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Multiple mechanisms of cytokine action in neurodegenerative and psychiatric states:

neurochemical and molecular substrates.

*S. Hayley & H. Anisman

Institute of Neuroscience, Carleton University, Ottawa, Ontario, Canada

Correspondence to: S.H., Carleton University, A511 Loeb Bld, 1125 Colonel By Drive, Ottawa,

ON, Canada, K1S 5B6;

Phone: (613) 520-2600x6314; FAX: (613) 520-4052; email: [email protected]

Key words: cytokine, sensitization, depression, Parkinsons disease, neurodegeneration,

microglia, neurochemical, neurotoxin


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Abstract

Neuroinflammatory processes appear to play a fundamental role in the pathology associated with

a number of neurodegenerative and psychiatric conditions. In this respect, the immunocompetent

brain microglia and peripheral macrophages release a host of proinflammatory cytokines that not

only modulate immunological processes but also influence neuronal functioning and even

survival. For instance, alterations of the cytokines, tumor necrosis factor-, as well as several of

the interferons and interleukins have been associated with Parkinsons disease (PD) and clinical

depression. Importantly, anti-inflammatory treatments that block these cytokines may impart

protection against behavioural pathology and neuronal damage in animal models of PD and

depression involving exposure to environmental toxins and stressors, respectively. The present

review highlights the involvement of inflammatory cells and cytokines in depression and PD and

explores some of the potential cellular and molecular mechanisms through which the

immunotransmitters affect neuronal functioning. Attention is also devoted to the possibility that

cytokines may sensitize neuroinflammatory pathways that, in turn, favour long-term pathology.


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Introduction

The articles presented in this issue of Current Pharmacological Therapeutics deals with the

influence of immune system messengers (cytokines) on central neuronal functioning, and

whether such effects are responsible for centrally mediated clinical conditions. Inasmuch as

major depressive illness and Parkinsons disease (PD) are highly comorbid, the present series of

articles focus on these states. It ought to be underscored from the outset that comorbidity may

occur for any number of reasons. For instance, one illness may directly or indirectly favour the

development of the second pathology, or alternatively, common processes give rise to both

illnesses. In the case of major depressive disorder and PD, inflammatory immune factors have

been implicated as a provocative factor, and there is reason to suspect that these come about

through a variety of environmental triggers, including physical or psychological distress. In the

present review we offer the position that the immune messengers, pro-inflammatory cytokines,

contribute to both these conditions (Figure 1), and that their sensitizing effects on central

processes may be particularly important.

We have argued that, like stressors, cytokine exposure or cytokine inducing immune

challenges can sensitize central nervous system (CNS) reactivity to subsequent insults. For

example, a single injection of either tumour necrosis factor- (TNF-) or the potent pro-

inflammatory bacterial endotoxin, lipopolysaccharide (LPS), sensitized CNS functioning, such

that later reexposure to these challenges provoked greatly augmented neurochemical and

behavioural disturbances [1,2,3]. Interestingly, many of these effects were reminiscent of the

neurovegatative and biological effects associated with depression and stressor induced disorders.

In addition to the potential cognitive and mood alterations elicited, cytokines and

neuroinflammatory processes have been implicated in the neurodegeneration observed in


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response to acute trauma as well as progressive disease states, including stroke, head injury,

seizure, Alzheimers disease, Multiple sclerosis (MS) and Parkinsons disease [4,5,6,7].

Furthermore, experimental findings and epidemiological studies have raised the possibility that

viral and/or bacterial insults encountered early in life may increase the likelihood of

subsequently developing PD [8,9], possibly by sensitizing neurons to the deleterious effects of

various environmental insults. In this regard, cytokines may be acting as a mediator of the

protracted consequences of diverse inflammatory challenges. It is our contention that if cytokine

exposure is coupled with other environmental insults, then neuronal systems may become

overly taxed (allostatic overload) favoring the evolution of neurodegeneration or behavioural

pathology. In this review we highlight some of the possible molecular pathways (e.g. MAP

kinases) involved in transducing cytokine signals into messages that influence the decision

making processes within cells, and which may be fundamental in promoting pathology related

to neuroinflammation.

Cytokines and psychiatric states: Depression

Stress-cytokine connection

Through disturbances of neuroendocrine and neurotransmitter functioning, stressors are

thought to be fundamental in the provocation or exacerbation of affective disorders [10,11]. Like

other stressors, cytokines such as IL-1, IL-6 and TNF- engender a host of behavioural,

neuroendocrine and central neurotransmitter changes, including increased plasma levels of

glucocorticoids and ACTH (adrenocorticotropin) coupled with augmented turnover of

monoamines within hypothalamic and extrahypothalamic regions [10, 12, 13]. It has been our

contention that by virtue of such neurochemical alterations, cytokines may contribute to

depressive illness.


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Given the similarity of the neurochemical changes elicited by cytokines and by traditional

stressors, Herman & Cullinan (1997) suggested that systemic insults, which include cytokines

and other insults that promote circulatory, respiratory or hemodynamic alterations, may have

stressor-like characteristics, and could potentially influence disease processes in ways that

stressors ordinarily have such effects. Of course, the effects of systemic and processive stressors

(the latter include events or stimuli that involve information processing) are not identical, and

cytokines may stimulate HPA activity and other central functions through pathways somewhat

different from those utilized by psychogenic (e.g. predator exposure, restraint) or neurogenic

(e.g., painful stimuli) stressors. In fact, both IL-1 and footshock provoked a similar activation (as

indicated by c-fos expression) of hypothalamic and cortical nuclei, however, the pattern of c-fos

expression varied greatly between the challenges within several limbic system regions, including

the amygdala and bed nucleus of the stria terminals [14].

Besides acting in a manner similar to that of stressors, cytokines may in fact mediate some

of the neurochemical changes elicited by stressors. For instance, administration of the IL-1

antagonist, IL-1ra, attenuated the hypothalamic NE, DA and 5-HT alterations, as well as the

ACTH elevation elicited by immobilization stress [15], suggesting that IL-1 mediates the effects

of stressors on these neuronal processes. Furthermore, hypothalamic levels of IL-1 are increased

in response to both immobilization and electric shock [15, 16] as well as in the hippocampus and

NTS in response to acute inescapable shock [17, 18] .

Given that proinflammatory cytokines alter monoamine functioning within several mood

regulatory brain regions, such as the hypothalamus, central amygdala and medial prefrontal

cortex, the proposition has been made that these factors may be fundamental in depressive

illnesses [19,20]. Interestingly, these monoamine changes are subject to the synergistic effects of


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stressors and cytokines, such that amine release from limbic neurons is dramatically increased in

IL-1 treated rodents also exposed to a mild stressor [21]. Curiously, however, the bulk of the

available data have been limited to the effects of acute cytokine/immune activation and limited

data are available concerning the influence of chronic activation of the inflammatory immune

system, despite the fact that immune activation rarely occurs on a transient basis.

Animal models: Sickness and Depression

Animal studies have shown that immunogenic challenges provoke a sickness syndrome that

is though to reflect many of the neurovegatative symptoms of depression. Along these lines,

Smith (1991) proposed that cytokines released from macrophages instigate the onset of

depression, and may account for the high comorbidity between depression and inflammatory

related illnesses, such as heart disease, arthritis, stroke, Alzheimers disease and Parkinsons

disease [22]. As well, the greater incidence of depression in women than men (3:1) may be

associated with the fact that estrogen is a potent macrophage activator [22]. Thus, it follows that

infections, tissue injury and environmental insults that promote inflammation may all impact

upon depression through macrophage related functioning.

Activation of macrophages and other inflammatory immune cells following systemic LPS

treatment results in the manifestation of a constellation of behavioral symptoms collectively

referred to as sickness behavior [23], which may mimic some of the neurovegatative symptoms

of depression. Symptoms include reduced locomotion, increased sleep and curled body posture

thereby minimizing energy expenditure, as well as elevated body temperature coupled with

anorexia, which may serve to make the physiological environment unfavourable to an invading

pathogen [23]. The cytokines IL-1 and TNF- also elicit sickness behaviors and IL-1 antagonists

attenuate these effects [12]. Moreover, cytokines disrupt operant responding for reinforcement


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and reduce social exploration, with the latter effects being secondary to illness or stemming from

changes in motivational state (e.g., anhedonia) [24]. However, studies employing progressive

ratio schedules of reinforcement (i.e., these tap into the degree of to which an animal is willing to

respond in order to gain a reward) indicated that cytokines act on incentive motivational forces

independent of effects related to sickness [24]. Moreover, antidepressant treatment effectively

attenuated the decreased responding for a palatable snack elicited by IL-1, but without

influencing the reduced chow intake ordinarily elicited by the cytokine [24].

Animal models: Neurochemical effects of cytokines

As indicated earlier, cytokine administration provokes hypothalamic pituitary adrenal

(HPA) activation as well as augmented monoamine utilization in several brain regions implicated

in depression. Indeed, systemic and central IL-1 administration increased the expression and

secretion of CRH and arginine vasopressin (AVP) from PVN neurons of the hypothalamus [25]

and direct infusion of IL-1 into the median eminence (site of CRH terminals from neurons

originating within the PVN) increased AVP and CRH secretion [26]. Correspondingly, IL-1

increased ACTH secretion from the pituitary corticotrophes and subsequent adrenal

corticosterone release [26]. Similar to IL-1, systemic TNF- elevated median eminence CRH

release, as well as circulating ACTH and corticosterone levels [1,2,3,20, 27]. Although central

infusion of TNF- was reported to dose-dependently increase circulating ACTH levels, this

cytokine had relatively modest effects on plasma corticosterone [2,3]. While much evidence

suggests that TNF- may stimulate HPA activity by direct actions upon the corticotropic cells

within the PVN [28,29], it was reported that the cytokine may affect HPA processes through


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actions outside the brain [30]. In this regard, we found that the vasoactive amine, histamine, may

be important for some of the protracted effects of TNF- upon HPA activity [31].

Like psychogenic and neurogenic stressors, subpyrogenic doses of IL-1 increased

accumulation of the 5-HT metabolite, 5-HIAA, within the PVN, central amygdala, and medial

prefrontal cortex [32]. Likewise, in vivo studies indicated that systemic IL-1 increased

hypothalamic NE release as well as that of 5-HT at the nucleus accumbens and the hippocampus

[21]. When centrally administered, IL-1 increased hypothalamic release of NE, 5-HT and DA,

as well as that of NE and 5-HT within the prefrontal cortex and hippocampus, respectively

[33,34]. Although less data are available concerning TNF-, systemic administration of the

cytokine was demonstrated to increase NE activity within the PVN, central amygdala, dorsal

hippocampus and locus coeruleus, while 5-HT utilization was increased within the PVN, medial

prefrontal cortex, hippocampus and central amygdala [35, 32]. In vivo, icv TNF- administration

increased plasma corticosterone, but did not influence hippocampal 5-HT release [36].

Clinical evidence for cytokines in depression

Depressed patients, particularly those presenting with melancholic illness, exhibit

disturbances of several aspects of immune functioning. For instance, severely depressed mood

was accompanied by altered circulating lymphocyte subsets, reduced mitogen-stimulated

lymphocyte proliferation, and impaired natural killer (NK) cytotoxicity, although equivocal

results have been reported [37]. However, depression was also associated with immune

activation reminiscent of an acute phase response, with increased plasma concentrations of

complement proteins, C3 and C4, IgM, and positive acute phase proteins, haptoglobin, 1-

antitrypsin, 1 and2 macroglobulin, and reduced negative acute phase proteins [38]. Thus,


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affective disturbances might be secondary to activation of some components of the immune

response. Consistent with this view, depressive illness was associated with elevated circulating

levels of cytokines and/or their soluble receptors, including IL-2, soluble IL-2 receptors (sIL-

2R), IL-1, IL-1 receptor antagonist (IL-1Ra), IL-6, soluble IL-6 receptor (sIL-6R), and -

interferon (IFN) [38, 39]. Moreover, increased production of IL-1, IL-6 and TNF- wasevident in mitogen-stimulated lymphocytes [40]. As already indicated, the cytokine changes may

have been related to severity of illness, and may have been a reflection of the duration of illness

or the age of illness onset [41].

While the relationship between cytokines and depression in the aforementioned studies are

based on correlational analyses, it has also been shown that direct administration of cytokines or

immune challenges to humans provokes depressive-like symptomatology. Indeed, healthy

volunteers administered a low dose of LPS or vaccinated with live attenuated rubella virus

displayed depressed mood up to 10 weeks following the challenge and in the case of the

endotoxin protracted anxiety and memory deficits were provoked [42]. Of considerable clinical

importance, is the use of cytokines as immunotherapeutic treatments for various cancers and

viral conditions. In this respect, cancer patients undergoing IL-2 or IFN- immunotherapy often

display depressive-like symptoms [43]. This point has been emphasized by Maes (1992,1999) in

which he indicates that IFN- treatment for hepatitis C or melanoma resulted in neuropsychiatric

symptoms, including fatigue, sleep disturbances, irritability, appetite suppression and depressed

mood. As well, IFN- as well as several other proinflammatory cytokines (TNF-, IL-1, IFN-)

were reported to increase levels of the 5-HT transporter which may decrease extracellular 5-HT

levels [44,45]. Alternatively, IFN- may reduce 5-HT levels through its enzymatic alterations

(e.g. stimulation of indoleamine 2,3-dioxygenase) that favour reduced plasma levels of the 5-HT


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precursor, trypthophan (see Figure 1) [46]. Hence, these cytokines can potentially mimic the

neurotransmitter changes observed in clinical depression. It is particularly significant, from both

a heuristic and a practical perspective, that depressive symptoms elicited by IFN- are attenuated

among patients that received conventional antidepressant treatments [43].

Neurodegenerative aspects of Depression

The enduring belief that depression is a biochemical disorder unrelated to neuronal survival

is changing with the demonstration of some degree of impaired neuronal survival evident in this

disorder. Indeed, imaging studies have demonstrated reduced hippocampal volume in the brains

of depressed patients and post-mortem analyses revealed a positive relationship between duration

of depression and atrophy of the hippocampus [47,48]. The exact mechanisms responsible for

such hippocampal atrophy are not clear, but several possibilities have been advanced, including a

reduction of neurogenesis, reduced dendritic branching as well as excitotoxic and apoptotic death

mechanisms. Although many of these effects are linked to the excessive glucocorticoid levels

associated with depression, cytokine mediated neuroinflammatory processes may also be

involved.

Neurodegenerative aspects of Depression: Mechanisms of death

Several studies reported a reduction of neurogenesis (or new cellular growth) within the

hippocampus of postmortem tissue obtained from patients suffering from major depression [49].

Similarly, rodents faced with chronic stressors or corticosterone treatments displayed impaired

hippocampal neurogenesis [50]. The fact that this impairment was reversed by chronic

antidepressant or a single electroconvulsive shock (ECS) treatment indicates that induction of

neurogenesis may be a clinically important event [51]. Although corticoids have been reported to

reduce neurogenesis [52], other factors may also provoke such effects in depressed individuals.


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Indeed, chronic ECS increased neurogenesis above basal levels, even in the presence of elevated

corticoids [53]. As well, the antidepressant, fluoxetine, normalized hippocampal neurogenesis in

animals exposed to inescapable electric shock independent of any actions upon corticosterone

[54]. Interestingly, drugs that inhibited either of the CRH1 and AVP1b receptors prevented the

reduction of neurogenesis evident in the chronic mild stress model of depression [55], suggesting

an important role for these neuropeptides in hippocampal changes evident in depression.

Hippocampal atrophy associated with clinical depression may be related to the direct actions

of glucocorticoids. Indeed, these hormones have been reported to impair glucose transport into

hippocampal pyramidal neurons resulting in an energetic compromise of these cells [56].

Likewise, glucocorticoids can elevate free cytocolic calcium concentrations and provoke

alterations of NMDA and/or AMPA glutamate receptors linked to excitotoxic processes [56].

Excessive glucocorticoid levels have also been reported to contribute to free radical

accumulation by reducing the capacity of antioxidant enzymes [57]. Each of these direct

corticoid mediated effects may lead to either hippocampal neuronal demise or a regression of

pyramidal neuron dendritic branching [57]. In any case, both situations would result in a

reduction of hippocampal volume similar to that observed in chronically depressed patients.

Indirect evidence has indicated the possibility that apoptotic processes may be operative in

depression. In this respect, postmortem analysis revealed that 11 out of 15 depressed patients

displayed, albeit modest, in situ end-labeling for DNA fragmentation [58]. However, data

concerning the expression of the prototypical apoptotic initiators, Fas, p53, Bax and downstream

caspases are lacking for clinical depression. A recent report did indicate an increased Bax/bcl-2

ratio in the brains of schizophrenic patients suggested in increased vulnerability to apoptotic

activation [59]. Animal studies have also indicated that stressor exposure reduced expression of


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the anti-apoptotic factor, bcl-2 [52,60], and that exposure to a severe stressor exacerbated infarct

size in response to ischemia through the suppression of bcl-2 expression [52].

Neurodegenerative aspects of Depression: Molecular pathways

Stressor provoked reductions of bcl-2 expression have been linked to altered neurotrophic

factor levels and activation of mitogen activated protein (MAP) kinase pathways [60]. In this

regard, the neurotrophic cytokine, brain derived neurotrophic factor (BDNF), was reduced in the

serum of depressed patients and the reduction negatively correlated with the degree of clinical

impairment, as determined by the Hamilton Rating Scale for Depression [61]. Likewise, rodents

exposed to a restraint stressor displayed reduced BDNF expression and this effect was prevented

by antidepressant treatment [62]. Attesting to the importance of BDNF in depression, it has

become clear that several clinically beneficial treatments for depression increase BDNF

expression, including selective serotonin reuptake inhibitors (SSRIs), tricyclics and ECS therapy

[63]. An upregulation of BDNF expression was also associated with improved performance of

rats in the forced swim test, which is used as a model of behavioural despair [63].

The underlying molecular pathways operative in models of depression are currently being

elucidated. As alluded to earlier, MAP kinase signaling pathways may be important for BDNF

and bcl-2 functioning in depression. The three MAP kinase pathways, (1) extracellular signal-

regulated kinase (ERK), (2) c-Jun N-terminal kinase (JNK; also know as stress-activated protein

kinase) and (3) p38, play an important role in responding to environmental events through the

transmission of synaptic signals to the nucleus [64]. Essentially, these pathways involve a series

of enzymes that sequentially phosphorylate each other to promote transcriptional activation and

synthesis of proteins important for cellular survival/death as well as inflammatory processes.

Importantly, BDNF and bcl-2 promote many of their physiological functions through stimulation


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of MAP kinase MAP signaling pathways [65,66]. Likewise, antidepressants, which can

normalize the stressor-induced reductions of BDNF, bcl-2 and cerebral catecholamines, also

modulate MAP kinase activity [67]. Thus, MAP kinase signaling has obvious implications for

depressive conditions, which are often precipitated by chronic stressors and characterized by

altered brain amine levels.

Neurodegenerative aspects of Depression: Cytokine involvement

As already indicated, it remains to be determined if true neurodegeneration occurs in

depression. Certainly, cytokines and other neuroinflammatory factors would be in a position to

influence such cellular viability. Cytokines may mediate neurodegeneration in depression

through the promotion of oxidative or excitototic factors derived from metabolic toxins [46]. In

this respect, IFN-, IFN- and TNF- have been reported to promote tryptophan metabolism

into kynurenine and subsequently into the oxidative metabolites, 3-hydroxy-kynurenine and

quinolinc acid, which themselves are increased in depression [68]. Chronic IFN- treatment

produced depressive symptomology that was associated with increased kynurenine and

decreased troptophan serum levels (Figure 1) [68]. Likewise, enhanced circulating IL-6 and IL-8

concentrations were correlated with elevated levels of kynurenine toxic metabolites [46].

Interestingly, these kynurenine metabolites can synergistically induce free radical generation and

have been implicated in a number of neurodegenerative disease including Huntingtons disease,

Parkinsons disease and AIDS dementia [46].

It will be recalled that macrophage activity was posited to influence mood states through

the release of cytokines following inflammatory challenges [22]. Accordingly, the common

myeloid lineage and striking similarity of functioning between peripheral macrophages and brain

microglia raise the possibility of common involvement of these cell types in depression. Indeed,


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microglia are the main central reservoirs of proinflammatory cytokines, and like macrophages,

microglia can act as antigen presenting cells within the CNS [69], thereby potentially

orchestrating central immune responses that may have deleterious consequences to local tissue.

Consistent with a role for microglia involvement in cognitive and mood processes, they release

several interleukins (IL-1, IL-6, IL-8), TNF- and IFN- in response to a host of stressful and

traumatic stimuli (e.g. stroke, head injury, seizure), as well during the course of

neurodegenerative diseases (e.g. PD, Alzheimers disease, MS) [69,70].

Unfortunately, postmortem analysis of cytokines and inflammatory factors in depressed

individuals is lacking. However, it is interesting that a strong link exists between

neurodegenerative disorders with an inflammatory component and depression. In fact, a high

degree of co-morbidity is evident between depression eeeand Parkinsons disease (~40%) [71].

Although it may be the case that the depressive symptomatology is a reaction to the stress and

uncertainty associated with facing and coping with a debilitating disease, degeneration of

neurons involved in regulation of mood may also be important. Indeed, epidemiological

evidence suggests that the onset of depression often precedes the diagnosis of PD [72]. Thus,

low levels of degeneration in monoaminergic regions important for emotionality and reward

processes (e.g. within locus coeruleus, ventral tegmental area) that occur long before the onset of

PD motoric disturbances may precipitate depressive pathology.

Neurodegenerative aspects of Depression: Conclusions

Although it seems likely that some degree of neuronal atrophy accompanies depression the

question still remains as to whether such changes are a cause or consequence of the disorder. In

this respect, it seems highly probable that the high circulating levels of glucocorticoids and


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sympathetic transmitters (epinephrine) associated with the disorder would eventually induce

some degree of cellular loss. Likewise, chronically dysregulated brain monoaminergic systems

would also be a potential candidate for eventual energetic, metabolic or other derangements that

would leave cells vulnerable to degeneration. In such situations the degeneration would be

secondary to the depressive condition, however, the possibility should not be dismissed that an

initial slowly developing, mild degree of degeneration would eventually produce disturbances in

mood regulatory circuits producing depressive symptomatology. In this respect,

neurodegenerative disorders, such as Parkinsons disease, are often associated with depression at

early stages before widespread degeneration and subsequent motor difficulties [72].

Cytokines and Neurodegeneration: Parkinsons disease:

Cytokines have been implicated in acute and chronic cell death [6,73]. Clinical studies

revealed increased levels of the proinflammatory cytokines in postmortem brain as well as in

blood of patients with stroke, head injury, multiple sclerosis, Alzheimers and Parkinsons

disease [6,73,74,75,76]. Although these findings have been recapitulated in animal models, as

indicated earlier it is still uncertain whether these cytokines play a neuroprotective or

neurodestructive role. It may be that relatively low endogenous cytokine levels act in a protective

capacity to buffer against damage related to death processes, whereas relatively high levels of

these factors may contribute to neuronal damage [76]. Indeed, low levels of cytokines can

provoke the release of potentially beneficial trophic factors and free radical scavengers, but

elevated levels may activate inflammatory cascades or even induce apoptotic death (self

destructive programmed death mechanism). For instance, mice genetically lacking TNF-

receptors (thereby removing the influence of endogenous TNF-) were more susceptible to

ischemic injury, but administration of exogenous TNF- at the time of ischemia exacerbated


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neuronal death [76]. Likewise, administration of the endogenous IL-1 antagonist, IL-1ra, reduce

infarct size in response to middle cerebral artery occlusion and prevented the accumulation of

inflammatory infiltrates within the area of damage [73], suggesting a prominent destructive role

for IL-1 in acute cerebrovascular insults. In effect, the concentration as well as timing of

cytokine exposure likely determines whether primarily protective or deleterious consequences

arise from these immunotransmitters.

Environmental stressors in PD: Animal models

Parkinsons disease is characterized by degeneration of the dopaminergic neurons within

the SNc thereby promoting a reduction of dopamine release from the terminals within the

striatum [77, 78]. The clinical features of PD, including bradykinesia, tremor and rigidity, stem

from the dysregulation of basal ganglia functioning associated with the reduced dopamine levels

[79]. Indeed, a dis-inhibition of striatal interneurons provides faulty input to the globus pallidus

and thalamus ultimately culminating in reduced drive to motor regulatory cortical regions [80].

The two most commonly used and widely validated animal models of PD are those involving

MPTP and 6-OHDA administration. Essentially, MPTP is a thermal breakdown product of a

meperidine-like form of synthetic heroin, that was accidentally discovered to induce

Parkinsonism in a group of drug users in the early 1980s [79,81]. Systemic exposure to MPTP

has been demonstrated in numerous studies over the past two and a half decades to provoke SNc

dopaminergic degeneration coupled with depletion of striatal dopamine in mice and primates

[79,82]. Although MPTP elicits behavioral disturbances (e.g. akinesia, tremor, impaired gait)

similar to those evident in clinical PD, a threshold of neuronal loss (estimated around 80%) may

have to be evident before such effects are manifested [83]. In contrast to MPTP, 6-OHDA is not

able cross the BBB and is consequently typically directly infused into the either the SNc or


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striatum where it exerts substantial destructive effects on local neurons and terminals [84]. 6-

OHDA is a hydroxylated analogue of dopamine that may be generated by auto-oxidation of

endogenous dopamine [85]. Consequently, the highly reactive metabolic nature of dopamine

itself may contribute to PD neurodegeneration.

Although rare familial forms of PD appear to have a strong genetic component [86], the

majority of cases are idiopathic and environmental events may act as causative agents. In this

respect, a provocative role of pesticides (e.g. rotenone and paraquat) as well as heavy metals (e.g.

iron, manganese) has been suggested [87,88,89]. In particular, epidemiological evidence

revealed a high incidence of PD in rural areas that use substantial amounts of agrochemicals

[90]. Consistent with a role for these chemicals in PD, all of the most commonly used pesticides,

namely, rotenone, paraquat and maneb have been linked to dopaminergic death in rodents

[88,89]. Indeed, continuous infusion of rotenone, using osmotic minipumps, elicited

degeneration of SNc dopaminergic neurons and destruction of non-dopaminergic neurons within

the basal ganglia and brainstem [84,91]. The fact that non-dopaminergic neurons were affected is

consistent with the modest degeneration observed within these areas in PD patients. Indeed, loss

of noradrenergic locus coeruleus neurons often occurs in PD and, as alluded to earlier, may

contribute to some of the depressive symptoms evident in PD [92].

Although less evidence is available concerning the impact of paraquat in PD, at least one

study demonstrated that repeated systemic administration of the pesticide provoked selective

destruction of SNc dopamine neurons [88]. However, striatal dopamine levels were not altered

by paraquat, suggesting that compensatory mechanisms may have been provoked by the

surviving neurons [88]. Although the fungicidal agent, maneb, alone did not influence

dopaminergic neurons, it did augment the neurodegenerative actions of paraquat [89], suggesting


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that potential synergistic interactions among these agents.

Environmental stressors in PD: Inflammatory death mechanisms

Three primary mechanisms may underlie the neurodegenerative actions of MPTP, 6-

OHDA and the pesticides: (1) inhibition of complex I of the mitochondrial respiratory chain, (2)

direct oxidative stress factors and (3) provocation of neuroinflammatory cascades. Each of these

chemicals shares the common property of being inhibitors of complex I of the mitochondrial

respiratory chain. Rotenone and MPTP are particularly potent complex I inhibitors, promoting

reduced oxidation of NAD+ substrates and a-ketoglutarate dehydrogenase, culminating in

decreased ATP levels, loss of mitochondrial membrane potential, faulty intracellular calcium

buffering and free radical generation [85]. Any one of these mitochondrial-mediated outcomes

could induce neuronal degeneration. For instance, reduced ATP levels may leave the cell unable

to meet energy demands and would be especially vulnerable to alternate metabolically

challenging insults (such as other toxins or stressors).

Although mitochondrial dysfunction itself can elicit oxidative neuronal damage, 6-

OHDA and MPTP have been demonstrated to provoke oxidative stress independent of the

mitochondria. Indeed, like endogenous dopamine, through auto-oxidation or through interactions

with the catecholaminergic enzyme, monoamine oxidase, 6-OHDA can generate toxic

metabolites, such as quinones, superoxide radicals, hydrogen peroxide and the hydroxy radical

which can directly damage neurons [85]. Likewise, rotenone damaged dopaminergic neurons

through the induction of free radicals and anti-oxidant treatments (e.g. alpha-tocopherol)

protected these neurons [93]. Systemic MPTP can induce superoxide as well as nitric oxide (NO)

formation, which together can create the incredibly reactive and destructive peroxynitrite radical

[94]. As well, MPTP may also impair the functioning of endogenous protective free radical


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scavengers, such as GSH, metallothionein and manganese superoxide dismutase [94].

Interestingly, as Czlonkowska and colleagues point out in this issue, inflammatory

processes associated with microglial activation likely contribute to the oxidative damage

provoked by MPTP [95,96]. In this respect, it is of interest that each of the environmental agents

used to induce experimental Parkinsonism also elicit profound immune activation and

neuroinflammation. This is not surprising given that a primary role of immunologial functioning

is to rid the body of such environmental antigens. Accordingly, excessive activation of central

and peripheral immune factors (such as cytokines) engendered by these challenges may

contribute to tissue damage evident in PD.

Microglial activation in PD neurotoxin models

Both in vivo and in vitro procedures have demonstrated that 6-OHDA, MPTP (or its

metabolite MPP+) and rotenone can induce substantial activation of microglia, the primary CNS

immunocompetent cell [95,96,97]. Indeed, systemic MPTP treatment promoted profound

microgliosis that was detected in the SNc of monkeys exposed to the toxin 5 to 14 years earlier,

suggesting a progressive, long term neuroinflammatory process was associated with relatively

brief toxin exposure [98]. Mice treated acutely with systemic MPTP (four 10 mg/kg doses

spaced 1 hr apart) displayed an increased number of microglia and morphological changes

indicative of activation (e.g. cellular thickening) that was evident for up to 4 and 14 days within

the striatum and SNc, respectively [96]. It of interest to note that microglial responses occurred

long before dopaminergic neuronal death was evident (14-21 days), providing evidence for a

primary role for the inflammatory process. Importantly, advancing age, which is a clear risk

factor for PD, has also been associated with profound microglia activation, with older mice (9-12

months) displayed greatly enhanced microglial reactivity following MPTP relative to young


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animals (3 months) [99].

In order to determine the mechanistic role of activated microglia in dopaminergic loss,

several co-culture systems have been established using embryonic ventral mesencephalic

neurons and postnatally obtained microglia. Using this approach, microglia (but not astrocytes)

co-cultured with mesencephalic neurons were found to contribute to MPTP provoked neuronal

injury [97]. Their deleterious effects were linked to NADPH oxidase, the main reactive oxygen

species-producing enzyme during inflammation [100]. In this regard, neurons obtained from

mice genetically lacking NADPH or treated with the pharmacological inhibitor, apocynin, were

largely resistant to MPTP toxicity [101]. Likewise, knockout mice lacking molecular subunits

(gp91pnox) required for functioning of NADPH oxidase, were resistant to rotenone induced

dopaminergic loss [101].

Corresponding to the in vitro data, NADPH oxidase is increased within the SNc of

human PD patients and MPTP treated rodents [100]. Likewise, NADPH oxidase deficient mice

displayed substantially less dopaminergic neuron loss in response to systemically delivered

MPTP compared to wild type animals [100]. Additionally, it has also become apparent that

systemic MPTP promotes microglial iNOS expression and that mice lacking this oxidative

enzyme displayed substantially reduced dopaminergic loss [82]. Treatment with minocycline, a

tetracycline derivative that inhibits microglial activation, prevented MPTP induced nigrostriatal

degeneration as well as NADPH oxidase, iNOS and nitrotyrosine (marker of NO activity)

expression [100]. Thus, inflammatory microglia reactivity likely contributes to ongoing

degeneration through the release of highly reactive oxidative species (see Figure 1).

It is exceedingly difficult to disentangle the interrelationships among inflammation,

oxidative stress, apoptosis and excitotoxic death mechanisms. From our perspective, it is


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important to note that microglia serve as pivotal regulators of each of these diverse molecular

processes. Thus, it should be underscored that microglia are the most prominent CNS cells

expressing proinflammatory cytokines, such as IL-1, IL-6, TNF- and IFN-, that have the

ability to influence death processes (see Figure 1). Indeed, TNF- and its related receptor family

member, Fas, have well established caspase mediated neuronal apoptotic consequences

stemming from their receptor linked intracellular death domain complexes [102]. Likewise, IL-

1 has been reported to promote neuronal apoptosis when found at elevated concentrations; in

fact, its synthetic enzyme, interleukin converting enzyme, is actually a member of the pro-

apoptotic caspase family [103]. In terms of excitotoxic death, IL-1 exacerbated the degree of

neuronal demise promoted by glutamate through NMDA and AMPA receptors [103], whereas

infusion of the endogenous IL-1 antagonist, IL-1ra, prevented striatal excitotoxicty [104].

Interestingly, TNF- was recently demonstrated to alter communication between microglia and

astrocytes to favor development of excitotoxicity [105]. Thus, cytokines may promote neuronal

death directly or though their impact upon glial cells. In fact, through their potent autocrine

stimulatory effects, cytokines may also amplify the release of any oxidative or other death

factors released from the microglia in which they originate.

Viral and bacterial involvement in PD

In addition to chemical agents, another environmental culprit has been implicated in PD,

namely pathogenic microorganisms. Cases of parkinsonian-like syndromes have been associated

with infections including, poliovirus, arbovirus, herpes simplex virus and encephalitis

[106,107,108]. A viral hypothesis proposed for PD has suggested the possibility that infection

prenatally or early in life with some (yet to be discovered) latent virus(es) may instigate the

disease [106]. The long incubation period and slow evolution of damage provoked by the virus


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could certainly be envisaged to correspond with the insidious time-course for PD onset. For

instance, cases of Parkinsonism associated with von Economo encephalitis have been reported to

occur years after infection [109]. Likewise, postencephalic cases of Parksinsonism that were

associated with the influenza epidemic of 1918 [108] have been attributed to cytotoxic effects of

the virus on the developing SNc within the intra-uterine environment [110]. In addition to

viruses, prenatal or early life exposure to a pathogen of bacterial origin may also play a role in

PD. In fact, as will be discussed shortly, recent animal studies have demonstrated that rats

receiving prenatal administration of the bacterial antigen, lipopolysaccharide (LPS), displayed

substantial degeneration of dopaminergic neurons [8]. It was also noted that rodents exposed to

low concentrations of pesticides early in life were much more susceptible to the neurotoxic

consequences of dopaminergic toxins later in life [89]. It may be that early exposure to

immunogenic events (viral, bacterial or chemical) provokes mild neuroinflammation (e.g.

microglial activation, cytokine release) that over time may cause neurodegeneration or render

dopamine neurons vulnerable to degeneration in response to normally low grade insults [8].

Cases of Parkinsonism have been reported in HIV infected individuals and it was

suggested that accompanying infections, such as toxoplasmosis, may exacerbate the impact of

the virus upon basal ganglia functioning [111]. HIV may directly impair dopamine neurons

through the envelope protein, gp120, or associated HIV protein, Tat, both of which inhibit

dopamine synthesis and have been found to promote nigrostriatal degeneration in exposed

rodents [111,112]. Another virus implicated in PD, the Japanese encephalitis virus, reduced the

number of dopamine neurons and provoked marked gliosis within the SNc of infected rats [113].

The PD-like behavioral symptoms provoked by the Japanese encephalitis virus, most notably

bradykinesia, were significantly improved by l-DOPA treatment [113].


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Neuroimmune mechanisms of PD

Examination of postmortem PD tissue revealed numerous signs of inflammation,

including microglial activation and increased levels of several proinflammatory cytokines (IL-1,

IL-2, IL-6 and TNF-), as well as expression of elements of the complement cascade; an

important mechanism mediating antibody dependent cytotoxicity [95,114]. In contrast to the role

of glial mediated inflammatory processes in PD, much less attention has focused upon the impact

of adaptive immune responses in the disease. In this respect, cytokines may orchestrate

lymphocyte activity and effector adaptive responses (e.g. antibody mediated complement

deposition, cell mediated cytotoxicity) that may damage CNS tissue.

Unlike several other neurological conditions, including MS, stroke and to a lesser degree

Alzheimers disease, definitive evidence of T lymphocytes within the PD brain is lacking.

However, reduced levels of T cells within the bloodstream and impaired proliferative responses

to mitogens in PD patients indicate some degree of altered peripheral lymphocyte immunity [95].

It has been estimated that 30% of PD patients have autoantibodies reactive against basal ganglia

neurons [115]. Likewise, examination of cases of Parkinsonism related to encephalitis revealed

that 95% of patients had autoantibodies reactive against basal ganglia antigens compared to 2%

of controls [116]. The suggestion has also been made that antibody dependent cell mediated

cytotoxicity mediated by natural killer (NK) cells may contribute to the pathogenesis of PD

[157]. This assertion largely stems from the finding that elevations of circulating NK cell activity

positively correlated with disease severity in PD patients [157].

One interesting emerging neuroimmune theory of PD suggests that formation of

neoepitopes (new antigens) within the basal ganglia recruits a specific destructive immune

reaction [117,118]. Specifically, accumulation of toxic dopamine auto-oxidation metabolites,


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particularly quinone, may cause tissue alterations favoring neoepitope creation [117]. Consistent

with this proposition, antibodies from a subset of PD patients (7/21) but not subjects with other

neurological diseases (0/21), recognized epitopes from dopamine quinone modified proteins

[118]. Thus, PD patients displayed antibodies against neoepitopes produced by altered dopamine

metabolism, which could contribute to or amplify ongoing inflammatory and degenerative

response [118]. Animal studies revealed that direct intra-SNc infusion of purified antibodies

from PD patients but not age matched disease controls, induced complement activation and

dopaminergic neuronal death in several rodent species [119]. Importantly, these effects were

prevented in mice lacking the Fc receptors, which are critical for antibody mediated activation of

microglial cells [119]. Indeed, through binding and clustering of the Fc membrane antibody

receptors, specific antibodies can provoke the release of oxidative species, such as superoxide

radicals, from microglia.

Neuroinflammatory models of PD: Cytokine involvement

Further support for a role of immune factors in PD comes from recent studies

demonstrating that central administration of the bacterial endotoxin, LPS, provoked a loss of

dopaminergic neurons within the SNc [120]. However, infusion of LPS into the hippocampus,

thalamus and cortex of rats did not induce substantial neuronal loss [120], suggesting that SNc

dopaminergic neurons are especially vulnerable to immunogenic insults. It was suggested that

the particularly high concentration of microglia within the SNc may contribute to the enhanced

vulnerability of these dopamine neurons [120]. In vitro experiments revealed that the

neurodegenerative consequences of LPS on mesencephalic neurons were only evident in the

presence of co-cultures including microglia [97,101]. Since LPS potently induces circulating

cytokine production and may also stimulate central cytokine expression [121], this may be one


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mechanism through which the endotoxin causes dopaminergic degeneration. In fact, as will be

discussed shortly, release of some of the typical proinflammatory cytokines elicited by LPS,

including IL-1, IL-6, TNF- and IFN-, can synergistically promote a variety of central

consequences, including cellular death [122,123].

As alluded to earlier, PD postmortem brain tissue often contains increased expression of

cytokines, such as IL-1, IL-6, IFN-, TNF-, as well as the TNF- receptor superfamily

member, Fas [124]. Likewise, cDNA microarray studies indicated that MPTP treated mice

displayed similar alterations of proinflammatory cytokine genes within basal ganglia brain

regions [125]. Although 6-OHDA stimulated TNF- and IL-1 expression within the basal

ganglia [124,126], there is a lack of data on the impact of pesticides, such as rotenone, on

cytokine levels.

Although correlative evidence exists, few studies have assessed the mechanistic role of

cytokines in PD; it is even unclear as to whether these immunotransmitters primarily act in a

protective or destructive capacity. However, two laboratories have recently reported altered basal

ganglia responses to MPTP in TNF- deficient knockout mice [127,128]. Although one report

indicated that TNF- deletion protected striatal terminals and normalized dopamine levels in

MPTP treated mice [127], the other found increased dopamine metabolism in the absence of any

evidence of neuroprotection in the MPTP null mice [128]. As depicted in Figure 2, our own

recent findings found that mice lacking the TNF- receptor superfamily receptor, Fas, displayed

attenuated dopaminergic neurodegeneration and associated microgliosis [129]. Interestingly, IL-

6 knockout mice displayed increased SNc dopaminergic soma and striatal terminal degeneration

following MPTP, suggesting enhanced sensitivity to the toxin in the absence of the cytokine

[130]. Thus, in keeping with the trophic actions reported for IL-6, endogenous levels of the


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cytokine may actually protect neurons against insults.

There are several mechanisms through which cytokines may influence the survival or

death of dopaminergic neurons. Although this section will evaluate some of these pro-death

mechanisms, it should be underscored that many cytokines (at least within the immune system)

act as growth factors promoting cellular differentiation and proliferation. However, it has been

well established that pro-inflammatory cytokines, such as TNF-, Fas and IFN- as well as

several chemokines (subcategory of chemoattractant cytokines), can promote cellular death

through apoptotic, excitotoxic or oxidative processes [131]. For instance, the recently reported

low levels of the intracellular Fas death domain (FADD) in PD patients prompted the assertion

that FADD expressing neurons may selectively die through apoptosis in PD [132]. Other

evidence for classical apoptotic pathways operative in PD includes reports of increased levels of

caspase-3 and 8 which act as downstream effectors of FADD, Bax and related death pathways

[133]. However, evidence also exists for inflammatory mechanisms of action for Fas. For

instance, as shown in Figure 2, we reported that Fas knockout mice displayed diminished

microglial activation within the SNc and striatum in response to MPTP compared to wild type

mice [129]. Fas also promoted central expression of the chemokine, IL-8 and has numerous

inflammatory effects in common with its superfamily member, TNF- [134].

Neuroimmune-neurotoxin interactions in PD: synergistic effects

The fact that PD often occurs in distinct clusters suggests that environmental factors (e.g.,

toxins) related to certain geographic areas may confer vulnerability to disease. Environmental

distribution of many chemical agents often overlap and it is certainly conceivable that multiple

toxins may synergistically influence neuronal processes. Likewise, one can surmise that the


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combination of various toxins (e.g. pesticides) with immunological events (e.g. viral, bacterial

pathogens) may interact in a similar fashion. Consistent with this proposition, co-administration

of LPS with rotenone synergistically augmented dopaminergic degeneration in mesencephalon-

microglia co-cultures, through the release of reactive oxygen species [97,101]. In vivo studies

indicated that although the pesticide, maneb, had no effect on dopaminergic neurons, its co-

administration with another pesticide, paraquat, synergistically enhanced the degree of

nigrostriatal damage and gliosis [89].

Interestingly, many of the behavioral deficits produced by MPTP are only evident in the

presence of other chemical agents or stressors [83], suggesting possible synergistic interactions

among these treatments. For instance, it has been reported that MPTP only induced akinesia and

cataplepsy deficits when co-administered with the pesticides, diethyldithiocarbamate (DDC) or

maneb [135, 136]. It was also reported that the imposition of a stressor (transportation stress)

was necessary to realize the behavioral effects of MPTP [83]. Taken together, these studies

indicate that consideration of the interactive effects of multiple factors is warranted when

considering the environmental triggers operative for PD.

Cytokine-provoked neuronal sensitization: Implications for Depression and PD

Cytokines may influence the neuronal responses to later challenges although the

processes leading to such outcomes do not involve recognition in the same way that typical

immune responses to antigens elicit such an effect. Sensitization from a neuronal perspective is a

fundamental process relevant to stressor related pathologies, and to neural plasticity in general.

Just as stressor experiences may increase the likelihood of a depressive episode developing given

a subsequent stressor event, cytokines, even when given at low concentrations that do not


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produce noticeable behavioural effects, may elicit central neuronal sensitization upon their

reexposure. From this perspective, stressors and cytokines have protracted effects on

psychological and neurological processes well after the initial actions of the treatment have

waned.

Cytokine induced HPA and behavioural sensitization: Peripheral mechanisms

Just as certain psychosocial and neurogenic stressors may provoke exaggerated

neurochemical responses upon subsequent stressor exposure [137,138,139,140,141], IL-1 and

TNF sensitized activity of the HPA axis so that later cytokine treatment provoked particularly

marked responses [1,142]. These cytokines induced a phenotypic change in the co-localization

of the hypothalamic neuropeptides, AVP and CRH, such that increased co-expression of AVP

occurred within CRH terminals in the external zone of the median eminence (ZEME). In keeping

with the synergistic impact of these neuropeptides on pituitary ACTH release, their increased co-

expression engendered by IL-1 was associated with enhanced ACTH and corticosterone

secretion [141,142].

Interestingly, the corticosterone and CRH/AVP sensitization effects of TNF- followed

different time courses. Although increased CRH and AVP co-storage within the ZEME peaked

7-14 days following the initial TNF- treatment, the sensitized corticosterone response was only

evident in mice re-exposed to the cytokine 28 days after pre-treatment with the cytokine [1].

Thus, mechanisms other than hypothalamic neuropeptide secretion may underlie the corticoid

sensitization. Indeed, we recently found evidence that TNF- may be acting as an adjuvant with

the well known immunogenic factor, bovine serum albumin (BSA), which was used as a carrier

protein in the injection mixture [143]. As well, systemic pre-treatment with an antihistamine


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cocktail (diphenhydramine + cimetidine, H1 and H2 antagonists, respectively) abrogated the

corticosterone sensitization, suggesting a histamine dependent mechanism was involved [31].

Paralleling the corticosterone sensitization, TNF- reexposure 28 days following initial

cytokine elicited profound sickness symptoms reminiscent of those associated with

systemic/anaphylactic shock [31]. Indeed, mice displayed pronounced reddening of the tail, ears

and nose (cyanosis; indicating increased accumulation of blood cells and inflammation), coupled

with a marked reduction of blood volume and pressure, as well as signs of hypothermia [31].

Although co-administration of the H1 and H2 antagonists, diphenhydramine and cimetidine, did

not appreciably influence the acute effects of TNF-, the sensitization of sickness associated

with reexposure to the cytokine was prevented [31]. Together, these findings raise the possibility

that events that induce TNF- or other cytokines (e.g. infections, stressors) may render

organisms vulnerable to subsequent exposure to the same or sufficiently similar insult. In the

case of TNF-, such reexposure may induce an acute phase reaction that could progress to full-

blown shock. Alternatively, exposure to relatively low endogenous levels of TNF- (as would be

expected in humans) over time may elicit modest acute phase reactions that could not only

influence visceral processes, but may also promote exaggerated corticoid release, inducing

detrimental effects as chronically high levels of the hormone may influence the development of

diabetes, heart disease, stroke and memory impairments.

Cytokine induced neurochemical sensitization: Central mechanisms

Although the corticosterone and sickness sensitization elicited by TNF- appears to be

related to peripheral immune processes, the cytokine also evoked sensitized brain neurochemical

responses independent of peripheral factors. Indeed, it will be recalled that the augmented


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CRH/AVP co-localization within the ZEME occurred independent of sickness and corticoid

variations. Likewise, reexposure to TNF- 1 day following pre-treatment with the cytokine

induced a sensitization of CRH expression within the central nucleus of the amygdala, at a time

when no signs of illness or corticoid activation were apparent [144]. These neuropeptide

variations presumably involved some sensitized central mechanism that was closely linked to the

timing of reexposure. Importantly, activation of CRH within the central amygdala contributes to

anxiety and responses to fear-related stimuli and it is thus possible that this mechanism

contributes to anxiety associated with endotoxin challenges (which induce TNF-) in humans

[145]. In fact, central infusion of TNF- was reported to induce anxiogenic effects in rodents

testing using an elevated plus maze [146].

Systemic TNF- markedly influenced monoamine activity within several brain regions [1,

32, 35], and re-exposure to the cytokine increased activity and/or levels of NE, DA and 5-HT in

a region-specific and time-dependent fashion [1, 3, 20]. As was the case for CRH/AVP, these

time-dependent brain neurochemical alterations occurred earlier than the sickness or HPA

changes [1]. Sensitized utilization of NE and 5-HT that was elicited by systemic TNF- were

reliably detected within the amygdala and prefrontal cortex [1], the former of which is important

for emotional responding and the latter involved in cognitive appraisal. Interestingly, as depicted

in Figure 3, when TNF- was central infused into the lateral ventricles, monoamine sensitization

effects were most apparent within the hypothalamus [147]. Such an effect may stem from region

specific difference in cytokine diffusion and uptake.

Cytokine sensitization and the development of depression.


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It will be recalled that cytokines and their soluble receptors are increased in depressive

conditions and antidepressant treatments often normalize these variations, at least for TNF-

[148]. As well, cytokine immunotherapy with IFN- or IL-2 induced depressive conditions that

were amendable to antidepressant treatments [68]. Interestingly, the assertion was made that

individuals vulnerable to developing depression have sensitized HPA responses when challenged

with immune agents [149]. In this regard, IFN- immunotherapy initially provoked a greatly

augmented ACTH responses in melanoma patients that subsequently developed depressive

pathology, relative to those that did not develop such symptoms [149]. Others have demonstrated

that daily IFN- treatment for three weeks reduced HPA responses to the cytokine but a greatly

augmented response to a sub-threshold challenge with CRH [150]. Thus, although repeated IFN-

administration may induce a tolerance to its own neuroendocrine actions may sensitize HPA

functioning to alternate challenges.

It is important to underscore that clinical cytokine administration involves quite high

concentrations of these immunotransmitters, certainly within the pathophysiological range [151],

unlike the relatively low levels detected in depressive individuals not undergoing such

treatments. We propose the possibility that modestly elevated cytokine levels that persist over

long time intervals may come to sensitize neural functioning in stressor sensitive brain regions.

Along these lines, it is also important to consider that the schedule or pattern of cytokine

exposure likely plays a critical role in its neurochemical consequences. Indeed,

immunotherepeutic cancer treatment schedules often involve repeated administration of the

cytokine for many months, often with delays of several days between injections. The chronicity

of such therapy would give ample time for the development of time dependent sensitization

effects. The intermittent nature of treatment may also favour the development of central


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sensitized effects, since we have found that TNF- sensitized CRH and monoamine activity

within brain regions controlling emotional, cognitive and endocrine responses when the cytokine

injections were spaced 1-7 days apart [144,147].

Like TNF-, administration of a bacterial endotoxin, such as LPS, may proactively influence

the HPA and central monoamine responses to subsequent challenges of a different sort (i.e.,

cross-sensitization developed). Unlike the effects of TNF-, however, the proactive effects of

LPS were relatively transient, being evident 1-day after the initial treatment but not at 28 days

[2]. Interestingly, the developmental timing of the immunological insult may be of relevance,

since rats exposed to endotoxin perinatally (1 and 3 days post partum) displayed sensitized CNS

activity in response to subsequent endotoxin or stressor exposure during adulthood [152]. These

data raise the possibility that early life infectious events may also impact upon psychological

responses to stressors encountered in adulthood [152].

One possibility that has been considered is that early insults may be affecting the plasticity of

neuronal processes. For example, as indicated earlier, recent theories of depression have

entertained the view that aberrant plasticity of certain neuronal pathways may be involved in the

etiology of depression and that the trophic cytokine, brain derived neurotrophic factor (BDNF)

may be important in this respect. Indeed, stressful events have been shown to reduce BDNF

expression, whereas chronic antidepressant treatments increased brain levels of BDNF and

attenuated the reduced BDNF ordinarily provoked by stressors [61,62,63]. While it is certainly

possible that the impact of stressors (and antidepressants) on BDNF and other cytokines, such as

IL-1 and TNF-, have additive or interactive effects with respect to depressive states, at present

data are unavailable concerning such potential interactions. Likewise, it is unclear whether cross-

sensitization occurs between these varied growth factors and stressors.


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Cytokine sensitization and the development of PD

Just as early life immune challenges may confer increased vulnerability to the

development stress-related anxiety, there is reason to believe that this treatment influences

vulnerability to other pathological conditions, such as PD. In this regard, prenatal exposure to

LPS at embryonic days 10-11 resulted in a substantial reduction of the number of SNc

dopaminergic neurons evident in adult rats [8]. In addition, prenatal LPS exposure engendered a

long-term increase of striatal TNF- that was even evident in mice 120 days of age [153]. As

the overall number of neurons within the SNc, as revealed by Map-2 immunoreactivity, was not

reduced by LPS, it appears that at the dose used the endotoxin selectively impacted dopamine

neurons. It was suggested that dopamine containing neurons may be particularly vulnerable to

the effects of early TNF- exposure, possibly through its inhibitory effects on important growth

factors, such as nurr-1 or sonic hedgehog [8].

Early exposure to proinflammatory environmental toxins may also act to sensitize

neurons to the deleterious actions of subsequent nigrostriatal insults. For instance, combined

treatment with the pesticides, paraquat + maneb, from postnatal days 5-19 sensitized rodents to

the damaging effects of these challenges months later [89]. Indeed, reexposure to the

combination of these pesticides in adulthood provoked a significantly greater SNc neuronal loss

and striatal dopamine depletion coupled with pronounced motor impairment relative to animals

not exposed to the toxins early in life [89]. Although not measured in this study, it will be

recalled that these pesticides readily instigate neuroinflammatory activation, particularly as

indicated by microgliosis.

The time course for neurodegeneration following inflammatory challenges is consistent

with the slow progressive nature of PD. For instance, continuous intra-SNc infusion of a low


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dose of LPS for several weeks (closely mimicking a typical neuroinflammatory state) elicited a

maximal microglia response after 2 weeks but SNc degeneration was not evident until 4-6 weeks

later [97]. Likewise, exposure to MPTP and closed head injury have been associated with

protracted elevations (often for years) of microglia and cytokines [154]. The latter may explain

cases of PD linked to repeated blows to the head, as observed in boxers. In any case, it is

conceivable that such ongoing neuroinflammation would sensitize individuals to the

degenerative consequences of subsequent environmental insults. We believe that microglia

activation can lead to cytokine release that contributes to pathology through two primary

mechanisms: (1) the promotion of glial derived reactive oxygen species and (2) activation of

intracellular neuronal apoptotic or excitotoxic death processes, possibly through stimulation of

MAP kinase pathways. Alternatively, the possibility should not be dismissed that low-grade

dopaminergic injury may recruit peripheral immune responses (e.g. antibody dependent

cytotoxicity) that may further amplify any ongoing degeneration.

In conclusion, as summarized in Figure 1, insults that affect immune and inflammatory

system functioning may have profound effects on CNS mechanisms implicated in the regulation

of neurochemical, behavioral and neurodegenerative processes. The fact that cytokines act as

common messengers between and within the CNS and immune system, coupled with the

possible involvement of these systems in both psychiatric and neurodegenerative conditions,

raises the possibility that manipulation of cytokine responses may have beneficial effects for

these clinical conditions. Indeed, anti-inflammatory clinical trials (e.g. using NSAIDs and

cytokine antagonists) are currently being explored for the treatment of MS and Alzheimers

disease [155]. Since cytokine immunotherapy (e.g. IFN- for melanoma) can induce depressive

symptomatology that is ameliorated by antidepressants, the possibility of using cytokine


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antagonists in certain cases of depression (particularly those associated with medical conditions)

should also be considered. In any case, the temporal pattern of exposure to stressors and immune

challenges over the life span sets the tone for whether or not such individuals develop a

sensitized state for CNS pathology.


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Acknowledgements:

Supported by the Canadian Institutes of Health Research and the Natural Science and Engineering

Research Council of Canada. S.H. and H.A. hold Canada Research Chairs in Neuroscience.

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