphate buffer saline (1X) was used

for all wash steps performed between incubation steps (Fisher Scientific). Visualization

of the primary antibody was performed by incubating sections in goat anti-mouse Texas

Red secondary antibody (1:300; Life Technologies), and goat anti-rabbit Alexa Fluor

488 secondary antibody (1:300; Life Technologies) for 2 hours at room temperature.

Sections were coverslipped with Vectashield mounting medium (Vector Laboratories).

Specificity of antibodies were tested by systematic omission of either primary or

secondary antibody and specificity of anti-GFP was further challenged by pre-incubating

the primary antibody with recombinant GFP protein prior to use in staining the protocol

(figure 3-1). All qualitative assessments of GFP staining in mice positive for the reporter

were made in comparison to GFP negative littermates.

Results

In this study we used fluorescent immunohistochemistry co-localization

techniques to determine the cellular origin of IL-2 in IL2p8-GFP transgenic mice that co-

express GFP reporter in cells that express IL-2. To test the specificity of the GFP

antibody, we pre-incubated the primary antibody with recombinant GFP prior to use in

our staining protocol to quench its ability to bind the antigen in tissue. Spleen tissue,

that reliably exhibits transgene expression from T cells, was used as a positive control

39

for each staining procedure (Figure 3-1). GFP expression was found throughout the

brain in discrete nuclei with apparent differences in relative staining intensity (Table 3-

1). In most cells expressing GFP, the reporter was co-localized with NeuN, a pan-

neuronal cell marker we used to confirm the neuronal phenotype of GFP positive cells

(Figure 3-2). The few cells stained positive for GFP but not NeuN were morphologically

and geographically identical to those expressing both markers.

Discussion

The IL2p8-GFP strain has proven to be a successful model for evaluating the

endogenous expression of IL-2 in the peripheral immune system in vivo. IL-2 is

implicated in a number of neurobiological processes and numerous studies have

attempted to determine the origin of brain-derived IL-2, however, the elusive properties

of IL-2 mRNA and protein have thwarted attempts using conventional methodologies.

Here we discuss our findings in the context of our previous work in the

septohippocampal system of IL-2KO mice and speculate on the significance of IL-2

expression in less familiar regions based on our histological data and known anatomical

and functional properties of regions positive for the reporter.

Multiple studies have shown that exogenously administered IL-2 to

septohippocampal neurons has potent effects on the release of acetylcholine from

septal neurons and trophic effects on both cell types in culture (Sarder et al., 1993;

Sarder et al., 1996). The known circuitry of the septohippocampal system, and

distribution of IL-2 receptors in the limbic system (Lapchak et al., 1991; Petitto et al.,

1995), corresponds well with the expression pattern of GFP we found in IL2p8-GFP

mice. The lateral septum, where robust expression of GFP was detected, projects

mainly to the medial septum and hippocampus and is therefore well positioned to

40

provide modulatory input to the septohippocampal system (Figure 3-3). We also

detected conservative expression of GFP from a subset of cells in the subiculum, the

main output structure of the hippocampus, that projects back to the septal nuclei (in

addition to other GFP positive regions i.e., prefrontal cortex, hypothalamus, mammillary

nuclei, entorhinal cortex and amygdala). We previously described the effects of brain-

derived IL-2 deficiency on cholinergic phenotype and neurotrophin expression in the

septohippocampal system of IL-2KO mice (Beck et al., 2005a; Meola et al., under

review). IL-2KO mice exhibit a significant loss of ChAT expression in the medial septum

and neurotrophin levels in both the medial septum and hippocampus are altered.

Because we did not detect GFP in the hippocampus, we suspect that these effects may

be due to lack of IL-2 signaling upstream in the septal nuclei that may interfere with the

impact of NGF arrival from the hippocampal projection field that can regulate the

expression of ChAT (Hagg et al., 1988). Because IL-2 receptors are expressed both in

the septum and hippocampus (Petitto et al., 1995), and neurotrophins in the

hippocampus are expressed independently of cholinergic innervation (Forster, Otten, &

Frotscher, 1993; Yu et al., 1995), further studies designed to identify a point of

convergence of the NGF and IL-2 signaling pathways are needed to determine how loss

of IL-2 signaling from the septal nuclei could result in these deleterious effects.

In addition to the neuropathology in the septum and hippocampus, we have

detected changes in several measures of behavior. IL-2KO mice exhibit markedly

impaired spatial learning and memory in the Morris water maze and while no differences

were found in reflexive startle responses to auditory stimuli, pre-pulse inhibition of

acoustic startle (PPI), a measure of sensorimotor gating, was increased significantly

41

(Petitto et al., 1999). The entorhinal cortex is well established as the seat of spatial

memory and navigation, whereas the dorsal endopiriform nucleus, lateral septum,

amygdala, and cingulate are known to be involved in sensorimotor gating and were

found to express GFP in ILp8-GFP reporter mice.

Further evidence of a pleiotropic role for IL-2 in the immune and nervous systems

has been demonstrated by systemic administration of IL-2. In studies evaluating the

neuropsychiatric symptoms of patients receiving IL-2 therapy, ~50% developed

cognitive and emotional symptomology as early as 2 days into therapy (Denicoff et al.,

1987; Capuron et al., 2000). Neurobiological side effects published by the manufacturer

of the IL-2 drug Proleukin include depression, hallucinations, memory deficits, dizziness,

fever, blurred vision and loss of taste. In this study, we found GFP to be expressed in

the septal nuclei, amygdala, median raphe nucleus, entorhinal cortex, vestibular nuclei,

solitary nucleus, olfactory bulb, and lateral geniculate nucleus of the thalamus, which

may help explain why those receiving IL-2 therapy report these disturbances.

Exogenously administered IL-2 also has multiple effects on the hypothalamic-

pituitary axis (HPA). Effects on pituitary cells include stimulation of cortisol production

and adrenal corticotropin releasing hormone (Hanisch et al., 1994), and increased

pituitary cell responsiveness to corticotropin-releasing hormone (Witzke et al., 2003).

IL-2 has also been shown to regulate the production and secretion of peptides from the

hypothalamus (Karanth et al., 1993; Lapchak and Araujo, 1993; Pardy et al., 1993). In

IL2p8-GFP mice, GFP was expressed from the paraventricular hypothalamic nuclei that

have direct projections to the pituitary gland, and the anterior/posterior hypothalamic

nuclei involved in thermoregulation.

42

Briefly, it is notable that many nuclei that were found to express GFP are

interconnected and have similar functional roles or modalities (Table 3-1). For example,

the motor cortex, anterior and lateral aspects of the striatum, interposed nuclei, red

nuclei, inferior olivary nuclei, and the gigantocellular reticular nucleus are all involved in

motor control; whereas the somatosensory cortex, fastigial nucleus of the cerebellum,

vestibular nuclei, and the mesencephalic nucleus of the trigeminal nerve are important

to proprioception. Lastly, many positive nuclei are involved in nociception and

analgesia: periaqueductal gray, central gray of the pons, and the raphe magnus

nucleus. Overall, the anatomical expression profile of GFP in this reporter model syncs

well with the existing literature and appears to be restricted to specific modalities. Our

data should help fill the gaps in our current knowledge of the origin of IL-2 in the brain,

as well as inspire novel inquiries as to its function in the nervous system. In this study

we evaluated the expression of GFP in the brains of ILp8-GFP mice under normal

physiological conditions. We did not detect GFP in any cell types other than neurons.

A few studies have reported on the expression of IL-2 from microglia in response to

lipopolysaccharide, a potent inducer of inflammation, and insults such as hypoxic

ischemia (Kowalski et al., 2004; Girard et al., 2008). In the future, it will be interesting to

test methods of microglia activation in the IL2p8-GFP transgenic reporter model.

43

Figure 3-1. GFP positive cells in the lateral septum (top) and in the spleen (bottom). Bottom panel illustrates specificity of GFP antibody. Reactivity of primary antibody was quenched with 10µl free recombinant GFP protein prior to use in staining protocol.

44

Figure 3-2. Representative micropictographs showing expression of GFP in the medial septum (top) and red nucleus (bottom). Arrows annotate cells positive for GFP (left), pan-neuronal cell marker NeuN (center), co-localization of both markers (right).

45

Figure 3-3. GFP positive cells in the lateral septum (left); fastigial nucleus and

interposed nucleus of the cerebellum (right). Shown here at 10X magnification.

Fastigial nucleus cerebellum

Posterior interposed nucleus

46

Table 3-1. List of nuclei positive for the IL-2 transgene throughout the rostral-caudal extent of the brain and brainstem. (+) symbol designates relative intensity of GFP staining as visualized by fluorescence immunohistochemistry

Nucleus Modality Relative staining intensity

Mitral cell layer olfactory bulb Olfactory ++

Granular cell layer olfactory bulb Olfactory +++

External piriform layer olfactory bulb Olfactory ++

Anterior olfactory nucleus (ventral and medial)

Olfactory ++

Ventral and lateral orbital cortices Limbic ++

Cingulate Limbic ++++

Motor 1 Motor ++

Motor 2 Motor ++

Dorsal endopiriform nucleus Sensory motor gating (SMG)

++++

Striatum Motor, SMG +

Lateral septum Limbic, SMG ++++

Medial septum Limbic ++

Horizontal limb diagonal band of Broca Limbic ++

Subiculum Limbic +

Paraventricular hypothalamic nucleus Limbic, Autonomic +++

Basal lateral amygdaloid nucleus Limbic +

Anterior and posterior hypothalamus Autonomic ++

Ventrolateral geniculate nucleus, parvocellular

Vision +++

Nucleus of the solitary tract Chemosensation ++++

Periaqueductal gray Analgesia ++

Median raphe nucleus Analgesia ++

Magnocellular reticular formation/ Gigantocellular reticular formation

Mixed ++++

Red nucleus Motor ++++

Entorhinal cortex (medial/lateral ) Spatial memory ++++

Mammillary bodies Memory ++++

Mesencephalic nucleus of 5 Proprioception, Motor ++

Pontine gray Relay ++

Lateral vestibular nucleus Proprioception, Motor +++

Fastigial nucleus cerebellum Proprioception, Motor ++++

Posterior interposed nucleus Proprioception, Motor ++++

Inferior olivary nucleus Proprioception, Motor +++

47

CHAPTER 4 EXPRESSION OF IL-2 IN RESPONSE TO SYSTEMIC LPS CHALLENGE AND

FACIAL NERVE AXOTOMY IN B6.CG- TG (IL2-EGFP) 17EVR TRANSGENIC MICE

Introduction

Interleukin-2 (IL-2), a well characterized immune system cytokine, has been

increasingly recognized as an endogenous brain-derived cytokine that may play a role

in the normal function of multiple CNS pathways. We have recently reported on the

neuronal expression of green fluorescent protein (GFP) in B6.Cg-Tg (Il2-EGFP) 17Evr

(IL2p8-GFP) transgenic mice, that reliably express the reporter in cells known to

produce IL-2 (Eizenberg et al., 1995; Meola et al., under review). As noted in the

previous study (Chapter 3), evaluation of GFP expression in IL2p8-GFP transgenic mice

indicate that resting microglia do not express IL-2 under normal physiological

conditions. Studies that report immunoreactive localization of IL-2 in rat microglia have

used measures of immune activation to induce microglia activation such as

intraperitoneal injection (i.p.) of lipopolysaccharide (LPS), a potent antigen from gram

negative bacteria, and physical manipulations such as hypoxic ischemia (Girard et al.,

2008). In this study we used two methods of microglia activation in ILp8-GFP mice to

assess the expression of IL-2 from activated microglia in vivo. Facial nerve axotomy, a

model of peripheral nerve injury we routinely use in our lab to evaluate

neuroinflammatory processes, results in the activation and proliferation of microglia in

the facial motor nucleus. Second, we used the standard procedure of i.p. injection of

LPS to induce systemic inflammation and activation of microglia in the brain.

48


Animals

Three B6.Cg-Tg (Il2-EGFP) 17Evr transgenic mice and two wild-type littermates

negative for the GFP reporter were used in all groups for each experiment (total of 30

mice). Transgene positive offspring were identified by PCR analysis of tail DNA. PCR

primers in the IL-2 proximal promoter (IL2-1F: 5′-CATCCTTAGATGCAACCCTTCC-3′)

and the GFP coding sequence (GFP-1R: 5′-GCTGAACTTGTGGCCGTTTAC-3′) were

used, amplifying a 830-bp product in transgene-positive mice. PCR conditions were as

follows: 94°C, 5 min, then 35 cycles of 93°C, 30 s; 62°C, 15 s; 72°C, 45 s, followed by a

final 5 min at 72°C, using an i cycler (BioRad).

Facial Nerve Axotomy

Animals were anesthetized with 4 % isoflurane. The right facial nerve was

exposed, and a portion of the main branch resected to prevent nerve reconnection as

described previously (Ha et al., 2007). The whisker response was assessed after

surgery to ensure complete whisker paralysis. Mice in each group were then sacrificed

at 7 and 14 days after resection injury to assess CD11b+ and MHC-II+ microglia in the

injured FMN.

Lipopolysaccharide

Animals received 1mg/kg LPS (Sigma) dissolved in 500uL of 1X PBS via i.p.

injection. Control animals received 1 volume of vehicle. Animals were sacrificed at 3,

6, and 26 hours after injection (Jeong et al., 2010; Terrando et al., 2010).

Tissue Preparation

Mice were anesthetized by a 0.5mg/ml ketamine cocktail in a 3:3:1 ratio

(ketamine/xylazine/acepromazine) and were perfused with 4% paraformaldehyde (PF).

49

Brains were dissected, post-fixed in 4% PF, and dehydrated in 30% sucrose overnight.


throughout the brain and brainstem at a thickness of 30μm. Sections were collected on

Superfrost/Plus slides (Fisher Scientific) and stored at −80°C until staining could be

performed.


Tissue sections were air-dried and were incubated in normal goat serum (Vector;

1:30 in PBS) for 1 hour at room temperature followed by overnight incubation at 4°C

with the primary antibodies rat anti-cd11b (MAB11372; Millipore), rat anti-MHC-II

(M5/114.15.2; PharMingen) or rabbit anti-GFP (A-11122; 1:5000; Life Technologies).

Phosphate buffer saline (1X) was used for all wash steps performed between incubation

steps (Fisher Scientific). Visualization of the primary antibodies for microglia markers

cd11b and MHC-II, was performed by incubating sections in goat anti-rat secondary

antibody (1:2000, Vector Labs) for 2 h at room temperature followed by incubation in

avidin-peroxidase conjugates (1:500, Sigma) for 1 h. No signal was obtained with each

of the primary or secondary antibodies alone. The chromagen reaction was revealed by

incubation in 3,3′-diaminobenzidine (DAB)-H2O2 solution (Sigma; 0.07% DAB/0.004%

H2O2). Sections were dehydrated in ascending alcohol washes, cleared in xylenes, and

coverslipped. (1:300; Life Technologies). Visualization of the primary antibody for GFP

was performed by incubating sections in goat anti-rabbit Alexa Fluor 488 secondary

antibody (1:300; Life Technologies) overnight at 4°C, washed, and coverslipped with

Vectashield mounting medium (Vector Laboratories). Spleens of transgene positive

animals were stained for GFP as positive controls.

50

Results

Groups that received facial nerve axotomy, assessed at either 7 or 14 days post-

surgery, exhibited robust activation of microglia as visualized by cd11b staining and

assessed by phenotypic changes in morphology, i.e. amoeboid vs. ramified phenotype,

and formation of phagocytic clusters around dying neurons (Ha et al., 2007). No GFP

staining was detected in activated microglia from either group (Figure 4-1).

Animals that received LPS showed a conservative increase in activation of

microglia at 26 hours after injection compared to those assessed at 3 and 6 hours (data

not shown). Sections stained for GFP did not reveal any expression in all animals

tested regardless of activation state. Spleens of transgene positive animals were

stained for GFP as positive controls.

Discussion

Previous studies that have reported the expression of IL-2 in microglia both in

vivo and in vitro have been conducted in rats (Girard et al., 2008; Kowalski et al., 2004).

Differences may exist between the two species in the expression of IL-2 or possibly in

the methods necessary to induce the expression of IL-2 in activated microglia. In

agreement with our findings, one study that incubated primary cultures of rat microglia

in a range of LPS concentrations found upregulation of a number of inflammatory

cytokines but failed to detect IL-2 during the 72 hour incubation period (Du and Li,

1998). We previous characterized the expression of GFP in the brains of naive IL-2p8-

GFP mice and found that all IL-2 expressing cells were neuronal with no indication of

expression from glial type cells. Pioneering studies done by the group that generated

the IL-2p8-GFP strain, report that GFP is reliably expressed by immune type cells in the

periphery that normally express IL-2. In this study, we used the spleens of transgenic

51

animals as positive controls due to the robust expression of IL-2 from T cells in the

white pulp of these tissues. Therefore, it does not appear that the GFP transgene

expressed in this model is restricted to neuronal subtypes or lacks the ability to be

expressed properly in cells of haematopoietic lineage. Together, these observations

and the data presented in Chapter 3 indicate that microglia in the mouse brain do not

express IL-2 in this model under any of the conditions tested.

Figure 4-1. IL-2 expression in injured facial motor nucleus (area indicated by

rectangular border). Representative section stained with anti-GFP primary antibody 7 days after periphery nerve axotomy. Note lack of IL-2 staining in area of interest in contrast to positive staining in the reticular nucleus above

52

CHAPTER 5 CONCLUSION

Summary of the Overall Findings

The overall purpose of this dissertation research was to investigate the

expression profile of IL-2 in the brain and relate those findings to our understanding of

how loss of IL-2 impacts the septohippocampal system. In Chapter 2, we aimed to

expand on our previous research and challenge the original postulation by our lab that

the observed loss of cholinergic somata in the medial septum of IL-2KO mice was due

to inflammatory processes. In these experiments, we tested the hypothesis that loss of

cholinergic staining in the medial septum was due to loss of brain-derived IL-2. To

accomplish this, we used congenic IL-2KO/RAG-2KO mice bred in our lab that lack the

T cell dependent autoimmune phenotype present in IL-2KO mice. We compared the

relative loss of ChAT positive cells in IL-2KO, IL-2KO/RAG-2KO, and IL-2WT animals

and found that both KO strains had a similar loss of cholinergic staining in the medial

septum compared to WT animals. We also evaluated the relative expression of

cytokines and chemokines in the medial septum of IL-2KO and IL-2WT mice. We found

no differences between groups. These data support the hypothesis that loss of ChAT

staining in the medial septum of IL-2 KO mice is due to loss of brain-derived IL-2 rather

than changes in cytokines associated with autoimmunity. To gain further insight into the

effect IL-2 deficiency has on septohippocampal cholinergic neurons, we compared

numbers of beta-III tubulin positive cells in medial septum of IL-2KO and IL-2WT mice.

There was no significant difference in the number of total neurons in the medial septum

of either group. This suggests that the loss of ChAT staining in the medial septum of IL-

2KO mice is due to a down regulation of ChAT and thus, a change in cholinergic

53

phenotype unrelated to cell death. This observation is in agreement with our previous

inability to detect any glial activation, indicative of cellular clearance of dead neurons in

these mice. Nerve growth factor (NGF) is well characterized as the trophic factor

responsible for maintaining cholinergic phenotype in a number of pathways. Here we

measured the relative expression of the neurotrophic factors, NGF and BDNF, in the

medial septum to compare to our previous data in the hippocampus. We found there to

be, similar to the hippocampus, a substantial dysregulation of NGF in the medial

septum. Further studies are necessary to determine how brain-derived IL-2 influences

the expression of neurotrophic factors in the septohippocampal system and how that

can affect the cholinergic phenotype of cells in the septal nuclei.

In Chapter 3, we described the expression of GFP in brains of IL-2p8-GFP

transgenic mice that express GFP in cells that normally express IL-2. This model offers

a clear interpretation of the cellular source of IL-2, more so than any method that was

previously available. Using fluorescent co-labeling techniques, we determined that IL-2

is selectively expressed by neurons in discrete nuclei throughout the brain and

brainstem. The expression profile of IL-2 in the mouse brain syncs well with the existing

literature and provides us the impetus and direction to investigate whether similar

pathologies exist in the multiple brain regions that express IL-2, similar to those we have

characterized in the septohippocampal system. Understanding how loss of IL-2 can

impact multiple systems can provide greater insight into the functional nature of IL-2 as

an apparent neuromodulator.

Finally, in Chapter 4 we investigated whether IL-2p8-GFP microglia activated in

vivo expressed GFP. Several studies in rats have concluded that microglia can be

54

activated to express IL-2 upon immune challenge both in vivo and in vitro (Kowalski et

al., 2004; Girard et al., 2008). We found, however, using two different methods of

microglia activation, that microglia did not express IL-2 at any of the time-points

evaluated. These findings are at odds with the current literature and may be due to

differences in rat versus mouse physiology or differences between experimental

conditions. In addition to activation and proliferation of microglia, in the facial nerve

axotomy injury model, there is an infiltration of T cells to the injured facial motor nucleus

(FMN). Not only did we fail to observe IL-2 expression in activated microglia in this

paradigm, most of the T cells present in the FMN did not appear to express IL-2

although we observed a strong baseline expression of IL-2 (GFP) in the spleens of

these mice. Although outside the scope of this study, it is interesting to entertain the

possibility of a mechanism that would restrict the expression of IL-2 in immune cells to

the peripheral compartment, considering the cross-talk possible between infiltrating

immune cells and brain circuits that rely on IL-2 signaling in normal brain physiology.

Implications

This series of studies is the first to demonstrate that endogenous levels of brain-

derived IL-2 may be an important factor in maintaining the cholinergic projections

between the septal nuclei and hippocampus. The impact on this pathway involves the

expression of neurotrophic factors proven necessary for the normal functionality of the

septohippocampal system. We postulate that this change in trophic factors contributes

to the observed alteration in cholinergic phenotype and that IL-2 exerts its modulatory

actions on converging pathways downstream of IL-2 and neurotrophin signaling.

Clinically, exogenously administered IL-2 is used to treat a number of disorders,

including cancer (Atkins et al., 1999; Davis and Gillies, 2003; Fyfe et al., 1995; Guirguis

55

et al., 2002; Parkinson et al., 1990; Rosenberg et al., 1985; Rosenberg et al., 1994),

and HIV infection (Armstrong and Kazanjian, 2001; Mitsuyasu, 2001; Smith, 2001). IL-2

treatments are not, however, without side effects. As noted earlier, a number of studies

have investigated the diverse neuropsychiatric and cognitive changes in patients

receiving IL-2 therapy (Denicoff et al., 1987; Capuron et al., 2000; Capuron et al., 2001;

Walker et al., 1997). While these clinical investigations identified cognitive dysfunction

associated with IL-2 therapy, they did not offer any insight to the physiological

mechanism underlying them. One hypothesis that fits well with our data is that chronic

IL-2 treatment may alter the function of brain pathways that use IL-2 in normal signaling.

IL-2 readily crosses the BBB and gross alterations in the level of circulating IL-2,

especially when well above that occurring naturally, can be expected to alter brain

systems that express the IL-2 receptor.

In addition to those receiving IL-2 therapy, alterations in IL-2 levels and

polymorphisms in the IL-2 gene have been reported in a number of patients with

neuropsychiatric and neurodegenerative diseases. The majority of studies interpret

these alterations as indicative of inflammatory processes related to the disease. As we

have shown here, in our work with IL-2KO mice and the IL-2p8-GFP reporter model,

altered levels of IL-2 detected in the cerebrospinal fluid and in post-mortem brain may

have a neuronal basis. The expression profile of IL-2 we reported in Chapter 3 strongly

suggests that alterations in IL-2 could have a number of multifaceted deleterious effects

on multiple pathways in the CNS. Future studies aimed at evaluating IL-2 as a brain-

derived cytokine integral to neuronal signaling, may find IL-2 to be an important player

in the pathogenesis and in the treatment of diseases involving these pathways.

56

Caveats and Future Directions

In Chapter 2, we examined the effects of IL-2 deficiency on the cholinergic

phenotype of septohippocampal neurons. We briefly described the alterations of

neurotrophic factors in the medial septum and hippocampus that may be associated

with the changes in cholinergic phenotype we observed. Further studies are needed to

determine how the alterations we measured are interrelated. Our new working

hypothesis is that IL-2 exerts its neurotrophic effects, and influence on acetylcholine

release, through indirect manipulation of classic neurotrophin signaling the in the CNS.

A quantitative comparison of RNA or proteins expressed in these two pathways in vivo,

accompanied by in vitro assays that would allow for the manipulation of these pathways

directly, would be useful in investigating this potential mechanism of action.

Though the majority of cholinergic neurons in the medial septum project to the

hippocampus (Schwegler et al., 1996), a small percentage also project to other areas,

including the mediodorsal nucleus of the thalamus (Gritti et al., 1998), parietal cingulate

(Gritti et al., 1997), and entorhinal cortices (Alonso and Kohler, 1984). Thus, to

accurately claim that the change in cholinergic phenotype in the medial septum of the

IL-2KO mouse corresponds to changes in neurotrophin expression, we would need to

expand our study to include other areas in evaluating changes in the molecular milieu of

projection fields in addition to the hippocampus. Likewise, in Chapter 3, we identified a

number of IL-2 positive neuronal populations that most likely are not involved in

septohippocampal signaling. Parallel studies should be conducted in these areas,

especially those that project to cholinergic regions, to test the generalization of future

claims.

57

Concluding Remarks

The complex array of various cytokines in the peripheral immune system and

CNS appear to play a major role in the intercommunication of these two systems. The

daunting nature of the questions unanswered in the field of neuroimmunology

necessitates the small strides made in studies such as those described in this

dissertation. As previously discussed, IL-2 has pleiotropic roles in the peripheral

immune system and in the CNS. Here we have identified the cellular source of IL-2 in

the brain. The systems that we found to express IL-2, and the modalities they serve, fit

well with what we already know about the disruptions that alteration of IL-2 can have on

experimental measures in the lab, and the neurobiological deficits exhibited by humans

receiving IL-2 in the clinic. Examination of the exact mechanisms underlying the

functional role of IL-2 in specific neuronal circuitry could prove to be a fruitful approach

in future research and may have important clinical utility. By understanding the role IL-2

plays in multiple systems, we can begin to explain how inflammation and/or alterations

of the cytokine environment can affect multiple pathways and contribute to the

symptomology and deleterious effects associated with the progression of disease.

58

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BIOGRAPHICAL SKETCH

Danielle Marie Meola was born in Langhorne, PA, to Judith L’Heureux and

Michael Meola. She lived in cahoots with her older sister and younger brother in Atco,

NJ until the perfect blend of divorce, and her mother’s hippie tendencies, led to escape

from a lifetime of tacky Italian-American customs to life on the road in a Type C Thomas

Built school bus they called home. The seemingly endless summer of traveling,

camping, and bathing in public park sprinklers came to an end in the Old Pueblo,

Tucson, AZ. Danielle’s roots in the southwest are evident in her spotty grasp of the

Spanish language and tolerance of spicy foods that may border on talent. Danielle was

always an enthusiastic student and although adventurous and willing, a mediocre

athlete. A creative bookworm by nature, she began writing stories and poetry at a

young age and was once awarded first place in a school district poetry contest. The

theme of her award winning masterpiece was nature and the change of seasons, a

source of beauty that continues to inspire Danielle’s extracurricular interests. Avid

nature enthusiasts, and lovers of anything free, Danielle’s family spent most vacations

in the great outdoors and participated in paintball wars, motor sports, hiking, and snow

skiing. The latter activity, matched with Danielle’s aforementioned athleticism, led to a

brief confrontation with a particularly stubborn Aspen and resulted in the paralysis of

Danielle’s right leg. Danielle’s struggle with her disability led to an interest in medicine

and a decade later she began her study of Neuroscience at the University of Florida.

Today, Danielle lives in McIntosh, FL with her beloved dog Prime and fiancé

Gustave Alan Kloes. They enjoy hiking, biking, and kayaking the many waterways

within a day’s trip of home and take advantage of any chance they get to pop a

tent…and a beer.

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