University of Groningen Chemokines in neuroimmunology ... · The immune system of the central nervous system Neurons control whole-body processes by signaling via complex neuronal

University of Groningen

Chemokines in neuroimmunologyDijkstra, Ina Miranda

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Publication date:2004

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Citation for published version (APA):Dijkstra, I. M. (2004). Chemokines in neuroimmunology: the role of CCL21 and its receptors in neuron-gliacommunication. Groningen: s.n.

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Partly published in Current Opinion in Pharmacology, 2002

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The immune system of the central nervous systemNeurons control whole-body processes by signaling via complex neuronal networksand are considered to be the most important cells of the central nervous system(CNS). Neurons are also the most vulnerable cells and cannot endure long-lastingdisturbances in their environment. Therefore, neurons depend on support from sur-rounding glial cells. Three types of glial cells have been identified: oligodendrocytes,astrocytes and microglia. Glial cells are “neuron-supportive” by maintaining an opti-mal environment and by producing neurotrophic factors. For a long time, the func-tion of glial cells, compared to neurons, was considered to be solely supportive andrather passive. However, experimental evidence from the last decades is now point-ing towards much a more active role of glial cells in neurodevelopment, signaling,neuronal plasticity, neurodegeneration and neuroinflammation.

From an immunological point of view, a delicate balance within the CNS exists.On the one hand, vulnerable neurons have to be protected from full-blown immuneresponses and their potential neurotoxic bystander effects. This is underscored by


Figure 1-1 Cellular composition of the blood-brain-barrier (BBB).

The BBB is formed by a tight interaction of three different cell types: endothelial cells, pericytes and foot-

processes of astrocytes. All three cell types are involved in regulating permeability of the BBB and form a

first-line defense against blood-borne elements and infectious agents.

the existence of a blood-brain-barrier (BBB), a physical barrier that prevents entryof blood-borne elements into the brain (fig. 1-1). On the other hand, invasion ofmicroorganisms into the CNS does occur occasionally and would be detrimental forneurons if there were no mechanisms to clear such infections.

For many years, the presence of the BBB has been one of the most important argu-ments in considering the CNS as an immune privileged site. Accordingly, immunereactions in the brain were thought to occur only in case of severe CNS damage oraccompanying brain infection by microorganisms, representing conditions duringwhich the BBB is disrupted. However, it is now evident that also in the normalhealthy brain, in the presence of an intact BBB, immune-related elements can befound. In the last decades it has become clear that particularly astrocytes andmicroglia exhibit immunological properties. Furthermore, it has become evidentthat these cells play an important role in neuroinflammation. Moreover, it is nowgenerally assumed that astrocytes and microglia are the endogenous immune cells ofthe CNS, participating in CNS immune surveillance and the initiation of neuroin-flammation during disease 18. Although this new knowledge has caused a majorchange in the view on the CNS as being immune-privileged, the CNS remains dif-ferent and is relatively limited in its immunological properties compared to periph-eral tissues.

Immune function of astrocytesAstrocytes constitute the major glial cell population in the CNS. Thus, in the humanbrain the number of astrocytes is tenfold higher than that of neurons. Previously,astrocytes were considered simply as “brain glue”. Nowadays, it is known that astro-cytes are involved in a plethora of brain-functions, including extracellular ion andtransmitter homeostasis, synaptogenesis, synaptic transmission and synaptic plastic-ity 129,159. Under neurodegenerative conditions, astrocytes proliferate and change theirmorphology. This process, called astrogliosis, is accompanied by an increased expres-sion of glial fibrillary acidic protein (GFAP). Astrogliosis can be both beneficial anddetrimental for neuronal survival. On the one hand, astrocytes are a source of neu-rotrophins and other growth and neuroprotective factors 42,52,61. Alternatively, astro-cytes can also produce neurotoxic substances. In addition, the formation of glial scartissue by astrocytes impairs neuronal function and prevents the outgrowth of newneuronal processes 11,40,46.

One aspect of astrocyte immune function that has been known for a long timeconcerns their contribution to the formation and maintenance of the BBB.Astrocyte foot processes are positioned very close to and surround the microvascu-lar endothelial cells (fig. 1-1). Thereby, astrocytes contribute to the structural andfunctional integrity of the BBB. It is thought that by this close association with the


BBB, astrocytes play an important role in regulating BBB permeability 1.Secondly, astrocytes can produce a wide variety of cytokines and chemokines,

immunological mediators that orchestrate immune reactions and leukocyte-infiltra-tion. Under normal conditions, cytokine and chemokine production in the CNS isvery limited. However, increased and abnormal expression of these immunologicalmediators is observed in various acute and chronic situations of neurodegeneration.Released cytokines and chemokines influence both neurotoxic and neuroprotectiveprocesses in the CNS. Thus, by producing cytokines and chemokines, astrocytesplay a central role in the type and extent of CNS immune and inflammatoryresponses 5,37.

Another, albeit controversial, immune-related feature of astrocytes is their abilityto regulate T-cell responses via presentation of antigen 40. Proper antigen presenta-tion, as observed in professional antigen presenting cells (APCs), requires the expres-sion of major histocompatibility complex (MHC) proteins and additional co-stimulatory molecules like B7-1 and B7-2. When both MHC-antigen complex andco-stimulatory molecules are presented to T-cells, proliferation and differentiation of T-cells towards memory and effector cells is initiated. Antigen presentation withoutproper costimulation leads to anergy or apoptosis of T-cells.

Astrocytes are generally considered as poor APCs. They do not constituitivelyexpress class II MHC molecules. However, in cultured astrocytes class II MHCexpression is induced by the cytokines interferon γ (IFN-γ) and tumor necrosis fac-tor α (TNF-α) 6. Although increased levels of these two cytokines are found in dif-ferent neuropathologies, conflicting results on the documentation of class II MHCpositive astrocytes in the diseased CNS have been reported 40,153,173. Also the findingsregarding astrocytic expression of the co-stimulatory molecules B7-1 and B7-2 andtheir ability to induce T-cell proliferation are inconsistent 6,35,144. Despite these con-tradictory reports, it is assumed that under certain circumstances astrocytes can pre-sent antigens. Moreover, it is thought that astrocytes can regulate T-cell responses bydifferential expression of co-stimulatory molecules.

Immune function of microglia: The macrophages of the brainMicroglia comprise about 10-20% of the total glia population in the adult humanbrain. These cells appear as morphological variable cells, depending on the place andconditions in the CNS. The nature and origin of microglia has been a matter ofdebate. In general, it is assumed that microglia are derived from mesodermal pro-genitors, most likely monocytes, which invade the developing brain.

In the healthy brain parenchymal microglia appear as cells with a small cell bodyand extensively branched processes. Although the function of microglia in the nor-mal CNS parenchyma remains elusive, it is thought that these cells, together with


astrocytes, constantly monitor their microenvironment and play a major role inhomeostatic and repair functions. This is in line with the fact that microglia are veryrapidly activated in response to neuronal damage 135,152. Such microglia activation isaccompanied by cell proliferation and an impressive change in morphology andimmunophenotype: from resting ramified cells into macrophage-like cells withphagocytic capabilities 19,149.

The immunophenotypical and functional alterations that accompany microgliaactivation include up-regulated expression of cell surface adhesion molecules andcomplement binding receptors, and increased motility. Moreover, as observed inastrocytes, activated microglia synthesize multiple factors, like nitric oxide, cytokinesand chemokines, which can exert immuno-and neuromodulatory effects 63,100.Whereas microglia produce numerous regulatory factors, their own activities are alsoregulated by various cytokines and growth factors 66,103,116.

Microglia are the most potent APCs in the CNS 4. It is known that microglia,once activated can process proteins and express class II MHC on their cell surface.In addition, activated microglia express co-stimulatory molecules and are very wellable to stimulate T-cell responses. Evidence is emerging that the antigen presentingability of microglia is crucial for proper T-cell-mediated post injury immuneresponses and subsequent recovery 140. During neuroinflammation microglia phago-cytose apoptotic T-cell infiltrates, a process critical in the resolution of the inflam-matory infiltrate. This is especially important in autoimmune T-cell inflammationas observed in the autoimmune disease multiple sclerosis (MS). Interestingly, phago-cytosis of apoptotic T-cells leads to a decrease in the secretion of inflammation sus-taining cytokines and chemokines induced by lipopolysaccharide (LPS) 92.

Phagocytosis of debris and dying cells by microglia in the CNS is regarded as abeneficial process. It has been demonstrated in in vivo axotomy models that activa-tion and recruitment of microglia contributes to repair and regeneration of neuraltissue 151. However, controversy exists on the nature of the other features associatedwith microglia activation. It is generally believed that the factors released by activat-ed microglia are predominantly neurotoxic and responsible for secondary bystanderneuronal death. Nevertheless, in vitro studies have demonstrated both neurotoxicand neuroprotective effects of microglia or microglia conditioned media on culturedneurons 44,79,122,165. An interesting theory proposes microglia activation to primarilyserve a beneficial purpose. According to this theory, production of neurotrophic fac-tors would help survive reversibly injured neurons, whereas irreversible injury wouldtrigger a neurotoxic microglial response. This immediately implies the existence of acommunication pathway between neurons and microglia. Since astrocytes are alsoparticipating in neuropathological processes, similar communication mechanismsbetween neurons and astrocytes and astrocytes and microglia are very likely. The



Figure 1-2 Working hypothesis of chemokines as mediators of several functions in the CNS.

Chemokines most likely mediate a variety of different functions in physiological and also in pathological

processes of the CNS. It is likely that at least three different endogenous brain cell types participate in

chemokine signalling: neurons (N), astrocytes (A) and microglia (MG).

(a) Several lines of evidence, including chemokine and chemokine receptor knock out models, suggest that

chemokines like CCL2, CCL3, CCL5 and CXCL10 (thought to be derived from glial cells) might be crucial

molecules for the attraction of blood leukocytes into the CNS. (b) Recent evidence has shown fast expres-

sion of neuronal chemokines like CCL2, CCL21 and CXCL10 in response to neuronal damage. It has been

suggested that these chemokines are involved in the communication between damaged neuron and sur-

rounding glial cells. (c) Chemokine and chemokine receptor expression in both astrocytes and microglia

also suggest chemokinergic communication between these two cell types. (d) In vitro experiments provide

evidence that several chemokines (CCL2, CCL5, CXCL8 and CXCL10) have direct neuroprotective prop-

erties and it is tempting to speculate that these chemokines released from glial cells may have similar

effects in vivo. (e) It has been shown that CXCL12 influences neurotransmission via direct and indirect

effects in vitro. If CXCL12 is released from astrocytes and has similar effects in vivo, this modulation of neu-

rotransmission may be another aspect of chemokine activity in the brain. (f) Chemokine receptors like

CXCR4 and CCR5, which are expressed in microglia and neuron, seem to be directly involved in the infec-

tion of microglia with HIV and in gp120 induced neurotoxicity.

factors involved in neuron-glia signaling pathways are still unknown, however,recent evidence points to chemokines as candidate communication molecules 152

(fig 1-2).In addition to parenchymal microglia, perivascular cells, that exhibit microglia-

like properties, are found in close proximity of cerebral vessels (see also fig. 1-1).Considering their morphology and immunophenotype, these cells very muchresemble blood-derived macrophages. In humans steady state turnover of perivascu-lar cells has been described during which perivascular macrophages are continuous-ly replaced by circulating monocytes. During pathologic circumstances, accumula-tion of perivascular cells in the perivascular space has been observed 123. Perivascularcells are known to constitutively express class II MHC molecules and chemokinereceptors. Moreover, class II MHC and B7 costimulatory molecules are upregulatedin inflammatory CNS conditions like MS. In combination with their capacity tophagocytose and their close interaction with the BBB, it is very likely that these cellsparticipate in antigen presentation to infiltrating T-cells during neuroinflam-mation 59,169.

In summary, glial cells play an important role in CNS homeostasis. It has becomeclear that these cells actively participate in processes related to neuronal develop-ment, -signaling, -plasticity and neurodegeneration and -regeneration. In addition,both astrocytes and microglia are “immunocompetent” cells that participate inimmune surveillance and the induction of neuroinflammation. Based on this widerange of glial activity, intimate neuron-glia communication pathways have beenassumed. The exact nature of this neuron-glia signaling remains to be further eluci-dated, but presumably cytokines and chemokines play an important role.

Chemokines in neuroimmunologyChemokines constitute a superfamily of small proteins (8-14 kDa) that are instru-mental for trafficking of leukocytes in normal immunosurveillance as well as thecoordination of infiltration of inflammatory cells under pathological conditions.

Due to extensive research and rapid progress in the field, a large number of reviewson chemokines has appeared, addressing their functions and localization includingthe central nervous system (CNS) 12,16,68,99,128. Research on chemokines in the brainhas primarily focussed on immune responses and local inflammation. Recently, newfunctions of chemokines like their involvement in neuronal development, nocicep-tion and synaptic transmission have been described 21,83,88,115,126,155. In the last fewyears, data on physiological and pathophysiological functions and localization ofchemokines and their receptors in the brain have been accumulating at high speed.


Chemokines and their receptors: Pharmacology and functionChemokines and their receptors constitute an elaborate intercellular signaling sys-tem. Currently, approximately 50 different human chemokines have been described.These chemokines interact with 18 different chemokine receptors 107,108,174. Becausechemokines and their receptors have been discovered and characterized by various


groups simultaneously, most chemokines are known by multiple names. Therefore,a new systematical nomenclature has recently been introduced (table 1-1).

Chemokines are classified by their structure, based on the number and spacing ofconserved cysteine motifs in the NH2-terminal. Thus, four groups named the C,CC, CXC and CX3C families have been distinguished. The classification of thechemokine receptor family parallels the four subgroups for chemokines; thus fourreceptor subgroups XCR, CCR, CXCR and CX3CR have been designated. Mostchemokine receptors recognize more than one chemokine ligand (see table 1-1).Since the different types of immune cells express multiple chemokine receptors,which overlap in their ligand specificities, chemokine receptor pharmacology is verycomplex. In addition, several naturally occurring chemokines can also function asreceptor antagonists, this suggests complex feedback in the regulation of leukocyterecruitment 85,86,171. Due to this promiscuity and the lack of both selective agonistsand antagonists the study of chemokine receptors in native systems is difficult.

Chemokines bind to seven transmembrane spanning receptors 108 and activate het-erotrimeric G-proteins. Generally, G-proteins activated by chemokine receptorsbelong to the Gαi family and are pertussis toxin sensitive. The signal transduction ofmost chemokine receptors often comprises inhibition of cyclic adenosinemonophosphate (cAMP) and transient increases in intracellular calcium.Furthermore, down-stream activation of mitogen-activated protein kinases(MAPK), phosphatidylinositol-3’ kinase (PI3K) and small GTP-binding proteinslike Rac, RhoA and Cdc42 presumably are involved in the chemokine-inducedcytoskeletal re-organization necessary for cell migration 133,156. In addition, activationof protein tyrosine Janus kinases (JAK) and the signal transducers and activators oftranscription (STAT) cascade has been observed after chemokine receptor activationand dimerization 146. As for other G-protein coupled receptors (GPCRs), chemokinereceptor mediated signaling can be modulated by several GPCR-regulating mecha-nisms. Among these, regulators of GPCR signaling (RGSs), receptor internali-zation and post-translational modifications (e.g. palmitoylation) have beendescribed 23,105,141.

As mentioned previously, the most studied response to chemokine stimulation ischemotaxis. Chemoattraction of different types of leukocytes is mediated by com-plex combinations of chemokine receptors. A clear distinction can be made betweenhomeostatic chemokines, which are mainly involved in physiological traffic likeimmune surveillance and inducible chemokines that are induced during inflamma-tion. It is now becoming increasingly clear that distinct migratory activity of lym-phocytes, and presumably also of monocytes and granulocytes at various stages ofactivation, are fine tuned by complex combinations of homeostatic and inducible


chemokines 104. In addition to chemotaxis, a number of other biological functions ofchemokines such as induction of cell adhesion, phagocytosis, T-cell differentiationand activation, apoptosis, angiogenesis, proliferation and cytokine secretion havebeen observed 90.

Chemokines and chemokine receptors in the CNS The first findings showing prominent expression of chemokines and their receptorsin brain tissue have been published approximately 10 years ago (see for example: 5 and 127). Meanwhile, numerous detailed studies on CNS chemokines andchemokine receptors have been published, and it is clear now, that the endogenouscells of the CNS synthesize distinct chemokines and might respond to chemokinestimulation by chemokine receptor expression 12,16,99,128. Interestingly, the evolution-ary most conserved chemokine CXCL12 and its exclusive receptor CXCR4, are cru-cial for normal CNS development 89. Therefore, it has been suggested that CXCchemokines actually originate from the CNS, where they were initially involved inprocesses related to CNS development e.g. chemotaxis of neurons. Later in evolu-tion, when the higher vertebrate systems required a more specialized immune sys-tem, CXC chemokines were recruited to perform similar chemotactic activities inthe periphery 72. An additional hypothesis further states that pirating of chemokinesand chemokine receptors has forced the expansion of chemokines to deal with thecontinuous threat of viral infections. Resulting in the highly redundant chemokinesystem we know today 72,106. On the other hand, the common ancestor of CXCchemokine receptors may initially have served an immune role with a high evolu-tionary rate that slowed down when this receptor acquired a role in CNS processes 142.

Several lines of evidence indicate that all types of endogenous cells of the CNS(astrocytes, oligodendrocytes, microglia and neurons) express functional chemokinereceptors 24,36,41,95,112. Numerous publications show that the expression of chemokinereceptors in CNS cells is regulated by inflammatory mediators like transforminggrowth factor (TGF) β1 or IFN-γ 62,76 but most reports indicate that there is alsochemokine receptor expression in “unchallenged” cells. The expression ofchemokines in the CNS is mostly inducible by inflammatory stimuli, whereas con-stitutive chemokine expression is hardly observed in normal brain. The twochemokines that are constituitively found in CNS are CX3CL1 and CXCL12,which are expressed by neurons and astrocytes respectively 17,65,94.

Compared to the large number of chemokines described in the periphery, evenunder pathological conditions relatively few chemokines have been found in brain.These few chemokines, however, seem to play a crucial role in neurodegenerationsince most neurodegenerative diseases known are accompanied by chemokineexpression. The most prominent chemokines found in the CNS are CCL2, CCL3,


CCL4, CCL5, CCL8, CXCL8 and CXCL10 12,16,68,99,128. Recent publications suggest,that glial chemokines are crucial mediators of infiltration in CNS inflammation15,48,70,73,143. This implies that inhibiting glial chemokine expression may provide novelsophisticated therapies for neurodegenerative diseases. Experiments performed incultured glial cells show that the expression of glial chemokines is regulated by dif-ferent pro- and anti-inflammatory factors, like cytokines, bacterial toxins, β-amyloidor viral proteins. In addition, induction of glial chemokine production by variousbacterial substances has recently been shown to be mediated by activation of Toll-like receptors 43.

Interestingly, these factors induce expression of specific chemokines, suggestingthat the expression of glial chemokines is dependent on distinct pro- and anti-inflammatory factors 69,74,87,95. Thus, chemokine expression and immune cell infiltra-tion in particular disease pathologies may depend on the pro- and anti-inflammato-ry factors associated with the disease. Unfortunately, little is yet known on the reg-ulatory mechanisms of glial chemokine expression in vivo.

CNS chemokines are not only expressed in glial cells. It has been shown recentlythat chemokines such as CCL2, CCL3, CCL21 and CXCL10 are also induciblyexpressed in neurons under conditions of neuronal degeneration 22,29,50,136,163.Induction of neuronal chemokines is generally fast compared to the expression ofthe same chemokine in glial cells. Whereas it often takes 1-3 days after neuronaldamage before glial chemokine expression is detectable, in neurons chemokines areexpressed within 6-12 hours 22,29,50,136,163. This rather early expression time point indi-cates a special function of neuronally derived chemokines. It has been suggested thatneuronal chemokines might contribute to the early communication system betweenneurons and glial cells after neuronal damage 152.

Functions of chemokines in the CNSIn the brain chemokines mediate early local immune responses and subsequentlyalso attract leukocytes, which are believed to migrate along a chemokine gradientacross the BBB to their target. As mentioned previously, astrocytes and microglia areCNS-resident immune cells, which can produce different kinds of chemokines.Since glial cells are closely associated with the BBB, it has been suggested that thesecells regulate leukocyte infiltration into the brain 18. In addition, chemokine bind-ing sites on human brain microvessels have been described 8.

As mentioned, almost all neurodegenerative diseases have been associated withexpression of chemokines (see table 1-2) and with attraction of local glia cells andleukocytes. The type of chemoattraction may specifically favor either local immunecells or predominantly blood leukocytes 57. Most likely, this depends on the types ofchemokines expressed upon different pathologies.


The most elaborately investigated neurodegenerative disease, characterized by leuko-cyte infiltration is MS. The pathology of MS is characterized by infiltration andaccumulation of macrophages and autoreactive T-cells, which are involved in break-down of the myelin sheats surrounding axons. In MS it has been demonstrated thatdistinct chemokines are expressed during the disease, which is accompanied byattraction and infiltration of leukocytes 145 (table 1-2). Similar expression andchemoattraction has also been observed in experimental autoimmune encephalitis(EAE), an animal model for MS 49,77. The prominent role for chemokines in thedevelopment of EAE has become clear from studies using knock out models. It hasbeen shown that CCR2 knock out mice develop reduced and delayed signs of MSpathology, which is accompanied by the absence of localized macrophage infiltration48,73. Similarly, CCL2 knock out mice show a clear reduction of EAE pathology anda strong reduction of macrophage infiltration into the brain 70. However, it should


be noted that the clinical outcome of EAE studies in chemokine/chemokine recep-tor knock out mice is also strongly dependent on the genetic background of the usedmouse-strain 56. Another interesting EAE study has been performed in CXCL10knock out mice. It was demonstrated that, although CXCL10 expression appears tobe a major attractant for activated CXCR3 positive T-cells in EAE, CXCL10 knock-out mice developed EAE to the same extent as wild type animals. An explanationmight be that these mice increased expression levels of an alternative CXCR3 ligand,e.g. CXCL11, suggesting a compensation mechanism. Another striking finding wasthe fact that CXCL10 knockout mice were actually even more susceptible to acquirethe disease, when the administered immunization dose was lower. In these mice anincreased antigen-specific Th1 response was observed in the lymph nodes, whichcorresponded with diminished expression of the immunosuppressor TGF-1β 80 .

A prominent example of localized neuroinflammation is Alzheimer’s’ disease 172. Itis suggested that chemokines expressed in the vicinity of amyloid plaques initiallyattract and/or activate local glia cells, whereas at a late stage infiltration of leukocyteshas been observed. The local inflammatory response of chemoattracted microgliaseemingly remains restricted to a small area around β-amyloid plaques.

In addition, involvement of chemokines in host defense against bacterial and viralinfections of the CNS has been suggested 13,78. The most prominent example ishuman immunodeficiency virus (HIV), which uses the chemokine receptorsCXCR4 or CCR5 as co-receptors to infect cells. The expression of these receptorsmost likely enables HIV to infect brain microglia, which are the brain endogenousvirus reservoir (see for review: 102). New results moreover indicate that chemokinereceptors are not only responsible for an HIV infection of target cells but might alsoplay an important role in the development of AIDS-related dementia 102. It is cleartoday that the glycoprotein gp120 from the envelope of HIV-1 has direct neurotox-ic effects 25,27,101,102. Since gp120 directly binds to chemokine receptors it has been sug-gested that they are also mediating the neurotoxic effects of gp120 102. This assump-tion has been corroborated by the findings that hippocampal neurons from patientssuffering from HIV encephalitis had higher expression levels of chemokine receptorsthan hippocampal neurons from HIV patients without encephalitis or controls 120.

Neuroprotective effects of chemokinesIn a few studies neuroprotective properties of chemokines have been demonstrated.For instance, it has been shown that co-stimulation with the chemokines CX3CL1,CXCL12, CCL3 and CCL5 prevents gp120-induced apoptosis in neurons 25,27,101.Recent data moreover indicate that chemokines like CXCL8, CXCL12, CCL5 andCCL2 protect neurons also from other forms of neuronal damage, like for exampleNMDA- or β-amyloid-induced neuronal death 26. Although there are conflicting


reports on the neuronal chemokine receptor subtype involved (CX3CR versusCXCR4 and CCR5: see for example 27,101), these results strongly indicate thatglial derived chemokines may have significant impact on the survival of neurons andunderlines the function of chemokines in the communication between glial cells andneurons. The fascinating results published by Bezzi and colleagues are in line withthis assumption. They could show that signaling based on CXCL12, CXCR4, TNF-α and glutamate between neurons, astrocytes and microglia had direct influence onthe viability of the neurons in the system 20.

Chemokines and neurodevelopmentIt has been suggested that chemokine receptor expression in neurons plays a role indevelopmental organization of the brain by regulating the migration of neuronalprogenitors. In rodents developmental expression of chemokines has been demon-strated 88,155. Accordingly, chemotactic recruitment of progenitor cells expressing thechemokine receptor CXCR4 seems to be crucial in CNS development, as CXCR4knock-out mice die soon after birth and show abnormalities in brain morphology 89.In humans, developmental expression of neuronal CCL2 98 and astrocytic CXCR3160 was demonstrated in fetal nervous tissue. The role of these proteins in neurode-velopment, however, remains to be established. Most recent studies also indicate thepresence of chemokine receptors on neural stem cells, indicating that these may playa role in the migration of stem cells to damaged places in the CNS 158.

Effects of chemokines on neurotransmissionMany astrocytes are intimately associated with synapses and the modulation ofsynaptic transmission by astrocytes has been studied extensively. It was thus reportedrecently that glutamate released from astrocytes controls the efficacy of synaptictransmission 21. Recent findings suggest that chemokines regulate glutamate releasefrom astrocytes. It has also been shown recently that the chemokine CXCL12 mod-ulates spontaneous synaptic activity in Purkinje cells in cerebellar slices 83,126. Thismodulation of synaptic activity was due to glutamate released from astrocytes afterstimulation with CXCL12 21,83.

Besides indirect modulation of synaptic activity by chemokines via release of astro-cytic neurotransmitter release, direct effects of chemokines on the electrophysiologyof neurons have recently been published. It has been shown that CXCL8 reducescalcium currents in cholinergic neurons by closure of N- and L-type calcium chan-nels 124. Another study demonstrated CXCL12-mediated inhibition of synchronizedcalcium spikes in hippocampal neurons 84. In addition, acute exposure of hip-pocampal neurons to CXCL10 has been reported to inhibit long-term potentiationvia its receptor CXCR3 162. Moreover, a direct effect of chemokines on chemokine


receptor expressing nociceptive neurons and subsequent enhanced sensitivity to painhas been claimed 115.

In summary, it is now well established that complex combinations of chemokinesare involved in specific recruitment of immune cells into the brain. The involvementof a large number of chemokines with overlapping biological activity suggests redun-dancy of this signaling system. However, specific knock out of chemokine genes hasshown to prevent pathological processes in disease models in experimental animals.In addition, novel functions concerning intercellular signaling may be involved inmaintaining CNS homeostasis, neurodevelopment, synaptic transmission, aging 47

and the development of tumors 131. Given the broad spectrum of chemokine func-tions in the periphery additional novel effects of chemokines in the brain may beexpected.

CCL21 and its receptors in the CNS:A possible neuron-glia communication pathwayThe chemokine CCL21 was described for the first time in 1997 by Hedrick andZlotnik and later in the same year by Nagira and co-workers 67,109. Regarding itsstructure CCL21 is unique since 6, instead of 4, cysteines are found near the N- ter-minus. CCL21 is expressed predominantly in the periphery, more specifically in theT-cell zone and high endothelial venules (HEV) of lymph nodes 109. Here CCL21regulates the homing of mature dendritic cells and naïve or activated T- and B-cells28,60,110,170 via activation of its receptor CCR7. Another peculiar feature of CCL21 isthe fact that besides activating CCR7, CCL21 can bind and activate CXCR3 areceptor of the CXC family, albeit with lower affinity than the other ligandsCXCL9, CXCL10 and CXCL11 (see table 1-1) 147,164. However, this only appears tobe the case in mice, as human CCL21 has not been found to bind and activatehuman CXCR3 in recombinant expression systems 75,164.

In 2000, Gosling et al. reported on another receptor able to bind CCL21, calledCCX-CKR 58. Two years later, the mouse homologue of this receptor was describedby Townson et al. 157. These receptors were, however, disqualified from the list ofofficial chemokine receptors, as no functional activity after ligand binding has beenobserved 107. It has been suggested that these receptors act as scavenger receptors,binding excess of chemokines in the extracellular space.

A few studies on expression of CCL21 in the CNS have been performed. Recently,induction of CCL21 and CCL19 was found in the endothelium of venules in theBBB during EAE. Expression of CCL21 was accompanied by infiltrates of mainlyCCR7 and CXCR3 positive T-lymphocytes 34. In the CSF of patients suffering from


MS elevated levels of CCL19 and CCL21 have also been found 7. Furthermore,transgenic mice overexpressing CCL21 in oligodendrocytes show a significantinflammatory response in the brain and spinal cord, accompanied by hypomyelina-tion, spongiform myelinopathy, myelin breakdown, reactive gliosis and infiltratesconsisting primarily of neutrophils and eosinophils. Interestingly, transgenic miceoverexpressing the chemokine ligand CCL19, which also activates CCR7, did notshow any of these characteristics 31.

A fast induced neuronal CCL21 expression in ischaemic mouse brain was recent-ly demonstrated 22. Based on this rapid induction of CCL21 in ischaemic neurons,it has been suggested that CCL21 might be a messenger that activates surroundingglial cells in order to trigger either neurotrophic or neurotoxic reactions. Indeed, cul-tured murine microglia respond to CCL21 with chemotaxis, raised intracellular cal-cium levels and altered electrophysiology 22,130. Nevertheless, the common receptorfor CCL21, CCR7 has never been found in resident cells neither in the healthy norin the diseased CNS. In a recent study, using knockout mice, the role of the “alter-native” receptor for CCL21, CXCR3, has been addressed. Since cultured microgliafrom CXCR3 knockout mice do not respond to CCL21, whereas CCR7 knockoutmicroglia remain unaffected in their responses, CXCR3 is suggested to be the recep-tor for CCL21 in the CNS 130. Thus a neuron-glia communication pathway involv-ing neuronal CCL21 and glial CXCR3 is suggested to play a regulative role in thefirst phase after neuronal damage.

Chemotaxis and its molecular basis:Directional sensing and polarization Chemotaxis is essential for all multicellular organisms, since it enables directedmigration of cells during development, immune reactions and wound healing. Twokey processes underlying chemotaxis have been identified: directional sensing (theability to sense chemoattractant gradients) and cell polarization. Detectable chemo-tactic gradients ranging from the front to the back of cells can be as small as 2%.Following gradient detection, complex signaling cascades, involving many differentintracellular molecules, enable cells to establish polarity and migrate. Most of therecent knowledge on chemotactic movement comes from studies in the amoeboideukaryote Dictyostelium discoideum, but similar mechanisms have also been investi-gated in leukocytes.

Directional sensingThe exact mechanism by which cells sense their direction to migrate and how theinteraction of a chemokine with its receptor eventually leads to migration is still notcompletely understood. Much progress, however, has been made in identifying the


signaling cascades involved. Several theories regarding directional sensing have beenproposed. It has long been assumed that the distribution of chemoattractant recep-tors would be polarized, e.g. more receptors at the leading edge, towards an increas-ing attractant concentration. In fact, asymmetrical membrane distribution has beenobserved for different chemoattractant receptors, including some chemokine recep-tors, such as CCR2, CCR5 and CXCR4 113,161. Front-rear polarity of receptors anddownstream mediators in migrating cells could be mediated by membrane raftmicro-domains that segregate raft and non-raft protein 93. However, more recentstudies have demonstrated homogeneous receptor distribution in the presence of achemoattractant gradient, indicating that asymmetrical receptor distribution is notrequired for establishing cell polarity. Therefore, it is now suggested that intracellu-lar components become polarized by a local activation-global inhibition mechanism,which amplifies the outside chemoattractant gradient 33,39.

Polarization and the cytoskeleton The ability of cells to assume a polarized morphology and subsequently to migrate,depends on the presence of a dynamic cytoskeleton (i.e. actin and microtubule net-works). According to most theories the first step in locomotion is the extension ofcell protrusions at the leading edge (see for recent review Raftopoulou and Hall2004). These protrusions (pseudopodia, lamellopodia and filopodia) are created bypolymerization of globular actin (G-actin) into filamentous actin (F-actin). In D.discoideum and leukocytes, analysis of actin polymerization has revealed the exis-tence of a biphasic response. During the first rapid phase, cells freeze motion andround up and in the second phase of actin polymerization new pseudopodia areformed and cells start moving 30.

In an immobile state, F-actin stress fibers are interacting with extracellular matrix(ECM) molecules via integrins at the so-called “focal complexes” or “focal adhe-sions”. These adhesion sites are composed of numerous proteins including paxillin,vinculin, and other tyrosine-phosphorylated proteins. The first morphologicalchanges that are observed during migration concern the formation of filopodia andsubsequent lamellopodia, which is accompanied by a decrease in stress fibers. Newlyformed protrusions are anchored to the ECM by formation of new focal complex-es; this appears to be a highly hierarchical mechanism. Next, focal complexes matureinto focal adhesions 81,167. At the rear edge, or uropod, adhesions become loosenedand the cell body is pulled towards the leading edge by contraction of the actin-myosin filament network 3. As adhesions break down at the rear of migrating cells,a fraction of the integrin remains associated with the substrate while the cytoplas-mic adhesion components slide along the edge of the cell 167.Stabilization of cell polarity is achieved through reorganization of the microtubule


cytoskeleton, which also becomes polarized during migration. The microtubulecytoskeleton originates in the microtubule organizing center (MTOC) that is local-ized near the nucleus. From the MTOC microtubules project towards the cellperiphery where they end and are capped by the actin network. In addition to re-orientation of the MTOC, accumulation and growth of microtubules at the leadingedge takes place 168.

Intracellular signaling cascades leading to migrationAs mentioned above, chemokines activate GPCRs that induce chemotaxis. Multipleevidence suggests that chemotaxis depends on βγ dimer release following activationof Gαi-coupled chemokine receptors 9,111,119. Release of βγ subunits results in the acti-vation of phospholipase C (PLC)β, which cleaves phosphatidyl-inositol-biphos-phate (PIP2) into diacylglycerol (DAG) and inositol-triphosphate (IP3).Subsequently, IP3 is the trigger for release of calcium from intracellular stores, bybinding to IP3 receptors. A role for calcium in regulating chemotaxis has been sug-gested 121. Besides IP3 mediated calcium release, the calcium-mobilizing metabolitecyclic ADP ribose (cADPR) regulates intracellular calcium release and a regulatoryrole of cADPR on chemokine receptor mediated chemotaxis in human monocyteshas been demonstrated 118. However, controversy exists on whether calcium releaseis actually crucial for chemotaxis, as PLCβ does not seem to be necessary for chemo-taxis 82.

Regarding glial cells, it has been demonstrated that microglial control of motilityand rearrangement of actin by complement 5a, requires a certain level of intracellu-lar calcium, but is not dependent on additional calcium transients 114. In addition tocalcium transients, chloride currents appear to play an important role in chemotaxismediated by the chemokine receptor CXCR3 in microglia 130.

Another mechanism associated with chemotaxis involves receptor dimerizationand activation of the tyrosine kinase pathway and JAK/STAT signaling cascade 139,146.This G-protein independent JAK/STAT activation appears to be crucial for theinduction of migration, as inhibition of JAK/STAT completely blocks chemotaxis96,97,146. However, the subject of most published reports on chemotaxis mechanismsconcerns two important classes of mediators: Phosphatidylinositol-3’ kinase (PI3K),its products and Rho GTPases.

Phosphatidylinositol-3’ kinase and its products: PI(3,4)P2 and PI(3,4,5)P3

Recently, it was discovered that during chemotaxis a number of pleckstrin homolo-gy (PH) domain containing proteins accumulate at the leading edge in D. dis-coideum. Among these proteins are calcium release activated calcium (CRAC),Akt/PKB and PH domain–containing protein A (PhdA) 138. It is known that PH


domains are important for protein-protein interactions. Moreover, PH domainsassociate with phospholipids in the cell membrane. Using this mechanism, PH-con-taining proteins are recruited in the proximity of their substrates present at the cellmembrane 2. The phospholipids phosphatidylinositol 3,4 biphosphate (PI(3,4)P2)and phosphatidylinositol 3,4,5 triphosphate (PI(3,4,5)P3) are the main candidatesfor binding of the PH domain containing proteins. A biphasic accumulation ofPI(3,4)P2 and PI(3,4,5)P3, that parallels biphasic actin polymerization, has beenobserved specifically at the front end of migrating D. discoideum 30,71. Temporal and spatial distribution of intracellular levels of PI(3,4)P2 and PI(3,4,5)P3

are regulated by two enzymes: phosphatidylinositol-3’ kinase (PI3K) and the tumor


Figure 1-3 Molecular basis of chemotaxis.

(a) Chemotaxis, initiated by occupation of GPCRs by chemotattractants along a concentration gradient is

associated with polarization of intracellular mediators like PI(3,4,5)P3. The amount of PI(3,4,5)P3 is deter-

mined by activity of PI3K and PTEN. During polarization, PI3K is localized to the leading edge, whereas

PTEN is confined to the lateral side and uropod. (b) PI(3,4,5)P3 accumulation at the leading edge directs

and activates PH-domain containing proteins and GTPase at the leading edge membrane. Thereby actin

polymerization and reorganization of microtubules and MTOC is directed and oriented towards the lead-

ing edge. N: Nucleus, M: MTOC.

suppressing enzyme PTEN, which acts as a PI3-phospatase 39. Both PI3K andPTEN are keyplayers in the process of actin polymerization and chemotaxis 33,38,51,150.In resting cells PI3Ks are localized in the cytosol, whereas PTEN is localized at themembrane. In migrating cells, PI3Ks are found at the membrane, specifically at theleading edge. Simultaneously, PTEN is transiently released from the membrane andis subsequently targeted to the rear- and lateral membrane compartments of the cell54 (fig. 1-3).

The importance of PI3K in the induction of actin polymerization and migrationis illustrated by the fact that a blockade of PI3K results in 70-80% loss of formationof protrusions 32,134,138. D. discoideum or neutrophils that lack PI3Ks are very defec-tive in temporal, spatial and quantitative regulation of F-actin and show “retarded”chemotaxis. In these cells PH domain containing proteins (Akt/PKB, CRAC andphdA) do not localize to the celmembrane at the leading edge 55,64,82,134.

The role of PTEN has been investigated in PTEN null- as well as in PTEN- over-expressing cells. Whereas PTEN null cells exhibit augmented PI3K signaling 14,71 andchemotaxis 51, overexpression of PTEN leads to decreased chemotaxis rates. It hastherefore been suggested that PTEN is involved in global inhibition by down-regulating PI3K pathways at the uropod, while PI3K plays a central role in localactivation of actin polymerization at the leading edge. Apparently, an appropriatebalance between PI3K and PTEN is required to establish cell polarization in achemoattractant gradient 33,54.

Rho GTPases Important targets of PI3Ks are proteins belonging to the Rho GTPase family. Rhoproteins act as molecular switches by cycling between active, GTP-bound and inac-tive, GDP-bound states. Rho GTPases are regulated by guanine-nucleotide-exchange-factors (GEFs), GTPase activating proteins (GAPs) and guanine-nucleotide-dissociation inhibitors (GDIs) 45,91,125 (fig 1-4). Rho-GTPases coördinatemany cellular responses by regulating the formation of different actin assemblies.Regarding chemotaxis, three Rho-GTPases: Rho, Rac and Cdc42 and the way theyregulate the actin skeleton have gained most attention (fig. 1-3 and 1-5) (for reviewsee 125,132. In polarized motile cells Cdc42 and Rac localize to the leading edge, where-as Rho localizes mostly to the cytosol 53.

Rho is involved in bundling of actin filaments into stress fibers, focal adhesionassembly and cell contractility. Rac plays a key role in migration by regulating denovo actin polymerization at the cell periphery and the formation of lamellipodialextensions and membrane ruffles. Cdc42 is involved in actin polymerization thatleads to filopodia formation. In addition, both Rac and Cdc42 are involved in reg-ulating the formation of focal complexes 3. The role of filopodia is not entirely clear;


they play a crucial role in defining cell polarity and controlling the direction ofmigration, but do not seem to be required for migration per se. Studies in D.melanogaster have shown that loss of Cdc42 function does not affect migration ofperipheral glial cells 137. Nevertheless, Cdc42 is necessary for the formation of a sta-ble pseudopod 148.

Rho GTPases are also involved in organizing the microtubule skeleton. Rho pro-motes microtubule stabilization, while Rac may promote tubule elongation. Inmigrating astrocytes and fibroblasts, Cdc42 regulates reorientation of microtubulesand the MTOC 45,53,132. For further information on Rho GTPase signaling cascadessee the more detailed legend of figure 1-5.


Figure 1-4 Regulators of GTPase activity.

Regulators of GTPase activity: GEFs, GAPs and GDIs interact with GTPases upon upstream signals leading

to active GTP-bound GTPase or inactive GDP-bound GTPase. GDI bound GTPases are removed from the

cell membrane and thereby not available for GTPase mediated signaling. (adapted from ref. 125).


Figure 1-5 Signaling cascades of Rho GTPases and their effects on the cytoskeleton.

Three important GTPase involved in chemotaxis are Rho, Rac and Cdc42. They influence migration by

affecting both the actin cytoskeleton (left) and microtubules (right). Targets of Rho are the serine/threo-

nine kinase p160ROCK and mDia. Activated p160ROCK activates LIM kinase, which phosphorylates and

inactivates cofilin. Cofilin severs actin filaments, inactivation of cofilin thus leads to stabilization of actin fil-

aments within actin-myosin bundles. In addition, p160ROCK phosphorylates the myosin binding subunit

(MBS) of myosin light chain (MLC) phosphatase. The subsequent inactivation of MBS results in increased

levels of myosin phosphorylation, cross-linking of actin filaments and contractile force generation. mDia

belongs to the formin homology containing protein family, which are cytoskeletal organizing proteins. mDia

binds both Rho-GTP and profilin, an actin binding protein that stimulates actin polymerization 125,166. In addi-

tion, Rho signaling via mDia results in direct stabilization of microtubules 117.

Similar to Rho, the GTPases Rac and Cdc42 affect both the actin cytoskeleton and microtubules. A target

of both Rac and Cdc42 is the serine/threonine kinase p65PAK, which phosphorylates and activates LIM

kinase, which in turn phosphorylates and inactivates cofilin, thereby inducing actin polymerization 10. In addi-

tion, Rac and Cdc42 activate members of the family known as the Wiskott-Aldrich syndrome protein

(WASP) family, which are a direct link between Rho GTPases and the actin cytoskeleton 154. The WASP fam-

ily proteins are classified into two structural groups: WASPs and WAVEs and both are key regulators of

actin polymerization. All of the family members contain domains that can bind both G-actin and Arp2/3 or

profilin. Arp2/3 and profilin are actin binding proteins that stimulate actin polymerization. In an activated

state, WASPs and WAVEs are able to stimulate Arp2/3 and profilin and thus initiate actin polymerization 154.

Regarding the microtubule-system, Rac may promote tubule elongation through p65PAK-mediated phos-

phorylation and inactivation of the microtubule destabilizing protein stathmin. Cdc42 is involved in reor-

ganization of microtubules and reorientation of the MTOC via the scaffold protein Par6 and an atypical pro-

tein kinase C, PKCζ 125.

In summary, chemotaxis starts with the detection of a chemoattractant gradient.After stimulation of GPCRs multiple signaling cascades are triggered that alter thecytoskeleton and result in cell polarization. Two important regulators of chemotaxis:PI3K and Rho GTPases have been identified. The products of PI3K direct migra-tion-related proteins to the leading edge, and Rho GTPases regulate changes of theactin and microtubule cytoskeleton. The mechanism by which PI(3,4)P2 andPI(3,4,5)P3 activate Rho GTPases is probably by a direct interaction with GEFs.During cell migration, Rac and PI3K can interact directly with each other and acti-vation of Rac leads to the production of PI(3,4,5)P3. Therefore, it is suggested thatPI3K and Rho GTPase provide a positive feedback loop, which contributes toamplification of the PI(3,4,5)P3 signal 53,125,148. In addition, it has been noticed thatcell adhesion to the ECM itself can activate Rac and Cdc42, which could providean additional positive feedback loop, carrying on migration even when receptor sig-naling is downregulated 132.


Outline of the thesisIt is known that stress and damage in neurons leads to early expression ofchemokines.

We propose a “chemokine-mediated communication mechanism between neuronsand glial cells”, since glial cells express multiple chemokine receptors and are capableto react to “neuronal” chemokines.

In this thesis a possible neuron-glia communication pathway mediated by thechemokine CCL21, was investigated. By using a number of molecular biologicaltechniques and functional assays, the presence, cellular source and functional activ-ity of both CCL21 and its receptors CCR7, CXCR3 and CCX-CKR was investi-gated in CNS cells.

The second chapter of this thesis describes the induction of CCL21 expression incultured mouse neurons and brain slices under excitotoxic conditions.

In the third chapter, the presence and functional activity of receptors for CCL21,CCR7 and CXCR3 have been examined. In this chapter we identify the presenceand functional activity of CXCR3 in cultured murine astrocytes and microglia. Inthis chapter we also describe the presence and functional activity of CXCR3 in cul-tured human astrocytes and microglia

Since expression of CXCR3 was found in human microglia, we investigated themigratory ability of human microglia in response to CCL21 and compared it withCCL21 effects on CXCR3 recombinant expression systems. The results of this studyare described in the fourth chapter

In the fifth chapter a closer look on the ability of mouse microglia to expressCCR7 has been taken. Expression of CCR7 was investigated in mouse microgliaafter stimulation with LPS and in brains of mice subjected to EAE.

The sixth chapter depicts experiments that were performed in order to investigatethe role of the CCL21-binding receptor, CCX-CKR on CXCR3 mediated signal-ing.

In the seventh and last chapter of this thesis an overview of the results has beenprovided and their interpretation is discussed.


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